HK1055991A - NOVEL DIOXYGENASES CATALYZING CLEAVAGE OF β-CAROTENE - Google Patents

Description

Dioxygenases catalyzing cleavage of beta-carotene

The present invention relates to the field of transforming bacterial, yeast, fungal, insect, animal, and plant cells, seeds, tissues, and whole organisms. More specifically, the present invention relates to the integration of recombinant nucleic acid sequences encoding one or more specific enzymes of the carotenoid/retinoid biosynthetic pathway into suitable host cells or organisms which, after transformation, exhibit the desired phenotype and which can be used, for example, in commercial production. In addition, the invention provides for the implementation of oxidative cleavage C by biotechnology₄₀Means and methods for the production of different characteristic metabolites of the carotenoid/retinoid pathway from carotenoids.

Background

Vitamin a (retinol) and its derivatives (retinal, retinoic acid) (the term "retinoids" is used throughout the specification) represent a group of chemical classes of compounds involved in a wide range of fundamental physiological processes in animals. They are essential in pattern formation during, for example, vision, reproduction, metabolism, cell differentiation, skeletal development, and embryogenesis. To study the effects of retinoids (such as vitamin a), several species (e.g., mouse, rat, chicken, and pig) have been used as vertebrate animal model organisms, while most studies in invertebrates were performed with Drosophila melanogaster (Drosophila melanogaster). The fly visual system has been a model for decades for studying receptor diversity and vitamin a utilization using electrophysiology, photochemistry, genetics, and molecular biology.

Vitamin A and its most important derivatives retinaldehyde and Retinoic Acid (RA) consist of 20 carbon atoms (C)₂₀) And belong to the chemical class of isoprenoids. Animals are generally unable to synthesize retinoids de novo. The biosynthesis of retinoids in animals depends on the ingestion of carotenoids with provitamin a activity from food. In those animals which are capable of synthesizing retinoids from carotenoids, enzymatic cleavage of provitamins is necessary. Such enzymatic activity has been described, for example, in mammals, in crude extracts derived from the small intestine and liver. This enzyme catalyzes the symmetric oxidative cleavage of beta-carotene to form two molecules of retinal, and is biochemically characterized as 15, 15' -beta-carotene dioxygenase (beta-diox I). These enzymes are involved in carotenoid metabolism/retinoid formation throughout the animal kingdom. For example, fig. 1 and 9 illustrate the biosynthetic pathway for the formation of retinoids described in mammals. Xanthophylls (oxygen-containing carotenoids) can be cleaved in addition to beta-carotene as long as they have an unsubstituted beta-ionone ring (e.g., beta-cryptoxanthin); also in different animal species, the ability to metabolize carotenoids other than β -carotene to form hydroxylated retinoids (e.g., zeaxanthin and lutein in the class insecta) has been reported. For further metabolism, it is necessary to enzymatically modify the resulting retinaldehyde to formRetinol (vitamin a) or retinoic acid.

Enzymatic oxidative cleavage of carotenoids is also found in bacteria and plants. Many examples of eccentrically cleaved carotenoids are found in higher plants. Examples include safron formation in crocus sativus, the formation of limonin and other apocarotenoids in citrus fruit, and most interestingly the phytohormone abscisic acid (ABA, a growth regulator involved in, for example, fall leaf and seed dormancy). ABA is derived from the oxidative cleavage of 9-cis-epoxy-carotenoids at the 11-12 carbon double bond. Recently, the study of the ABA biosynthesis-deficient maize mutant vp14 provided a better molecular understanding of this cleavage reaction, and the cloning and molecular characterization of the first carotenoid cleavage enzyme (β -diox I) of animal origin. The problem raised by this finding is how similar enzymes participate in carotenoid/retinoid metabolism in animals, catalyzing oxidative cleavage of carotenoids with provitamin a activity. In subsequent experiments, similar enzymes (β -diox II) were indeed identified and characterized, which are also involved in the carotenoid/retinoid pathway and specifically cleave β -carotene to form β -apocarotenal (β -apocarotenol, a precursor of retinoic acid). Thus, in addition to β -diox I being a novel class of β -carotene specific enzymes, another novel class of enzymes (β -diox II) that can be identified in accordance with the present invention also performs oxidative cleavage of the same substrate, i.e., β -carotene.

The function of these important classes of enzymes in carotenoid metabolism/retinoid formation has been studied in vitro in animals for nearly 40 years. However, all attempts to isolate and purify such proteins and to identify their molecular structures have failed. The disclosure of the molecular structure of these enzymes, including their nucleotide sequences (cdnas) and their amino acid sequences, will be important for a number of fields in which the role of vitamin a/retinoids in animals and in medicine is being investigated. In addition, the genetic material can be used to transform intact living organisms, thereby allowing the production of retinoids (such as vitamin a and retinoic acid) in, for example, plants and microorganisms to increase their nutritional value.

In vertebrates, the symmetrical/asymmetrical cleavage of beta-carotene in the biosynthesis of vitamin a and its derivatives has been discussed controversially. In addition to β -diox I, the present invention also provides the identification of cdnas from mouse, human, and zebrafish that encode a second carotenoid dioxygenase, designated β -diox ii, which specifically catalyzes the asymmetric oxidative cleavage of β -carotene to form β -apocarotene aldehyde and β -ionone (known as floral aromas from, e.g., roses). In addition to beta-carotene, the enzyme oxidatively cleaves lycopene. Its deduced amino acid sequence shares significant sequence identity with β, β -carotene-15, 15' -dioxygenase, and both classes of enzymes, β -diox I and β -diox II, have several conserved motifs. As regards their function, apocarotene aldehyde formed by the enzyme acts as a precursor (and possibly other physiological effects) for retinoic acid biosynthesis. Thus, in contrast to drosophila, both (symmetrical and asymmetrical) cleavage pathways for carotene exist in vertebrates, revealing here a higher complexity of carotene metabolism.

In humans, as is generally known, retinal (i.e., the cleavage product of β -diox I) is the determining factor in vision. It is also clear that the enzymes that determine the availability of the retinoic acid direct precursor in whole organisms or individual cells will have a broad effect on the retinoic acid signaling pathway and the cellular responses mediated thereby.

Retinoids have several medical applications, such as cancer treatment. As an active ingredient in a (prophylactic or therapeutic) pharmaceutical preparation, retinoids can be used for the prevention and/or treatment of different types of cancer. For example, animal models show that retinoids can regulate cell growth, differentiation, and apoptosis, and suppress carcinogenesis in several tissues (such as lung, skin, breast, prostate, and bladder). The latter also finds application in clinical studies in patients exhibiting premalignant or malignant lesions of the oral cavity, cervix, bronchial epithelium, skin, and other tissues and organs. Some retinoids exhibit antitumor activity in vitro even on highly malignant cells, as evidenced by inhibition of proliferation and induction of differentiation or apoptosis. A prominent example of therapeutic effect is the differentiation of promyelocytic leukemia cells into granulocytes by all-trans retinoic acid, which is currently successfully used for the treatment of this type of cancer (Nason-Burchelal and Dmrosysky, in Retinoids, p 301, 1990; Xu and Lotan, in Retinoids, p 323, 1999).

The invention provides for the first time a complete molecular characterization of enzymes involved in the carotenoid/retinoid metabolism of animals, catalytic oxidative cleavage of carotenoids with provitamin a activity. The achievements of the present invention, including the discovery of complete nucleotide sequences encoding these gene types, can improve nutritional status, particularly in underdeveloped countries, by providing plants or parts thereof transformed according to the present invention. In accordance with the present invention, a novel class of enzymes, designated β -diox II, is provided which also performs oxidative cleavage of β -carotene. But in contrast to beta-diox I, it produces a second known precursor of beta-apocarotenal, retinoic acid. Thus, the present invention provides two novel classes of enzymes that specifically oxidatively cleave beta-carotene and accumulate retinoic acid precursors.

For example, vitamin a deficiency is a very serious health problem that causes severe clinical symptoms in the part of the world population where cereals are raw (such as rice as the main or almost sole staple food). In south-east asia alone, it is estimated that 500 million children develop dry eye disease each year, with 25 million eventually blinding. In addition, while vitamin a deficiency is not the ultimate determinant of death, it is associated with increased susceptibility to potentially fatal afflictions such as diarrhea, respiratory diseases, and childhood diseases (such as measles). According to statistical results compiled by UNICEF, improved provitamin nutrition can prevent the death of 100-200 million people in children aged 1-4 years each year and 25-50 million people in later childhood. For these reasons, it is highly desirable to increase vitamin a levels in the staple food.

Vitamin deficiency no longer forms a general problem in developed countries, since plant foods provide sufficient provitamin a and vitamin a can be obtained directly from animal products. However, for prophylactic reasons or in case of certain clinical and/or genetic disorders or dysfunctions that afflict e.g. resorption or correct cleavage of provitamin into vitamin a, it may be desirable to provide retinoids as functional ingredients e.g. so-called "functional foods".

Despite numerous publications and patents relating to the complete chemical synthesis of retinol and its analogues, there is a strong need for biotechnological production of these substances with high value in nutritional, diagnostic and pharmaceutical/therapeutic applications.

Summary of The Invention

The present invention provides means and methods for transforming bacteria, yeast, fungi, insects, animal and plant cells, seeds, tissues, and whole organisms to produce transformants capable of expressing asymmetrically cleaved beta-carotene dioxygenase (beta-diox II) polypeptides or functional fragments thereof and accumulating beta-apocarotene aldehyde and beta-ionone as well as apolycopene aldehyde (apo1 ycopenal). The invention also provides means and methods for producing retinoids by biotechnology using cells, tissues, organs, or whole organisms that naturally or post-transform accumulate beta-carotene or that take up beta-carotene from culture media. The invention also provides DNA molecules encoding said novel beta-carotene dioxygenases derived from different sources and taxonomic groups of living organisms and designed to be suitable for carrying out the method of the invention, and plasmids or vector systems comprising said molecules. In addition, the invention provides transgenic bacteria, yeast, fungi, insect, animal, and plant cells, seeds, tissues, and whole organisms that exhibit improved nutritional quality or physiological condition and that comprise the above-described DNA molecules and/or are produced using the methods of the invention. In addition, the invention provides antibodies that exhibit specific immunoreactivity for beta-diox II polypeptides and are useful for diagnostic, therapeutic, and screening purposes as well as for isolating and purifying such polypeptides. Finally, the invention provides means and methods for using the DNA molecules according to the invention in the field of gene therapy.

Thus, the present invention provides for the de novo introduction and expression of β -carotene cleaving enzymes in organisms that are not themselves retinoid-like, such as plant materials, fungi, and bacteria, as well as modification of existing retinoid biosynthesis to regulate accumulation of retinoids of certain purposes. In addition, the present invention provides DNA probes and sequence information that enable one of skill in the art to clone the corresponding genes and/or cDNAs from other sources, such as animal species not disclosed throughout the specification.

In addition, the present invention provides pharmaceutical formulations comprising the gene product or functionally active fragments thereof as active ingredient, as well as simple and suitable diagnostic test systems to further demonstrate the functionality of these molecules.

Brief description of the drawings

Figure 1 shows the major steps in the formation of retinoids in animals. The key steps in vitamin a formation are highlighted by thick arrows; only the all-trans isomer of the retinoid is shown.

FIG. 2 shows Escherichia coli (Escherichia coli) producing and accumulating beta-carotene⁽⁺⁾Strain) caused by expression of Drosophila melanogaster beta-carotene dioxygenase relative to a control (E.coli)^(-)Strain) from yellow (β -carotene) to almost white (retinoids).

FIG. 3 shows beta-carotene-producing E.coli (E.coli) transformed with a plasmid expressing Drosophila beta-carotene dioxygenase cDNA⁽⁺⁾Strain) against E.coli transformed with vector control (pBAD-TOPO)^(-)HPLC analysis and spectral identification of the strains. The scale bar indicates an absorbance of 0.01 at 360 nm. A. Escherichia coli⁽⁺⁾Strain (top line) and Escherichia coli^(-)Formaldehyde/chloroform extract of strain (lower line). B. Hydroxylamine/formazans producing the corresponding oximes (cis and trans) from the corresponding retinal isomersAnd (3) extracting with alcohol. In the online, the real standards are separated. Coli in the middle line⁽⁺⁾Isomeric composition of the extract of the strain. Coli in the lower line^(-)HPLC profile of the strain extract.

FIG. 4 illustrates the reaction of Escherichia coli⁽⁺⁾Absorption spectra (n-hexane) of the main substances extracted by the strain with respect to the true standard (dotted line).

FIG. 5 shows the enzymatic activity of β -diox-gex fusion protein under different conditions. The fusion protein β -diox-gex was incubated under different conditions in a buffer containing 50mM Tricine/NaOH (pH7.6) and 100mM NaCl. The reaction was started by adding 5. mu.l of beta-carotene (80. mu.M in ethanol). The reaction was terminated and extracted after 2 hours of incubation at 30 ℃. HPLC analysis was performed and an HPLC profile at 360nm was shown. The scale bar indicates an absorbance of 0.005 at 360 nm. A. In the presence of 5. mu. MFeSO₄And 10mM L-ascorbic acid; B. in the absence of FeSO₄Keeping temperature during ascorbic acid; C. incubate in the presence of 10mM EDTA; D. the fusion protein was incubated at 95 ℃ for 10 minutes before incubation.

FIG. 6 depicts the cDNA sequence and deduced amino acid sequence of β -diox II from Drosophila melanogaster.

FIG. 7 is a linear alignment of deduced amino acid sequences of vp14 (maize), RPE65 (retinal pigment epithelium, bovine), and β -dioxI (Drosophila). Identities are indicated in black and conserved amino acids according to the PAM250 matrix are indicated in grey. We used visual alignment and procedural alignment. Highly conserved regions can be found, for example, at position 549-. All β -diox homologs identified to date share this common motif, which characterizes the enzyme according to the invention.

FIG. 8 is a graph illustrating mRNA levels of β -diox I in different parts of the body. The expression pattern of β -diox mRNA was investigated by RT-PCR. Beta-diox mRNA was detectable only in the head. cDNA was synthesized from total RNA preparations derived from adult drosophila (female and male) head, chest, and abdomen. A set of intron-spanning primers was used to study mRNA levels of ribosomal protein rp49(FLYBASE accession FBgn0002626) in the same RNA samples as controls.

FIG. 9 is a schematic of mammalian beta-carotene/retinoid metabolism. Solid arrows indicate the formation of vitamin a by a symmetrical cleavage pathway. The formed retinal can be further metabolized to retinol and retinyl esters (stock) or oxidized to retinoic acid. The broken line arrows indicate the formation of β - (8 ', 10 ', 12 ') -apocarotene aldehyde by asymmetric cleavage of β -carotene. To form retinoic acid, it is necessary to shorten β -apocarotene aldehyde by a mechanism similar to that of β -oxidation of fatty acids.

FIG. 10 is a comparison of the deduced amino acid sequences of two carotenoid dioxygenases in mice. Linear alignment of deduced amino acid sequences of mouse beta-diox I (mouse-1) and mouse beta-diox II (mouse-2). Identities are indicated in black and conserved amino acids according to the PAM250 matrix are indicated in grey. The asterisk marking may involve binding of the cofactor Fe²⁺6 conserved histidine residues.

FIG. 11 shows an analysis of the products formed in an in vitro assay for β -diox II enzyme activity. Crude extracts of E.coli expressing beta-diox II were incubated in the presence of beta-carotene for 2 hours. The compounds formed were then extracted and analyzed by HPLC. A. A formaldehyde/chloroform extract; B. hydroxylamine/methanol extract. Compounds that stayed 4.6 minutes after extraction in the presence of formaldehyde/chloroform could be detected; whereas in the presence of hydroxylamine/chloroform its residence time became 16 minutes. C. UV/VIS spectrum of peak 1; D. UV/VIS spectrum of peak 2.

FIG. 12 shows the color of E.coli strains that synthesize and accumulate beta-carotene and lycopene after expression of beta-diox I or beta-diox II, respectively. A. A large intestine bacillus control strain accumulating beta-carotene; B. a beta-carotene accumulating E.coli strain expressing beta-diox; C. a beta-carotene accumulating escherichia coli strain expressing beta-diox II; D. a beta-diox II-expressing lycopene-accumulating E.coli strain; E. a control strain that accumulates lycopene.

FIG. 13 shows the detection of carotene cleavage products by HPLC analysis on E.coli extracts. HPLC analysis of the carotene cleavage products formed in the β -carotene-producing E.coli strain. The bacterial cells were extracted by the hydroxylamine/methanol method (von Lintig J and Vogt K, J.biol.chem., 275: 11915-11920, 2000). A. Extracts of β -diox I expressing e.coli strains (top line) relative to control strains (bottom line). The composition of the retinoids found is indicated in the figure. B. Extracts of β -diox II expressing e.coli strains (top line) relative to control strains (bottom line). 6 species can be detected and separated into two different types of compounds according to their UV/VIS spectra (class 1: peaks 2, 5, and 6; class 2: peaks 1, 3, and 4). C. The UV/VIS spectrum of peak 2 is representative of compounds of group 1; D. the UV/VIS spectrum of peak 4 is representative of compounds of group 2.

FIG. 14 is a linear alignment of deduced amino acid sequences of Drosophila (Drosophila β -diox I, SEQ ID NO: 2), mouse-2 (Mus musculus, SEQ ID NO: 17), human-2 (human, SEQ ID NO: 21), and zebrafish-2 (Danio rerio, SEQ ID NO: 19). Identity is indicated in black. The arrows indicate regions of putative homology with drosophila β -diox. Highly conserved regions can be found, for example, at position 549-. All β -diox homologs identified to date share this common motif, which characterizes the enzyme according to the invention.

FIG. 15 is a phylogenetic tree calculation of metazoan polyene chain dioxygenase and plant VP 14. Phylogenetic Tree calculation the adjacency (NJ) algorithm for deducing amino acid sequences based on the sequence distance method and using all metazoan polyene chain dioxygenases and the plant VP14 (Saito N and Nei M, mol. biol. Evol., 4: 406-K425, 1987). Two different types of vertebrate carotene dioxygenases are indicated by numbers 1 and 2 following the organism name. In addition to the sequences reported herein, the following sequences were used: human-1 (AAG15380), mouse-1 (Redmond TM, Gentlemann S, Duncan T, Yu S, Wiggert B, Gantt E, and Cunningham FXJr., J.biol.chem., Online, 2000), RPE65 (XP001366), RPE65 cattle (A47143), Drosophila (von Lintig J and Vogt K, J.biol.chem., 275: 11915-11920, 2000), VP14(AAB 62181).

FIG. 16 shows an assessment of the steady state mRNA levels of two carotenoid dioxygenases in different tissues of mice. The mRNA levels of β -diox I, β -diox II, and β -actin were analyzed by RT-PCR in various tissues of mice. The reaction products were loaded on a TBE-agarose (1.2%) gel for analysis. The gel was stained with ethidium bromide and the photograph was displayed. Each sample was analyzed in the presence (+) and absence (-) of reverse transcriptase to demonstrate that the PCR product was derived from mRNA.

Detailed Description

The present invention provides isolated novel beta-carotene dioxygenase (beta-diox II) polypeptides or functional fragments thereof having the biological activity of specifically cleaving beta-carotene and lycopene to form beta-apocarotene aldehyde and beta-ionone, respectively, and apolycopene aldehyde. According to a preferred embodiment of the present invention, based on the sequence information obtained from the mouse, the β -diox ii polypeptide or a functional fragment thereof comprises one or more amino acid sequences selected from the group consisting of: SEQ ID NO: 17 at positions 29-47, 96-118, 361-368, and 466-487, wherein the second and fourth portions are preferred. These regions are particularly identified by SEQ ID NO: the regions listed at positions 96-118 and 466-487 of 17 are of particular interest because they have been shown to be highly conserved in nature. Thus, one skilled in the art can readily design, synthesize, and use sequences derived from SEQ ID NOs: 16 and comprising one or more nucleic acid sequences selected from the group consisting of: SEQ ID NO: 16at positions 115-141, 286-354, 1081-1104 and 1396-1461, wherein the second and fourth are preferred, as suitable screening means for expression analysis or for revealing further members of such novel enzymes having the above-mentioned enzymatic activities and thus being encompassed by the present invention. Obviously, as depicted in fig. 14, the same applies to the homologous β -diox II sequences provided herein. For example, the β -diox II polypeptide or functional fragment thereof comprises, for example, SEQ ID NO: 19 (zebrafish) at positions 55-63, 112-: 21 (human) at positions 59-67, 116-138, 385-392, and 490-511, wherein the corresponding second and fourth terms are preferred. Thus, as described above, the nucleic acid sequences derived from SEQ ID NOs: 18 and/or 20 and comprising one or more nucleic acid sequences selected from the group consisting of: SEQ ID NO: 18 at position 191-217, 362-430, 378-385 and 482-503, and SEQ ID NO: 20at positions 175-201, 346-414, 1153-1176 and 1468-1533, wherein the corresponding second and fourth portions are preferred. All of these β -diox II homologs, as well as others from a number of different sources, can be readily identified and used in accordance with the principles of the present invention.

The present invention is based, in part, on the fact that essentially all plants, fungi, and bacteria do not themselves contain retinoids. While all plants, some fungi, and many bacteria are capable of synthesizing beta-carotene, they generally do not have enzymes capable of cleaving beta-carotene into retinoids. These organisms can thus be used according to the invention as a source of beta-carotene for the synthesis of retinoids after the introduction of, for example, cDNA coding for beta-carotene dioxygenase type II. In addition, such organisms that accumulate geranyl-diphosphate (GGPP) but do not produce substantially beta-carotene naturally or otherwise in the absence of downstream enzymes may also be used in the context of the present invention. The synthesis of beta-carotene requires a phytoene synthase (psy) which participates in the first carotenoid specific reaction involving two steps of reaction leading to the head-to-head condensation of two molecules of GGPP to form the first, yet colorless, carotene product phytoene. In addition, the further enzymatic pathway necessitates the complementation of three additional plant enzymes: phytoene Desaturase (PDS) and zeta-carotene desaturase (ZDS), each catalyzing the introduction of two double bonds, and lycopene beta-cyclase. In order to reduce the work of transformation, bacterial Carotene desaturases which are able to introduce all four double bonds required for a complete desaturation sequence and to convert phytoene directly into lycopene, such as the CrtI derived from Erwinia (cf. Xudong Ye et al, "Engineering the proyitinan A (. beta. -Carotene) Biosynthetic Pathway inter (Carotenoid-Free) riceEndosperm" (introduction of the provitamin A (. beta. -Carotene) Biosynthetic Pathway into the rice endosperm (which does not contain carotenoids)), Science, 287: 303-305, 2000, can be used in a preferred embodiment of the invention. For example, vectors capable of preferentially expressing both plant phytoene synthase (psy) (GenBank  accession number X78814) and bacterial phytoene desaturase (crtI) (GenBank  accession number D90087) can be used to direct the formation of lycopene in, for example, plastids that are typically substantially carotenoid-free. In addition, a second vector capable of expressing lycopene beta-cyclase (GenBank  accession number X98796) can be easily designed and used for co-transformation. However, as transformation experiments may prove, the introduction of a nucleic acid sequence encoding the lycopene beta-cyclase may not be essential, as the use of one transformation comprising a psy and crtI combined expression cassette resulted in transformants that showed accumulation of beta-carotene as well as lutein and zeaxanthin. In order to allow this pathway to proceed to the formation of retinoids such as retinoic acid or vitamin a and derivatives thereof, a nucleic acid sequence encoding a polypeptide according to the invention or a functional fragment thereof may be introduced alone or in combination with any of the other enzymes mentioned above. Thus, the present invention enables the complete introduction or complementation of the carotenoid/retinoid pathway in a given host suitably selected according to the present invention.

The term "comprising a carotenoid" or "substantially carotenoid-free" as used throughout the specification to distinguish certain target cells or tissues means that the corresponding plant or other material not transformed according to the invention is known to be generally substantially carotenoid-free, as is the storage organ (such as rice endosperm, etc.). The absence of carotenoids does not exclude cells or tissues that accumulate carotenoids in barely detectable amounts. Preferably, the term should be defined as a plastid containing material having a carotenoid content of 0.001% w/w or less.

With regard to the selection of suitable sources for the production of enzymes that cleave carotenoids, it is understood that, in addition to the β -diox I sequences disclosed herein from drosophila and β -diox II sequences from human (Homo sapiens), Mus musculus (Mus musculus), and zebrafish (Danio rerio), one skilled in the art can readily find, isolate, and use all functionally equivalent DNA molecules and fragments thereof, such as those described with respect to SEQ ID NO: 1. 16, 18, and/or 20, a sequence from an existing organism encoding an enzyme or functional fragment thereof exhibiting the same desired activity, i.e., asymmetric cleavage of beta-carotene into retinoids, and a sequence substantially homologous to a partial or complete sequence of Drosophila melanogaster (SEQ ID NO: 1), Musca musculus (SEQ ID NO: 16), Danio rerio (SEQ ID NO: 18), and/or human (SEQ ID NO: 20), for example for ensuring the expression of a beta-diox II polypeptide or a functional fragment thereof having the desired biological or enzymatic activity, i.e.specifically cleaving beta-carotene and lycopene to form beta-apocarotenal and beta-ionone and apolycopene aldehyde, respectively, or for determining the presence of a nucleic acid characteristic of said polypeptide or functional fragment thereof. For example, by using the sequence information of Drosophila melanogaster (SEQ ID NO: 1), vertebrate β -diox II homologs from human (SEQ ID NO: 20), Danio rerio (SEQ ID NO: 18), and rattus norvegicus (SEQ ID NO: 16), which are also encompassed by the present invention, can be identified by conventional screening procedures known in the art and described in further detail below.

Thus, these DNA sequences are preferably selected from the group consisting of:

(1) SEQ ID NO: 16 and/or SEQ ID NO: 18 and/or SEQ ID NO: 20 or a complementary strand thereof; and

(2) SEQ ID NO: 16at the 115-141 position, the 286-354 position, the 1081-1104 position and the 1396-1461 position or complementary strands thereof; and

(3) SEQ ID NO: 18 at positions 191-217, 362-430, 1160-1183 and 1472-1537 or complementary strands thereof; and

(4) SEQ ID NO: 20at the 175-201 position, the 346-414 position, the 1153-1176 position and the 1468-1533 position or complementary strands thereof; and

(5) a DNA sequence or a functional fragment thereof which hybridizes under high stringency conditions with the DNA sequence or the complementary strand thereof defined in (1), (2), (3), and (4); and

(6) DNA sequences which hybridize to the DNA sequences defined in (1), (2), (3), (4) and (5) if the degeneracy of the non-genetic code is not present.

Hybridization stringency refers to the conditions under which a nucleic acid hybrid behaves stably. These conditions will be apparent to those of ordinary skill in the art. As is known to those skilled in the art, the stability of hybrids is reflected by the melting temperature (Tm) of the hybrid, which decreases by about 1-1.5 ℃ for every 1% decrease in sequence homology. The stability of hybrids is generally a function of sodium ion concentration and temperature. Hybridization reactions are typically performed under higher stringency conditions, followed by washes of different stringency.

As used herein, high stringency means allowing only 1M Na⁺Those nucleic acid sequences which form stable hybrids at 65-68 ℃ are subjected to hybridization conditions. High stringency conditions can be provided by, for example, hybridization in an aqueous solution containing 6 XSSC, 5 XDenhardt's solution, 1% SDS (sodium dodecyl sulfate), 0.1M sodium pyrophosphate, and 0.1mg/ml denatured salmon sperm DNA as a non-specific competitor. After hybridization, high stringency washes can be performed in several steps, with the last wash (approximately 30 minutes) being performed in 0.2-0.1x SSC, 0.1% SDS at the hybridization temperature.

Moderate stringency refers to conditions corresponding to hybridization in the above-described solution, but at about 60-62 ℃. In this case, the last wash is performed in 1 XSSC, 0.1% SDS at the hybridization temperature.

Low stringency refers to conditions that correspond to hybridization in the above solutions, but at about 50-52 ℃. In this case, the last wash was performed in 2x SSC, 0.1% SDS at hybridization temperature.

It will be appreciated that a variety of buffers (e.g., formaldehyde-based buffers) and temperatures may be used to modify and repeat these conditions. Denhardt's solution and SSC are well known to those skilled in the art, as are other suitable hybridization buffers (see, e.g., Sambrook et al, Molecular Cloning, Cold spring harbor laboratory Press, 1989; or Ausubel et al, eds Current Protocols in Molecular Biology, John Wiley & Sons, 1990). Optimal hybridization conditions must be determined empirically, since the length and GC content of the probe also have some effect.

It should be mentioned herein that the term "substantially homologous DNA sequence" to a DNA sequence encoding β -diox II refers to a DNA sequence encoding a nucleotide sequence identical to SEQ ID NO: 17. 19, and 21, and a DNA sequence representing a polypeptide or a functional fragment thereof having the biological activity of specifically cleaving β -carotene to form β -apocarotene aldehyde and/or having the ability to specifically bind to an antibody raised against the polypeptide or the functional fragment thereof of the present invention.

According to a preferred embodiment, these DNA sequences are in the form of cDNA, genomic or artificial (synthetic) DNA sequences and can be prepared as known in the art (see, e.g., Sambrook et al, supra) or as specifically described below.

The nucleic acids of the invention can be obtained according to methods well known in the art by guidance provided herein. For example, the DNA of the present invention may be obtained by chemical synthesis, using Polymerase Chain Reaction (PCR), or by screening a genomic library or an appropriate cDNA library constructed from a source identified as having β -diox II or expressing β -diox II at a detectable level.

Chemical methods for synthesizing nucleic acids of interest are known in the art and include triester, phosphite, phosphoramidite, and H-phosphate methods, PCR and other self-primer methods, and oligonucleotide synthesis on solid supports. These methods can be used if the entire sequence of the nucleic acid is known, or if a nucleic acid sequence complementary to the coding strand is available. Alternatively, if the target amino acid sequence is known, the known and preferred coding residues for each amino acid residue can be used to infer the likely nucleic acid sequence.

Other methods for isolating the gene encoding β -diox II are the use of PCR techniques as described (Sambrook et al, chapter 14, 1989). The method requires the use of oligonucleotide probes capable of hybridizing to the beta-diox II nucleic acid. Strategies for selecting oligonucleotides are described below.

Libraries are screened with probes or analytical tools designed to identify the gene of interest or the protein encoded by it. For cDNA expression libraries, suitable methods include monoclonal or polyclonal antibodies that recognize and specifically bind to β -diox II; an oligonucleotide of about 20-80 bases in length encoding a known or suspected β -diox II cDNA from the same or a different species; and/or complementary or homologous cdnas or fragments thereof encoding the same or a hybrid gene. Probes suitable for screening genomic DNA libraries include, but are not limited to, oligonucleotides, cDNAs or fragments thereof encoding the same or hybrid DNA, and/or homologous genomic DNA or fragments thereof.

Nucleic acids encoding beta-diox II can be isolated by screening an appropriate cDNA or genomic library with a probe, i.e., a nucleic acid disclosed or referred to herein (including oligonucleotides derivable from the sequences set forth in SEQ ID NOs 1, 16, 18, and/or 20) under appropriate hybridization conditions. Suitable libraries are commercially available or can be prepared from, for example, cell lines, tissue samples, and the like.

As used herein, a probe refers to a nucleic acid molecule having, for example, a nucleotide sequence comprising 10 to 50, preferably 15 to 30, most preferably at least 20 consecutive bases that is identical to, for example, the nucleotide sequence of SEQ ID NO: 1. 16, 18, and/or 20, or a greater number of identical (or complementary) single-stranded DNAs or RNAs of identical (or complementary) contiguous bases. The length and certainty of the nucleic acid sequence selected as a probe should be sufficient to minimize false positive results. The nucleotide sequence may be based on a conserved or highly homologous nucleotide sequence or region in β -diox II as described above. The nucleic acid used as a probe may be degenerate at one or more positions. The use of degenerate oligonucleotides may be particularly important where the preferred codon usage of the species from which the library is screened is unknown.

Preferred regions for constructing probes include 5 'and/or 3' coding sequences, sequences predicted to encode ligand binding sites, and the like. For example, full-length cDNA clones disclosed herein, or fragments thereof, can be used as probes. Preferably, the nucleic acid probes of the invention are labeled with suitable marker means that are readily detectable after hybridization. For example, a suitable means of labeling is a radioactive label. A preferred method for labeling DNA fragments is to incorporate alpha with the Klenow fragment of DNA polymerase in a random priming reaction^32PdATP, as is well known in the art. Usually with gamma^32PLabeled ATP and polynucleotide kinase end-labeled oligonucleotides. However, other methods (e.g., non-radioactive) may also be used to label the oligonucleotides or fragments, including, for example, enzyme labels, fluorescent labels of suitable fluorophores, and biotinylation.

Positive clones are identified, for example, by detecting hybridization signals after screening the library with a DNA portion comprising substantially the entire β -diox II coding sequence or with suitable oligonucleotides based on said or an equivalent DNA portion; characterizing the identified clones by restriction enzyme mapping and/or DNA sequence analysis; tests are then performed, for example by comparison with the sequences listed herein, to determine whether they comprise a DNA sequence encoding the complete β -diox II (i.e. whether they comprise transcription start and stop codons). If the selected clones are incomplete, they can be used to re-screen the same or different libraries to obtain overlapping clones. If the library is a genomic library, overlapping clones may contain exons and introns. If the library is a cDNA library, overlapping clones will contain an open reading frame. In both cases, the entire clone can be identified by comparison to the DNA and deduced amino acid sequences provided herein.

In order to detect any abnormality of endogenous β -diox II, genetic screening can be performed using the nucleotide sequence of the present invention as a hybridization probe. Likewise, antisense or ribozyme type therapeutics can be designed based on the nucleic acid sequences provided herein.

It is contemplated that the nucleic acid of the present invention can be readily modified by nucleotide substitution, nucleotide deletion, nucleotide insertion, or inversion of a nucleotide fragment, and any combination thereof. These mutants can be used, for example, to generate beta-diox II mutants having an amino acid sequence that differs from that found in nature. Mutagenesis may be either pre-determined (site-specific) or random. Mutations that are not silent mutations should not alter the reading frame of the sequence and preferably do not produce complementary regions that can hybridize to form secondary mRNA structures, such as loops or hairpins.

In addition, the present invention contemplates and enables genetic and functional genomic studies using the sequence data provided herein. Genetic studies are performed as an aid to sequencing and mapping and are designed to provide interesting and potentially important clues about biological functions including, for example, homology searches, secondary structure associations, differential cDNA screening, expression cloning, genetic linkage analysis, positional cloning, and mutagenesis analysis. In contrast to genetic studies, functional studies often use cells or animals in an attempt to understand the more direct association of sequences with biological functions, including, for example, screening for phenotypic changes in systems such as yeast, drosophila, mitochondria, human tissues, mice, and frogs, using gene "knockouts" or other methods aimed at controlling gene expression or protein action to provide useful information relating sequences to function. These techniques are well known in the art.

The use of the above method should preferably meet one or more of the following criteria: (1) inhibition of gene sequences should be sequence specific, thereby substantially eliminating false positive results; (2) should have broad applicability, i.e., it should be possible to work with both high and low abundance genes, as well as sequences whose products are intracellular, membrane-bound, or extracellular; (3) should be applicable to models predicting the condition of interest (human); (4) should enable dose-response studies to determine the most affected dose of interest; (5) the amount of information required for the target validation study should preferably be minimal, i.e. the technology is capable of direct study of ESTs without the need to obtain full-length gene sequences, promoters and other regulatory information, or preconditions for protein sequence/structure; (6) should be available for high throughput mode.

Thus, the present invention provides sufficient guidance for the application of all of the methods and techniques described above, including "knockouts", intrabodies, aptamers, antisense oligonucleotides, and ribozymes. In a preferred embodiment of the invention, beta-diox II specific antisense oligonucleotides derived from any of the above beta-diox II sequences (such as those set forth in SEQ ID NOs: 1, 16, 18 and/or 20) are useful in dose-response studies in a retinoid/vitamin A deficiency-associated model at any stage of development of an organism. In another preferred embodiment, a specially designed ribozyme is used to perform optimized sequence-specific inhibition by manipulating elements inherent to its mechanism of action. For example, ribozymes can be designed that bind only their target, and by selecting a target sequence of 15 nucleotides (well within the information limits of typical ESR), it is statistically ensured that the target sequence will only appear once in the genome. Thus, the present invention generally provides specifically designed ribozymes that interact only with its target (which is expected to occur only once in the genome), with a high degree of assurance that only a particular target is inhibited. More specifically, the invention provides ribozymes uniquely equipped to perform several types of important controls that can demonstrate that inhibition of a particular mRNA target is the true cause of a condition or phenotypic change mediated by β -diox II. For example, it is known to mutate the catalytic core of a ribozyme so that it is unable to cleave but still functions with respect to its target with high specific binding. These "inactivated" ribozymes produce no or only substantially reduced targeted inhibition relative to active ribozymes-making them very effective negative controls. Alternatively, the catalytic core may be maintained in an active form, but the targeting arms modified such that they no longer bind to the target sequence. If non-specific cleavage occurs, such a construct should exhibit activity. Since ribozymes contain discrete binding arms, the two binding arms of the ribozyme will bind separately and add selectivity to the ribozyme while maintaining specificity. Since the binding strength of such discontinuous binding arms is relatively low compared to, for example, continuous antisense binding, any mismatch between the ribozyme and the target sequence is not expected to bind effectively, thereby allowing target shedding prior to cleavage.

For the methods and techniques exemplified above, the complete sequence and (functional) fragments thereof, in particular the fragments described above, may be used.

If desired, nucleic acids encoding beta-diox-related proteins or polypeptides can be cloned from cells or tissues using probes derived from beta-diox II according to established protocols. Specifically, these DNAs can be prepared as follows:

1) isolating mRNA from the appropriate cell or tissue, e.g. by hybridization of a DNA probe or by selection of the desired mRNA by expression in an appropriate expression system and screening for expression of the desired polypeptide, preparing single-stranded cDNA complementary to the mRNA and thereby preparing double-stranded cDNA, or

2) E.g. using DNA probes or using a suitable expression system and screening for expression of the desired polypeptide, isolating cDNA from a cDNA library and screening for the desired cDNA, or

3) Incorporating the double-stranded DNA of step 1) or 2) into a suitable expression vector,

4) appropriate host cells are transformed with the vector and the desired DNA is isolated.

The polyadenylated mRNA is isolated by known methods (step 1). Isolation methods include, for example, homogenizing the cells in the presence of a detergent and a ribonuclease inhibitor (e.g., heparin, guanidinium isothiocyanate, or mercaptoethanol), extracting the mRNA with chloroform/phenol mixtures (optionally in the presence of salts and buffers, detergents, and/or cation chelators), and precipitating the mRNA from the remaining salt-containing aqueous phase with ethanol, isopropanol, or the like. The isolated mRNA is further purified by cesium chloride gradient centrifugation followed by ethanol precipitation and/or chromatography (e.g. affinity chromatography, such as oligo (dT) cellulose or oligo (U) sepharose chromatography). Preferably, the purified mRNAs are separated by size by gradient (e.g., linear sucrose gradient) centrifugation or chromatography on a suitable size fractionation column (e.g., agarose gel).

The desired mRNA is selected by direct screening of the mRNA with DNA probes or by translation and screening of the resulting polypeptide in a suitable cellular or cell-free system. Preferably, a DNA hybridization probe is used to achieve selection of the desired mRNA, thereby avoiding the additional step of translation. Suitable DNA probes are DNA of at least 17 nucleotides of known nucleotide sequence derived from DNA encoding beta-diox II or related proteins. Alternatively, EST sequence information can be used to generate suitable DNA probes.

The synthetic DNA probes are synthesized according to known methods described in detail below, preferably using stepwise condensation of solid phase phosphotriester, or phosphoramidite methods, such as condensation of dinucleotide coupling units by the phosphotriester method. These methods are suitable for the synthesis of mixtures of the desired oligonucleotides by using mixtures of two, three, or four nucleotides dA, dC, dG, and/or dT in protected form or corresponding dinucleotide coupling units in appropriate condensation steps, as described (Ike Y et al, Nucleic Acids Research, 11: 477, 1983).

For hybridization, the DNA probe is labeled, for example, by radiolabeling by the well-known kinase reaction. Hybridization of size-separated mRNA to a DNA probe containing a label is carried out according to known procedures, i.e., in a buffer and salt solution containing additives (e.g., calcium chelators, viscosity modifying compounds, proteins, unrelated DNA, etc.) at a temperature that facilitates selective hybridization (e.g., about 0-80 ℃, e.g., about 25-50 ℃ or 65 ℃, preferably about 20 ℃ below the melting temperature of the hybridized double-stranded DNA).

The fractionated mRNA may be translated in cells (e.g., frog oocytes) or in cell-free systems (e.g., reticulocyte lysate or wheat germ extract). The resulting polypeptide is screened for β -diox II activity or for reaction with antibodies raised against β -diox II or a protein related thereto (e.g., in an immunoassay such as a radioimmunoassay, enzyme immunoassay, or fluorescent marker immunoassay). The preparation of such immunoassays and polyclonal and monoclonal antibodies is well known in the art and applies accordingly. In accordance with the present invention, polyclonal antibodies are provided.

The preparation of single stranded cDNA from a selected mRNA template is well known in the art, as is the preparation of double stranded DNA from single stranded DNA. The mRNA template is incubated with a mixture of deoxynucleoside triphosphates (optionally radiolabeled with deoxynucleoside triphosphates to enable screening of the reaction results), primer sequences (such as oligo dT residues capable of hybridizing to the poly (a) tail of the mRNA), and a suitable enzyme (such as reverse transcriptase, e.g. from Avian Myeloblastosis Virus (AMV)). After degradation of the template mRNA, for example by alkaline hydrolysis, the cDNA is incubated with a mixture of deoxynucleoside triphosphates and a suitable enzyme to produce double stranded DNA. Suitable enzymes are, for example, reverse transcriptase, the Klenow fragment of E.coli DNA polymerase I, or T4 DNA polymerase. Typically, a hairpin loop structure formed spontaneously from a single-stranded cDNA serves as a primer for the synthesis of the second strand. The hairpin structure was removed by digestion with S1 nuclease. Alternatively, the 3' end of the single-stranded DNA is first extended by a homopolydeoxynucleotide tail before hydrolysis of the mRNA template, followed by synthesis of the second cDNA strand.

Alternatively, double-stranded DNA is isolated from a cDNA library and the desired cDNA is screened (step 2). The cDNA library was constructed as follows: mRNA is isolated from suitable cells (e.g., chicken embryo cells, human mononuclear leukocytes, or human embryonic lung epithelial cells) as described above and single-and double-stranded cDNA is prepared therefrom. The cDNA is digested with appropriate restriction endonucleases following an established protocol and incorporated into lambda phage (e.g., lambda charon4A or lambda gt 11). The cDNA libraries replicated on nitrocellulose membranes are screened using DNA probes as described above, or expressed in a suitable expression system and the resulting polypeptides are screened for reaction with specific antibodies to the desired β -diox II.

Various methods are known in the art for incorporating double stranded DNA into an appropriate vector (step 3). For example, complementary homopolymers can be added to double-stranded DNA and vector DNA by incubation in the presence of the corresponding deoxynucleoside triphosphates and an enzyme (such as a terminal deoxynucleotidyl transferase). The vector and double-stranded DNA are then ligated together by base pairing between complementary homopolymeric tails, and finally ligated by a specific ligase (such as ligase). Other possibilities are to add synthetic linkers to the ends of the double stranded DNA, or to incorporate the double stranded DNA into the vector by blunt end or sticky end ligation.

Transformation of an appropriate host cell with the obtained hybrid vector (step 4) and selection of a transformed host cell (step 5) are well known in the art. Hybrid vectors and host cells may be particularly useful for the production of DNA or for the production of the desired β -diox II.

In addition to being useful for the production of recombinant β -diox II proteins, these nucleic acids may also be used as probes, thereby allowing one skilled in the art to readily identify and/or isolate nucleic acids encoding β -diox II. The nucleic acid may be unlabeled or may have been labeled with a detectable moiety. In addition, the nucleic acids according to the invention can be used, for example, in a method for determining the presence or even the amount of a β -diox II-specific nucleic acid, which comprises hybridizing a β -diox II-encoding (or complementary) DNA (or RNA) to a test sample nucleic acid and determining the presence and (optionally) the amount of β -diox II. In another aspect, the invention provides a nucleic acid sequence which is complementary to a nucleic acid sequence encoding a β -diox II or which hybridises under stringent conditions. These oligonucleotides are useful in antisense and/or ribozyme methods, including gene therapy.

The invention also provides a method for amplifying a nucleic acid test sample comprising priming a nucleic acid polymerase (chain) reaction with a nucleic acid (DNA or RNA) encoding (or complementary to) beta-diox II.

Thus, the DNA sequences of the present invention can be used as standards to identify novel PCR primers for cloning substantially homologous DNA sequences from other sources. Alternatively, they and these homologous DNA sequences can be incorporated into vectors for expression or over-expression of the encoded polypeptide in a suitable host system by methods known in the art, e.g., as described in Sambrook et al, supra. However, it is known to the skilled person that the DNA sequence itself may also be used to transform a suitable host system of the invention to obtain overexpression of the encoded polypeptide.

As indicated above, the present invention thus provides specific DNA molecules, as well as plasmids or vector systems comprising said DNA molecules, which comprise within an operable expression cassette a DNA sequence capable of directing the production of functionally active β -carotene dioxygenase II, i.e.directing the production of retinoids from β -carotene. Preferably, the DNA molecule further comprises at least one selectable marker gene or cDNA operably linked to a constitutive, inducible, or tissue-specific promoter sequence that allows its expression in bacterial, yeast, fungal, insect, animal, or plant cells, seeds, tissues, or whole organisms. If plastid-containing material is selected for transformation, the coding nucleotide sequence is preferably fused to a suitable plastid transit peptide coding sequence, both of which are preferably expressed under the control of a tissue-specific or constitutive promoter.

Polypeptides according to the invention include β -diox II and derivatives thereof that retain at least one common structural determinant of β -diox II.

By "common structural determinant" is meant that the derivative under investigation has at least one structural feature of β -diox II. Structural features include having an epitope or antigenic site that is capable of cross-reacting with antibodies made against naturally occurring or denatured β -diox II polypeptides or fragments thereof, having amino acid sequence identity with β -diox II, and having a shared structural/functional association. Thus, the β -diox II provided by the present invention includes splice variants, amino acid mutants, glycosylation variants encoded by mRNA resulting from other splicing of the primary transcript, and other β -diox II covalent derivatives that retain the physiological and/or physical properties of β -diox II. Exemplary derivatives include molecules obtained by covalently modifying a protein of the invention with a moiety of a non-naturally occurring amino acid by substitution, chemical, enzymatic, or other suitable means. Such a module may be a detectable module, such as an enzyme or a radioisotope. Also included are naturally occurring variants or homologs of β -diox II found by a particular species, preferably a mammal. Such variants or homologues may be encoded by related genes of the same gene family, allelic variants of a particular gene, or represent otherwise spliced variants of the β -diox II gene.

Derivatives that retain a common structural feature may be fragments of β -diox II. Fragments of β -diox II include its individual domains, as well as smaller polypeptides derived from the domains. Preferably, the smaller polypeptides derived from β -diox II according to the invention define one characteristic feature of β -diox II. In theory, fragments can be almost any size as long as they retain one of the characteristics of β -diox II. Preferably, the fragments are 5-200 amino acids in length. Longer fragments are considered to be truncated forms of full-length β -diox II and are generally encompassed by the term "β -diox II". Exemplary fragments of β -diox II polypeptides are SEQ ID NOs: 17, positions 39-47, 96-118, 361-368 and 466-487, SEQ ID NO: 19, positions 55-63, 112-134, 378-385 and 482-503 of SEQ ID NO: 21 at positions 59-67, 116-138, 385-392, and 490-511.

Derivatives of β -diox II also include mutants thereof, which may comprise amino acid deletions, additions, or substitutions, as long as the requirements for maintaining at least one characteristic feature of β -diox II are met. Thus, conservative amino acid substitutions may be made without substantially altering the nature of β -diox II, as may 5 'or 3' truncated forms. In addition, deletions and substitutions may be made to the β -diox II fragment encompassed by the present invention. Mutants of beta-diox II can be generated from DNA encoding beta-diox II, i.e. the DNA is subjected to in vitro mutagenesis resulting in, for example, addition, exchange, and/or deletion of one or more amino acids. For example, substitution, deletion, or insertion variants of β -diox II can be made by recombinant methods and screened for immunological cross-reactivity with the native form of β -diox II.

The invention also provides beta-diox II polypeptides and derivatives thereof which retain at least one common antigenic determinant of beta-diox II.

By "shared antigenic determinant" is meant that the derivative under investigation has at least one antigenic function of β -diox II. Antigenic functions include having epitopes or antigenic sites capable of cross-reacting with antibodies raised against naturally occurring or denatured β -diox II polypeptides or fragments thereof.

Derivatives that retain a common antigenic determinant may be fragments of β -diox II. Fragments of β -diox II include its individual domains, as well as smaller polypeptides derived from the domains. Preferably, the smaller polypeptides derived from β -diox II according to the invention define an epitope characteristic of β -diox II. In theory, fragments can be almost any size as long as they retain one of the characteristics of β -diox II. Preferably, the fragments are 5-500 amino acids in length. Longer fragments are considered to be truncated forms of full-length β -diox II and are generally encompassed by the term "β -diox II".

The present invention provides a process for producing a β -diox II polypeptide comprising (1) expressing in a suitable host a polypeptide encoded by the DNA described above, and (2) isolating said β -diox II polypeptide according to conventional techniques well known in the art. In addition, a protein obtained or obtainable by the above process is provided.

Preferably, the protein of the invention or a derivative thereof is provided in isolated form. By "isolated" is meant a protein or derivative thereof that is identified as free of one or more components of its natural environment. Isolated β -diox II includes β -diox II in recombinant cell culture. The presence of beta-diox II in organisms expressing recombinant beta-diox II genes, whether the beta-diox II protein is isolated "or otherwise, is included within the scope of the present invention.

If desired, retinoids (such as β -apocarotene aldehyde, β -ionone, and apolycopene aldehyde) formed in any of the systems (bacteria, fungi, plants, animals, etc.) can be further metabolized into retinol, retinyl esters, retinoic acid, and its corresponding stereoisomers. Those modifications can be used to improve the efficiency of the cleavage reaction and/or to accumulate the desired retinoids. Accumulation of specific retinoids may be useful because retinoids exert different biological functions depending on their oxidation state (alcohol, aldehyde, and acid) and stereoisomeric forms, e.g., retinal/retinol function in vision, retinoic acid function in developmental processes and differentiation, and retinyl esters are the normal reservoir of vitamin a in animals. Accumulation of the desired retinoid derivative may be achieved by co-expression of a retinoid-modifying enzyme with β -diox II. By those functional associations, for example, accumulation of retinyl esters can be achieved in plants and/or bacteria as feed, food, and/or feed and food additives, or biosynthesis of specific retinoids (e.g. 9-cis retinoic acid, a ligand for RXR transcription factor) can be achieved. In addition, co-expression of a retinoid binding protein of animal origin may improve the yield of the desired retinoids.

According to a preferred embodiment of the invention, the following enzymes or combinations of enzymes are co-expressed together with β -diox ii. For example, if it is desired to convert retinal to retinol, an alcohol dehydrogenase (e.g., AF059256) and/or a retinal dehydrogenase/reductase (e.g., AW211228) may be used. Where retinyl esters are intended to be produced from retinol, a retinol acyltransferase (e.g., AF071510) may be used. If retinoic acid should be generated from retinal, a retinal oxidase (e.g., AB017482) will be selected. Alternatively, if it is desired to co-express a retinoid-binding protein, it is envisaged to select a retinol-binding protein (e.g. AJ 236884). Finally, different isomerases that convert the all-trans structure of the above compounds to 13-cis, 11-cis, 9-cis, or-cis isomers can be co-expressed.

In accordance with the present invention, means and methods are provided for transforming plant cells, seeds, tissues, or whole plants and for transforming microorganisms, such as yeast, fungi, and bacteria, to produce transformants capable of mediating retinoid synthesis. According to another aspect of the invention, the method may also be used to modify retinoid metabolism in an animal.

The host material selected for transformation should express the introduced genes and their expression is preferably homozygous. Typically, the gene will be operably linked to a promoter that is functionally active in the target host cell of a particular plant, insect, animal, or microorganism (such as fungi and bacteria, including yeast). The expression level should be sufficient to obtain the desired characteristics from the gene. For example, expression of a selectable marker gene should provide for the appropriate selection of transformants produced according to the methods of the invention. Similarly, expression of a gene encoding an enzyme for enhancing nutritional quality that exhibits the desired activity of cleaving β -carotene into carotenoids/retinoids should result in transformants having a relatively higher content of the encoded gene product relative to the same species that have not been subjected to the transformation method of the invention. On the other hand, it is often desirable to limit the over-expression of the gene of interest in order to avoid significantly adversely affecting the normal physiology of plants, insects, fungi, animals, or microorganisms, i.e., to an extent that their cultivation becomes difficult.

The gene encoding beta-carotene dioxygenase can be used in expression cassettes for expression in transformed prokaryotic or eukaryotic host cells, seeds, tissues, or whole organisms. To achieve the object of the present invention, i.e., to introduce the ability to cleave beta-carotene to form retinoids in the target host of interest, transformation is preferably performed using an operable expression cassette comprising a transcription initiation region (linked to the gene encoding beta-carotene dioxygenase II).

The transcriptional initiation region may be native or analogous to the host, or foreign or heterologous. Exogenous means that the transcriptional initiation region is not found in the wild-type host into which it is introduced.

In plant material, those transcription initiation regions which are associated with storage proteins (such as gluten, patatin, napin, cruciferin, β -conglycinin, phaseolin, etc.) are of particular interest.

The transcription cassette will comprise (in the 5 '-3' direction of transcription) a transcription and translation initiation region, a DNA sequence encoding beta-carotene dioxygenase II or a functional fragment thereof (retaining its specific enzymatic, immunogenic, or biological activity), and a transcription and translation termination region functional in the targeted host material, such as a plant or microorganism. The termination region may be native with respect to the transcriptional initiation region, may be native with respect to the DNA sequence of interest, or may be derived from other sources. Suitable termination regions for Plant material, such as octopine synthase and nopaline synthase termination regions, can be obtained from the Ti plasmid of Agrobacterium tumefaciens (see Guerineau et al, mol.Gen.Genet., 262: 141-144, 1991; Proudfeot, Cell, 64: 671-674, 1991; Sanfacon et al, Genes Dev., 5: 141-149, 1991; Mogen et al, Plant Cell, 2: 1261-1272, 1990; Munroe et al, Gene, 91: 151-158, 1990; Ballas et al, Nucl. acids Res., 17: 7891-7903, 1989; Joshi et al, Nucl. acids Res., 15: 96969639, 1987).

For expression of β -carotene dioxygenase II in plants or in plastid-containing material, the coding sequence is preferably fused to a sequence encoding a transit peptide which, upon expression and translation, directs the transport of the protein from which the transit peptide is cleaved to (plant) plastids, such as chloroplasts, in which carotenoid biosynthesis takes place. For example, a β -diox II cDNA can be fused at the translational level to a sequence encoding a ribulose-1, 5-bisphosphate carboxylase (ribosomal bisphosphate carboxylase-oxygenase) small subunit transit peptide or to a sequence encoding other plastid protein transit peptides. Such transport peptides are known in the art (see, for example, Von Heijne et al, Plant mol. biol. Rep., 9: 104. sup. 126, 1991; Clark et al, J. biol. chem., 264: 17544. sup. 17550, 1989; Della-Cioppa et al, Plant physiol., 84: 965. sup. 968, 1987; Romer et al, Biochim. Biophys. Res. Commun., 196: 1414. sup. 1421, 1993; and Shah et al, Science, 233: 478. sup. 481, 1986). Any gene useful for carrying out the present invention may utilize native or heterologous transit peptides.

The construct may also comprise any other essential regulatory genes, such as plant translational consensus sequences (Joshi, 1987, supra), introns (Luehrsen and Walbot, mol.Gen.Genet., 225: 81-93, 1991), and the like, operably linked to a nucleotide sequence encoding beta-carotene dioxygenase II. It is expected that the intron sequence within the introduced coding gene will increase the expression level by stabilizing the transcript and allowing it to be efficiently transported out of the nucleus. Included among these known intron sequences are the introns of Plant ubiquitin genes (Cornejo, Plant mol. biol., 23: 567-one 581, 1993). In addition, it has been observed that the insertion of the same construct into different loci of the genome can alter expression levels in plants. It is believed that this effect is due in part to the location of the gene on the chromosome, i.e., different isolates will have different expression levels (see, e.g., Hoever et al, Transgenic Res., 3: 159-166, 1994). Other regulatory DNA sequences which may be used to construct the expression cassette include, for example, sequences which are capable of regulating (inducing or repressing) transcription of the associated DNA sequence in plant tissue.

It is known that, for example, certain plant genes are induced by various internal or external factors, such as plant hormones, heat shock, chemicals, pathogens, hypoxia, light, stress, and the like.

Another group of DNA sequences which can be regulated comprises the chemical driver sequences which are present in the tobacco PR (pathogenesis-related) protein gene and are induced by means of chemical regulators, such as described in EP-A0332104.

Another consideration for the expression of exogenous genes in plants, animals, insects, fungi, or microorganisms is the stable level of the transgenic genome, i.e., the tendency of the exogenous gene to segregate from the population. If a selectable marker is linked to the gene or expression cassette of interest, selection can be applied to maintain the transgenic host organism or portion thereof.

It may be advantageous to include a 5' leader sequence in the expression cassette construct. These leader sequences may enhance translation. Translation leader sequences are known in the art and include: picornavirus leaders, such as the EMCV leader (5' non-coding region of encephalomyocarditis virus; Elroy-Stein et al, Proc. Natl. Acad. Sci. USA, 86: 6126-6130, 1989); potyviruses, such as the TEV leader sequence (tobacco etch Virus; Allisson et al, Virology, 154: 9-20, 1986); human immunoglobulin heavy chain binding protein (BiP; Macejak and Sarnow, Nature, 353: 90-94, 1991); untranslated leader sequences from alfalfa mosaic virus coat protein mRNA (AMV RNA 4; Jobling and Gehrke, Nature, 325: 622-; tobacco mosaic virus leader sequence (TMV; Gallie et al, Molecular Biology of RNA, 237-; and maize chlorotic mottle virus leader (MCMV; Lommel et al, Virology, 81: 382-.

Depending on where the DNA sequence encoding beta-carotene dioxygenase II is expressed, it may be desirable to synthesize a sequence having host-preferred codons or chloroplast or plastid preferred codons. Plant-preferred codons can be determined from the highest frequency codons in the proteins expressed in the greatest amounts in the particular plant species of interest (see EP-A0359472; EP-A0386962; W091/16432; Perlak et al, Proc. Natl. Acad. Sci. USA, 88: 324-3328, 1991; and Murray et al, Nucl. acids Res., 17: 477-498, 1989). In this way, the nucleotide sequence can be optimized for expression in any targeted host. It will be appreciated that all or any portion of the gene sequence may be optimized or synthesized. I.e.synthetic or partially optimized sequences can also be used. For the construction of chloroplast preference genes, see USPN 5,545,817.

Expression systems encoding beta-diox II are useful for studying beta-diox II activity, particularly in transgenic cells, tissues, or animals. Preference is given to systems in which the expression of β -diox II has been attenuated, in particular by means of transposon insertion. Mutant cells, tissues, or animals according to the invention have impaired β -diox II expression. Especially those expressing severely attenuated but unlimited expression mutants can be used to study beta-diox II activity. They show increased sensitivity to the modulated interaction of putative upstream signaling agents with specific target domains of β -diox II and to modifications of downstream targets predicted to mediate their biological responses. Accordingly, the invention also provides a method for assessing the ability of an agent to target β -diox II activity, comprising exposing a β -diox II mutant as described herein to the agent and determining the effect on the biological activity of β -diox II.

In preparing a transcription cassette, a variety of DNA fragments can be manipulated to provide DNA sequences in the correct orientation and in the correct reading frame. For this purpose, adaptors or linkers may be employed to ligate the DNA fragments, or other manipulations may be included to provide convenient restriction sites, remove excess DNA, remove restriction sites, and the like. For this purpose, in vitro mutagenesis, primer repair, restriction digestion, annealing, excision, ligation, etc., may be employed, wherein insertions, deletions, or substitutions (e.g., transitions and transversions) may be involved.

Expression cassettes carrying cDNA or genomic DNA encoding a native or mutant β -carotene dioxygenase can be placed in expression vectors by standard methods. As used herein, a vector (or plasmid) refers to a discrete element used to introduce heterologous DNA into a cell for expression or replication. The selection and use of such vectors is well within the skill of the art. Many vectors are available, and the selection of an appropriate vector will depend on the intended use of the vector (i.e., whether it is for DNA amplification or for DNA expression), the size of the DNA to be inserted into the vector, the type of host (plant, animal, insect, fungal, or microbial) to be transformed with the vector, and the method of introducing the expression vector into a host cell. Each vector contains a variety of elements, depending on its function (DNA amplification or DNA expression) and compatible host cells. Typical expression vectors typically contain, but are not limited to, prokaryotic DNA elements encoding a bacterial origin of replication and an antibiotic resistance gene, providing for the growth and selection of the expression vector in a bacterial host; a cloning site for insertion of a foreign DNA sequence, herein a DNA sequence encoding an enzyme capable of cleaving β -carotene to form a carotenoid/retinoid; eukaryotic DNA elements that control the initiation of transcription of foreign genes, such as promoters; and DNA elements that control transcript processing, such as transcription termination/polyadenylation sequences. It may also contain sequences required for eventual integration of the vector into the chromosome of the targeted host.

In a preferred embodiment, the expression vector further comprises a gene encoding a selectable marker (functionally linked to a promoter), such as hygromycin phosphotransferase (van den Elzen et al, Plant mol. biol., 5: 299-392, 1985). Other examples of genes which confer antibiotic resistance and are therefore suitable as selectable markers include the neomycin phosphotransferase gene (Velten et al, EMBO.J., 3: 2723-2730, 1984); the kanamycin resistance (NPT II) gene derived from Tn5 (Bevan et al, Nature, 304: 184-187, 1983); the PAT gene (Thompson et al, EMBO J., 6: 2519-2523, 1987); and chloramphenicol acetyltransferase gene. For a general description of plant expression vectors and selectable marker genes suitable for use in the present invention, see Gruber et al, Methods in plant molecular Biology and Biotechnology (Methods in plant molecular Biology and Biotechnology), pp.89-119, CRC Press, 1993. As the selection marker suitable for yeast, any marker gene that facilitates selection of transformants due to phenotypic expression of the marker gene can be used. Suitable markers for yeast are, for example, genes that confer resistance to the antibiotics G418, hygromycin, or bleomycin, or genes that provide prototrophy in auxotrophic yeast mutants (e.g., URA3, LEU2, LYS2, TRP1, or HIS3 genes).

Suitable selectable markers for use in mammalian cells are genes that enable identification of cells competent to take up β -diox nucleic acid, such as dihydrofolate reductase (DHFR, methotrexate resistance), thymidine kinase, or genes that confer G418 or hygromycin resistance. Mammalian cell transformants are placed under selection pressure where only transformants that have taken up and expressed the marker are uniquely suitable for survival. In the case of DHFR or Glutamine Synthase (GS) as markers, selection pressure can be applied by culturing the transformants under conditions of gradually increasing pressure, thereby resulting in amplification of both the selection gene and the associated DNA encoding β -diox II (at its chromosomal integration site). Amplification refers to the process of repeated tandem within a recombinant cell chromosome of a gene that is more desirable for producing a protein necessary for growth, with a closely linked gene encoding the desired protein. The desired protein increment is usually synthesized from the thus amplified DNA.

The promoter element used to control the expression of the gene of interest and the marker gene, respectively, can be any plant compatible promoter. They may be plant gene promoters, such as the promoter of the small subunit of ribulose-1, 5-bisphosphate carboxylase (ribosomal bisphosphate carboxylase-oxygenase) or the promoters from agrobacterium tumefaciens tumor inducing plasmids, like the nopaline synthase and octopine synthase promoters; or viral promoters such as the cauliflower mosaic virus (CaMV)19S and 35S promoters or the figwort mosaic virus 35S promoter. For a review of known plant promoters suitable for use in the present invention, see for example international application WO 91/19806.

"tissue-specific" promoters provide particularly high accumulation of the desired gene product in tissues expressing the carotenoid or xanthophyll biosynthetic pathway products; although some expression may occur in other parts of the plant. Examples of known tissue-specific promoters include the gluten 1 promoter (Kim et al, Plant Cell physiol., 34: 595-603, 1993; Okita et al, J.biol.chem., 264: 12573-12581, 1989; Zheng et al, plantaH., 4: 357-366, 1993), the tuber-directed type I patatin promoter (Bevan et al, Nucl.acids Res., 14: 4625-4638, 1986); a promoter linked to the ADPGPP gene of potato tubers (Muller et al, mol.Gen.Genet., 224: 136-146, 1990); the soybean promoter of beta-conglycinin (also known as the 7S protein, driving seed-directed transcription) (Bray, Planta, 172: 364-; and seed-directed promoters from the maize endosperm zein gene (Pedersen et al, Cell, 29: 1015-1026, 1982). Another type of promoter useful in the present invention is the plant ubiquitin promoter. Plant ubiquitin promoters are well known in the art, as described by Kay et al, Science, 236: 1299, 1987 and EP-A0342926. Also suitable for use in the present invention are the actin promoter, the histone promoter, and the tubulin promoter. EP-A0332104 describes an example of a preferred chemically inducible promoter, such as the tobacco PR-1a promoter. Another preferred class of promoters are the wound-inducible promoters. Preferred promoters of this class include the following: stanford et al, mol.gen.genet., 215: 200-208, 1989; xu et al, Plant mol. biol., 22: 573 588, 1993; logemann et al, Plant Cell, 1: 151-158, 1989; rohrmeier and Lehle, plantamol. biol., 22: 783 792, 1993; firek et al, Plant mol. biol., 22: 192-; and Warner et al, Plant j, 3: 191-201, 1993.

According to a preferred embodiment, the cassette for expression of β -carotene dioxygenase II comprises a β -diox II cDNA and is translationally fused to a sequence encoding a transit peptide for plastid import, a polyadenylation signal, and a transcription terminator, all of which are operably linked to a suitable constitutive, inducible, or tissue-specific promoter that permits expression of the desired protein in plant cells, seeds, tissues, or whole plants.

Furthermore, the β -diox II gene according to the present invention preferably comprises a secretion sequence to facilitate secretion of the polypeptide by a bacterial host, such that it is produced as a soluble native peptide rather than as inclusion bodies. Depending on the case, the peptide may be recovered from the periplasmic space or the culture medium of the bacterium.

Suitable promoter sequences for yeast hosts may be controlled or constitutive, and are preferably derived from highly expressed yeast genes, especially the Saccharomyces cerevisiae gene. Thus, a promoter of TRP1 gene, ADHI or ADHII gene, acid phosphatase (PHO5) gene; a promoter of a yeast mating pheromone gene encoding an alpha or a factor; a promoter derived from a gene encoding a glycolytic enzyme, such as a promoter of an enolase, glyceraldehyde-3-phosphate dehydrogenase (GAP), 3-phosphoglycerate kinase (PGK), hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, or glucokinase gene; or a promoter from the TATA Binding Protein (TBP) gene. In addition, it is possible to use hybrid promoters comprising an Upstream Activating Sequence (UAS) of one yeast gene and a downstream promoter element (including a functional TATA box) of another yeast gene, for example a UAS comprising the PHO5 gene and a hybrid promoter comprising a functional TATA box of a yeast GAP gene (PHO5-GAP hybrid promoter). A suitable constitutive PHO5 promoter is, for example, the shortened acid phosphatase PHO5 promoter lacking upstream regulatory elements (UAS), such as the PHO5(-173) promoter element (beginning at nucleotide 173 and ending at nucleotide 9 of the PHO5 gene).

Transcription of the β -diox II gene from a vector in a mammalian host may be controlled by promoters derived from the genome of viruses such as polyoma, adenovirus, fowlpox, bovine papilloma, avian sarcoma, Cytomegalovirus (CMV), retroviruses, and simian virus 40(SV40), from heterologous mammalian promoters such as the actin promoter or a strong promoter such as the ribosomal protein promoter, or from promoters normally associated with β -diox sequences, provided that these promoters are compatible with the host cell system.

Transcription of DNA encoding β -diox II in higher eukaryotes may be increased by inserting an enhancer sequence into the vector. Enhancers are relatively orientation and location independent. Many enhancer sequences are known from mammalian genes such as elastase and globin. However, enhancers from eukaryotic cell viruses are often employed. Examples include the SV40 enhancer and the CMV early promoter enhancer on the late side of the replication origin (bp positions 100-270). Enhancers may be spliced into the vector 5 ' or 3 ' to the β -diox II DNA, but are preferably located at the 5 ' site of the promoter.

Advantageously, the eukaryotic expression vector encoding β -diox II may comprise a Locus Control Region (LCR). LCRs are capable of directing high levels of hydration site independent expression of transgenes integrated into the chromatin of host cells, which is important, inter alia, for expression of β -diox II genes in permanently transfected eukaryotic cell lines in which chromosomal integration of the vector has occurred, in the design of vectors for gene therapy applications, or in transgenic animals or other hosts disclosed herein or known in the art.

According to a preferred embodiment of the invention, the expression cassettes and plasmids or vector systems disclosed herein additionally comprise a nucleic acid sequence encoding a specific retinoid modifying enzyme and/or retinoid binding protein, preferably co-expressed with a polypeptide according to the invention, as described above.

Eukaryotic host cells suitable for expression of β -diox II, including fungal (including yeast), insect, plant, animal, human, or nucleated cells from other multicellular organisms, will also contain sequences necessary for termination of transcription and stabilization of mRNA. These sequences are typically obtained from the 5 'and 3' untranslated regions of eukaryotic or viral DNA or cDNA. These regions include nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding β -diox II.

Prokaryotic or eukaryotic host cells, seeds, tissues, and whole organisms encompassed by the present invention can be obtained by several methods. One skilled in the art will appreciate that the method may be selected based on the type of host targeted for transformation, such as a plant, i.e., monocot or dicot. Such methods typically include direct gene transfer, chemically induced gene transfer, electroporation, microinjection (Crossway et al, BioTechniques, 4: 320-334, 1986; Neuhaus et al, Theor. appl. Genet., 75: 30-36, 1987), Agrobacterium-mediated gene transfer, microprojectile acceleration using devices such as those available from Agracenus, Inc. (Madison, Wis.) and Dupont, Inc. (Wilmington, Delaware) (see, e.g., Sanford et al, U.S. Pat. No. 4,945,050; and Mc Cabe et al, Biotechnology, 6: 923-926, 1988), and the like.

One method for obtaining a transformed plant or part thereof of the invention is direct gene transfer, wherein plant cells are cultured or otherwise grown under suitable conditions in the presence of a DNA oligonucleotide comprising the nucleotide sequence desired to be introduced into the plant or part thereof. The source of donor DNA is typically a plasmid or other suitable vector containing the desired gene. For convenience, reference is made herein to plasmids, it being understood that other suitable vectors containing the desired gene are also contemplated.

Any suitable plant tissue that takes up the plasmid can be treated by direct gene transfer. These plant tissues include, for example, those in early stages of development, in particularIs the reproductive structure before meiosis, especially 1-2 weeks before meiosis. Typically, the pre-meiotic reproductive organ is soaked in a plasmid solution, such as by injecting the plasmid solution directly into the plant at or near the reproductive organ. The plants are then self-pollinated or cross-pollinated with pollen from another plant treated in the same manner. The plasmid solution typically contains about 10-50. mu.g DNA in about 0.1-10ml for each flower structure, but can be above or below this range depending on the size of the particular flower structure. The solvent is typically sterile water, saline, or buffer, or a conventional plant culture medium. If desired, the plasmid solution may also contain agents to chemically induce or enhance plasmid uptake, such as PEG, Ca²⁺And so on.

After exposure of the reproductive organs to the plasmid, the flower structure is grown to maturity and the seeds are harvested. Transformed plants having the marker gene are selected by their germination or growth in marker sensitive or preferably marker resistant medium, based on the plasmid marker. For example, seeds obtained from plants treated with a plasmid having a kanamycin resistance gene will remain green, while plants not having this marker gene are albinism. The presence of mRNA transcription and peptide expression of the desired gene can be further demonstrated by conventional Southern, Northern, and Western blot techniques.

In another method suitable for carrying out the present invention, plant protoplasts are treated to induce uptake of a plasmid or vector system according to the invention. The preparation of protoplasts is well known in the art and generally involves digesting plant cells with cellulase and other enzymes for a sufficient time to remove the cell wall. Protoplasts are usually isolated from the digestion mixture by sieving and washing. The protoplasts are then suspended in a suitable medium, such as F medium, CC medium, etc., usually at a concentration of 10⁴-10⁷Individual cells/ml. Then to this suspension were added the plasmid solution and an inducing agent (such as polyethylene glycol, Ca) as described above²⁺Sendai virus, etc.). Alternatively, the plasmid may be encapsulated in a liposome. The solution of plasmids and protoplasts is then incubated at about 25 deg.CWhen it is time, it is usually about 1 hour. In some cases it may be desirable to heat shock the mixture by briefly heating to about 45 c, for example for 2-5 minutes, and rapidly cooling to the holding temperature. The treated protoplasts are then cloned and the expression of the desired gene is selected by, for example, expression of a marker gene and conventional blotting techniques. The whole plant is then regenerated from the clones in the conventional manner.

Electroporation techniques are similar except that polyethylene glycol, Ca, are typically absent or present in the electroporation chamber²⁺Isochronous application of current to a mixture of naked plasmid and protoplast. Typical electroporation consists of 40-10,000DC volts in 1-10 pulses, with a duration of 1-2000 microseconds, with an interval between pulses of typically 0.2 seconds. Alternating current pulses of similar intensity may also be used. More specifically, the charged capacitor is discharged in an electroporation cuvette containing a suspension of plasmids and protoplasts. This treatment leads to a reversible increase in the permeability of the biological membrane, so that the DNA according to the invention can be inserted. Electroporated plant protoplasts regenerate their cell wall, divide, and form callus (see, e.g., Riggs et al, 1986).

Another method suitable for transforming target cells involves the use of Agrobacterium. In this method, agrobacterium containing a plasmid with the desired gene or gene cassette is used to infect plant cells and the plasmid is inserted into the target cell genome. Cells expressing the desired gene are then selected and cloned as described above. For example, one method of introducing a gene of interest into a target tissue (e.g., tuber, root, grain, or legume) by means of plasmids (e.g., Ri plasmids) and Agrobacterium (e.g., Agrobacterium rhizogenes or Agrobacterium tumefaciens) is to utilize a small recombinant plasmid suitable for cloning in E.coli, into which T-DNA fragments have been spliced. This recombinant plasmid is cut at a site within the T-DNA and a "passenger" DNA is spliced at the opening. The passenger DNA consists of the gene of the present invention to be introduced into the plant DNA and a selection marker (e.g., a gene for antibiotic resistance). This plasmid was then re-cloned into a large plasmid and introduced into an agrobacterium strain carrying the unmodified Ri plasmid. During the growth of bacteria, rare double recombination sometimes occurs, resulting in the T-DNA in the bacteria comprising an insert, i.e., passenger DNA. These bacteria were identified and selected by survival on antibiotic-containing media. These bacteria are used to insert their T-DNA (modified with passenger DNA) into the plant genome. This procedure using Agrobacterium rhizogenes or Agrobacterium tumefaciens produces transformed plant cells that can be regenerated into healthy, viable plants (see, e.g., Hinche et al, 1988).

Another suitable method is the bombardment of the cells with microprojectiles which are coated with the transforming DNA (Wang et al, Plant mol. biol., 11: 433-439, 1988) or are accelerated in the direction of the cells to be transformed in a DNA-containing solution by pressure impact, whereby the solution is finely dispersed in mist form by the pressure impact (EP-A0434616).

Microprojectile bombardment has been developed as an effective transformation technique for cells, including plant cells. Sanford et al (Particulate Science and Technology, 5: 27-37, 1987) reported efficient delivery of nucleic acids to the cytoplasm of onion (Allium cepa) plant cells using microprojectile bombardment. Christou et al (Platn Physiol., 87: 671-A674, 1988) reported the stable transformation of soybean callus with a kanamycin resistance gene by microprojectile bombardment. The same authors reported that approximately 0.1-5% of the cells were penetrated and found observable levels of NPTII enzyme activity and kanamycin resistance up to 400mg/L in transformed calli. McCabe et al (1988, supra) reported the use of microprojectile bombardment to stably transform soybeans (Glycine max). McCabe et al also reported that R is a member of the group₀Recransformation of chimeric plants by R₁The discovery of plants (see also Weissenger et al, Annual Rev. Gene., 22: 421-.

Alternatively, plant plastids can be directly transformed. Stable transformation of chloroplasts has been reported in higher plants (see, e.g., Svab et al, Proc. Natl. Acad. Sci. USA, 90: 913-917, 1993; Staub and Maliga, EMBO J., 12: 601-606, 1993). The method relies on particle gun delivery of DNA containing a selectable marker and targeting of the DNA to the plastid genome by homologous recombination. In these methods, plastid gene expression can be achieved through the use of a plastid gene promoter or through transcriptional activation of a plastid-transmitted transgene positioned to facilitate silencing by a selective promoter sequence (such as a promoter sequence recognized by T7 RNA polymerase). Silencing a plastid gene is activated by a specific RNA polymerase expressed from a nuclear expression construct and targeting the polymerase to the plastid through the use of a transit peptide. Tissue-specific expression can be achieved in this way by a nuclear-encoded, plastid-directed specific RNA polymerase expressed from a suitable plant tissue-specific promoter. McBride et al (Proc. Natl. Acad. Sci. USA, 97: 7301-.

All plant transformation systems produce a mixture of transgenic and non-transgenic plants. Selection of transgenic plant cells can be achieved by introducing antibiotic or herbicide genes that enable selection on media containing the corresponding toxic compounds. In addition to those marker systems for the selection of transgenic plants, new so-called "positive selection systems" have been successfully used for plant transformation (PCT/EP94/00575, WO 94/20627). In contrast to antibiotic or herbicide resistance selection systems, where transgenic cells acquire the ability to survive on a selection medium, but kill non-transgenic cells, this approach facilitates the regeneration and growth of transgenic plant cells and starves them rather than kills non-transgenic plant cells. Therefore, this selection strategy is referred to as "positive selection". Vector systems for Agrobacterium-mediated transformation have been constructed and successfully used, for example, to transform potato, tobacco, and tomato (Haldrup A, Petersen SG, and Ollels FT, Platn mol. biol., 37: 287-296, 1998). Transformation systems based on such positive selection systems can be used according to the invention for introducing constructs comprising beta-diox II to obtain plants expressing beta-diox II polypeptides and thereby capable of enzymatically cleaving beta-carotene to form beta-apocarotene aldehyde. In addition, the use of those selection systems would have the advantage of overcoming the disadvantages of using antibiotic or herbicide genes in selection systems, such as toxicity or allergenicity of the gene product and interference with antibiotic treatment, as is generally known in the art.

The list of possible transformation methods listed above by way of example is not to be construed as exhaustive and is not intended to limit the subject matter of the invention in any way.

The present invention therefore also encompasses prokaryotic or eukaryotic host cells, seeds, tissues or whole organisms transformed or transfected with the DNA molecules according to the invention as listed above or with plasmids or vector systems in a suitable manner such that said host cells, seeds, tissues or whole organisms are capable of expressing a polypeptide or a functional fragment thereof having the biological activity of specifically cleaving beta-carotene and lycopene to form beta-apocarotene aldehyde and beta-ionone and apolycopene aldehyde, respectively, and/or having the ability to specifically bind antibodies prepared against said polypeptide or functional fragment thereof.

According to the invention, the prokaryotic or eukaryotic host cell, seed, tissue, or whole organism is selected from the group consisting of: bacteria, yeast, fungi, insects, animal, and plant cells, seeds, tissues, or whole organisms. With respect to prokaryotic taxa, the host may be selected from the group consisting of: probacteria (proteobacteria) including members of the alpha, beta, gamma, delta, and epsilon subgenus; gram-positive bacteria, including Actinomycetes (Actinomycetes), Firmicutes (Firmicutes), clostridia (Clostridium) and its relatives, yellow bacteria (flavobacteria), cyanobacteria (cyanobacteria), green sulfur bacteria (greensulfur bacteria), green non-sulfur bacteria (greennon-sulfur bacteria), and archaea (archaea). Suitable protobacteria belonging to the sub-alpha phylum may be selected from the group consisting of: agrobacterium (Agrobacterium), Rhodospirillum (Rhodospirillum), Rhodopseudomonas (Rhodopseudomonas), Rhodobacterium (Rhodobacter), Rhodomicrobium (Rhodomicbium), Rhodococcus (Rhodococcus), Rhizobium (Rhizobium), Nitrobacter (Nitrobacter), Oenospirium (Aquapirium), Micrococcus (Hyphomicium), Acetobacter (Acetobacter), Arthrobacter bailii (Beijerinckia), Paracoccus (Paracoccus), and Pseudomonas (Pseudomonas), preferably Agrobacterium and Rhodobacter, most preferably Agrobacterium (Agrobacterium) and Rhodobacter, respectively. Suitable protobacteria belonging to the beta subdivision may be selected from the group consisting of: rhodococcus (Rhodocyclus), Rhodococcus (Rhodopherax), Rhodovivax, Spirillum, Nitrosomonas (Nitrosomonas), Coccidium (Spherotilus), Thiobacillus (Thiobacillus), Alcaligenes (Alcaligenes), Pseudomonas (Pseudomonas), Bordetella (Bordetella), and Neisseria (Neisseria), preferably ammonia-oxidizing bacteria such as Nitrosomonas, most preferably Nitrosomonas ENI-11. Suitable protobacteria belonging to the subgenus γ may be selected from the group consisting of: chromobacterium (Chromatium), Thiospirillum (Thiospirillum), Beggitaatoa (Beggiatoa), Leuconostoc (Leucothrix), Escherichia (Escherichia), and Azotobacter (Azotobacter), preferably Enterobacteriaceae (Enterobacteriaceae) such as Escherichia coli (Escherichia coli), and most preferably Escherichia coli K12 strain such as M15 (described as DZ 291, Villarejo et al, J.Bacteriol., 120: 466-474, 1974), HB101(ATCC No. 33649), and Escherichia coli SG13009(Gottesman et al, J.Bacteriol. 265, 148: 273-273, 1981). Suitable protobacteria belonging to the δ subgenus may be selected from the group consisting of: bdellovibrio (Bdellovibrio), Desulfovibrio (Desulfovibrio), Desulfomonas (Desulfuromonas), and Myxobacteria (Myxobacteria) such as Myxococcus (Myxococcus), preferably Myxococcus xanthus (Myxococcus xanthus). Suitable protobacteria belonging to the epsilon subdivision may be selected from the group consisting of: ohiobacillus (Thiorulum), Wolinella (Wolinella), and Campylobacter (Campylobacter). Suitable gram-positive bacteria may be selected from the group consisting of: actinomycetes such as Actinomycetes (Actinomycetes), Bifidobacterium (Bifidobacterium), Propionibacterium (Propionibacterium), Streptomyces (Streptomyces), Nocardia (Nocardia), Actinoplanes (Actinoplanes), Arthrobacter (Arthrobacter), Corynebacterium (Corynebacterium), Mycobacterium (Mycobacterium), Micromonospora (Micromonospora), Frankia (Frankia), Cellulomonas (Cellulomonas), and Brevibacterium (Brevibacterium), Thenobacterium (Firmicutes) including Clostridium and relatives thereof such as Clostridium, Bacillus (Bacillus), Desulfuricus (Desulomalum), Thermomyces (Streptococcus), Lactobacillus (Lactobacillus), Streptococcus (Streptococcus), Streptococcus (Streptococcus), Streptococcus (Streptococcus), and Streptococcus (Streptococcus), Streptococcus (Streptococcus), and Streptococcus (Streptococcus), and Streptococcus (Streptococcus), Streptococcus (Streptococcus), and Streptococcus (Streptococcus) including Streptococcus), and Streptococcus (Streptococcus) including Streptococcus (Streptococcus), and Streptococcus (Streptococcus), can be strain, and Bacillus) including Bacillus, and Bacillus, such as a strain (Lactobacillus) including Bacillus, such as, Mycoplasma (Mycoplasma), acheoloplasma (Acheoleplas), and Spiroplasma (Spiroplas), preferably Bacillus subtilis and Lactococcus lactis. Suitable xanthines may be selected from the group consisting of: bacteroides (Bacteroides), Cytophaga (Cytophaga), and Flavobacterium (Flavobacterium), preferably Flavobacterium such as Flavobacterium ATCC 21588. Suitable cyanobacteria can be selected from the group consisting of: chlorococcales, including Synechocystis and Synechococcus, preferably Synechocystis sp and Synechococcus sp PS 717. Suitable green sulfur bacteria may be selected from the group consisting of: chlorella (Chlorobium), preferably Chlorella limanita (Chlorobium limicola f. thiosulfophilum). Suitable green non-sulfur bacteria may be selected from the group consisting of: chloroflexaceae (Chloroflexaceae) such as Chloroflexus (Chloroflexus), preferably orange Chloroflexandrius (Chloroflexarataceae). Suitable archaea may be selected from the group consisting of: halobacteriaceae (Halobacteriaceae) includes the genus Halobacterium (Halobacterium), preferably Halobacterium salinum.

With respect to eukaryotic taxa of fungi (including yeast), the host may be selected from the group consisting of: ascomycota (Ascomycota) includes Saccharomyces species (Saccharomyces) such as Pichia and Saccharomyces, and Anamorphic Ascomycota (Anamorphic Ascomycota) includes Aspergillus, preferably Saccharomyces cerevisiae and Aspergillus niger (ATCC 9142).

Eukaryotic host systems include insect cells preferably selected from the group consisting of: SF9, SF21, Trychplusiani, and MB 21. For example, a polypeptide according to the invention can be advantageously expressed in an insect cell system. Insect cells suitable for use in the method of the invention include in principle any lepidopteran (lepidopteran) cell capable of being transformed with an expression vector and expressing a heterologous protein encoded thereby. In particular, Sf cell lines such as the Spodoptera frugiperda (Spodoptera frugiperda) cell line IPBL-SF-21 AE (Vaughn et al, In Vitro, 13: 213-217, 1977) are preferably used. Particularly preferred is the derived cell line Sf 9. However, other cell lines may be used, such as Trichoplusia ni 368(Kutstack and Marmoroch, Invertebrate Tissue Culture Applications in medicine, Biology, and Agriculture, Academic Press, New York, USA, 1976). These cell lines, as well as other insect cell lines suitable for use in the present invention, are commercially available (e.g., Stratagene, Lajoria, Calif., USA). As well as expression in cultured insect cells, the present invention also includes expression of heterologous proteins such as β -diox II in intact insect organisms. The use of viral vectors such as baculoviruses enables infection of whole insects, which in some respects is easier to grow than cultured cells, since they require less special growth conditions. Large insects such as silk moths can provide high yields of heterologous proteins. Proteins can be extracted from insects according to conventional extraction techniques. Expression vectors suitable for use in the present invention include all vectors capable of expressing foreign proteins in insect cell lines. In general, vectors that are useful for mammalian and other eukaryotic cells are equally applicable to insect cell cultures. Baculovirus vectors specifically intended for use in insect cell culture are particularly preferred and are widely available commercially (e.g., Invitrogen and Clontech). Other viral vectors capable of infecting insect cells are also known, such as the Sindbis virus (Hahn et al, PNAS USA, 89: 2679-. The baculovirus vector of choice (reviewed in Miller, Ann. Rev. Microbiol., 42: 177-179, 1988) was Autographa californica (Autographa californica) polynuclear polyhedrosis virus (AcMNPV). At least part of the polyhedrin gene of AcMNPV is typically replaced with a heterologous gene, as polyhedrin is not essential for viral propagation. For insertion of heterologous genes, transfer vectors are advantageously used. Preferably, the transfer vector is prepared in an E.coli host and the DNA insert is then transferred to AcMNPV by a process of homologous recombination.

The eukaryotic host system further comprises animal cells, preferably selected from the group consisting of: baby Hamster Kidney (BHK) cells, Chinese Hamster Ovary (CHO) cells, Human Embryonic Kidney (HEK) cells, and COS cells, with 3T3 and 293 cells being most preferred.

The host cells referred to in this disclosure include cells cultured in vitro and cells within a host organism.

The invention also provides transgenic plant material selected from the group consisting of: protoplasts, cells, calli, tissues, organs, seeds, embryos, ovules, zygotes, and the like, in particular whole plants which have been transformed by a method according to the invention and which comprise the recombinant DNA of the invention in expressible form, and methods for the production of said transgenic plant material.

As used herein, the term "plant" generally includes eukaryotic algae, embryonated plants including the phylum Bryophyta, the phylum Pteridophyta, and the phylum Spermatophyta (stertophyta), such as the subphylum Gymnospermae (Gymnospermae) and the subphylum Angiospermae (Angiospermae), the latter including ligniopsida, Rosopsida (true- "dicotyledonous"), and the class Liliopsida ("monocotyledonous"). Representative and preferred examples include cereal seeds such as rice, wheat, barley, oats, amaranth, flax, triticale, rye, corn, and other grasses; oilseeds such as canola seeds, cotton seeds, soybeans, safflower, sunflower, coconut, palm, and the like; other edible seeds or seeds containing edible parts, including pumpkin, squash, sesame, poppy, grape, mung bean, peanut, pea, bean, radish, alfalfa, cocoa, coffee, hemp; tree nuts such as walnuts, almonds, pecans, chickpeas, and the like. Other examples include potato, carrot, sweet potato, sugar beet, tomato, pepper, cassava, willow, oak, elm, maple, apple, and banana. In general, the present invention is applicable to the cultivation of species for food, drugs, beverages, and the like. Preferably, the target plant selected for transformation is cultivated for a food product, such as a grain, root, legume, nut, vegetable, tuber, fruit, condiment, or the like.

Positive transformants generated according to the invention can be regenerated into plants following procedures well known in the art (see, e.g., McCormick et al, Plant Cell Reports, 5: 81-84, 1986). These plants are then grown and the progeny, after pollination with the same transformed species or different species, can be evaluated for the presence of the desired trait and/or the extent of expression of the desired trait and the resulting hybrid identified as having the desired phenotypic characteristic. The first assessment may include, for example, the level of bacterial/fungal resistance of the transformed plant. Two or more generations may be grown to ensure that the phenotypic feature of interest is stably maintained and inherited, and then the seeds harvested to ensure that the desired phenotype or other characteristic has been achieved.

Also included within the scope of the invention are transgenic plants, particularly transgenic fertile plants transformed by the methods of the invention and asexually and/or sexually propagated progeny thereof, which still exhibit new and desirable characteristics resulting from transformation of the parent plant.

The term "progeny" is understood to include progeny of transgenic plants resulting from "asexual propagation" and "sexual propagation". This definition is also intended to include all mutants and variants obtainable by known methods, such as cell fusion or mutant selection, and all crossing and fusion products of transformed plant material, which mutants and variants still exhibit the characteristic properties of the originally transformed plant.

Plant parts, such as flowers, stems, fruits, leaves, roots, which originate from the transgenic plants previously transformed by the method of the invention or their progeny and thus consist at least in part of the transgenic cells, are also subject of the invention. Another aspect of the invention relates to diagnostic means and methods for measuring, analyzing and assessing the qualitative and quantitative properties inherent to nucleic acid and/or amino acid molecules according to the invention. For example, suitably designed oligonucleotides (representative examples of sequences disclosed herein) enable, for example, tissue typing, expression mapping, and allele determination (SNP analysis), preferably in high throughput devices such as DNA and protein microarrays, and the like. Other fields of application include the generation of specific constructs as gene therapy tools, and the generation of antibodies for, e.g., purification, therapeutic, or diagnostic purposes.

In accordance with yet another embodiment of the present invention, there are provided antibodies that specifically recognize and bind to β -diox ii. For example, a sequence for a polypeptide having SEQ ID NO: 17. 19, or 21, or a pharmaceutically acceptable salt thereof. Alternatively, a β -diox II or a β -diox II fragment such as described above (which may also be synthesized by in vitro methods) is fused (by recombinant expression or in vitro peptide bond) to an immunogenic polypeptide and this fused polypeptide is then used to generate antibodies against the β -diox II epitope.

Anti-beta-diox II polypeptides can be recovered from the serum of the immunized animal. Monoclonal antibodies can be prepared in a conventional manner from cells from the immunized animal.

The antibodies of the invention are useful for studying the localization of β -diox II, screening expression libraries to identify nucleic acids encoding β -diox II or functional domains, and purifying β -diox II, among others.

The antibodies according to the invention may be whole antibodies of the natural type, such as IgE and IgM antibodies, but preferably IgG antibodies. In addition, the invention includes antibody fragments, such as Fab, F (ab')₂Fv, and scFv. Small fragments (such as Fv and scFv) have advantageous properties for diagnostic and therapeutic applications due to their small size, i.e. superior tissue distribution.

Diagnostic and therapeutic applications of the antibodies according to the invention are particularly indicated. Thus, they may be antibodies that have been altered to contain effector proteins such as toxins or markers. Especially preferred are markers that are capable of imaging antibody distribution in tumors in vivo. These markers may be radioactive or radiopaque markers, such as metal particles, that are readily visualized in the patient. In addition, they may be fluorescent or other markers that can be visualized on tissue samples taken from patients.

Recombinant DNA technology can be used to improve the antibodies of the invention. Thus, chimeric antibodies can be constructed to reduce their immunogenicity in diagnostic or therapeutic applications. In addition, immunogenicity can be minimized by humanizing antibodies by CDR-grafting (see EP-A-239400, Winter) and optionally framework modification (see WO 90/07861, Protein Design Lab).

The antibodies according to the invention may be obtained from animal sera or, in the case of monoclonal antibodies or fragments thereof, may be produced in cell culture. Recombinant DNA technology can be used to produce antibodies in bacterial or preferably mammalian cell culture according to established protocols. The selected cell culture system preferably secretes the antibody product.

Accordingly, the invention includes a method for producing an antibody according to the invention, comprising culturing a host (e.g., an E.coli or mammalian cell) that has been transformed with a hybrid vector comprising an expression cassette comprising a promoter operably linked to a first DNA sequence encoding a signal peptide and linked in frame to a second DNA sequence encoding an antibody, and isolating the antibody.

The in vitro proliferation of hybridoma cells or mammalian host cells is carried out in a suitable medium, which is a conventional standard medium, such as Dulbecco's Modified Eagle's Medium (DMEM) or RPMI 1640 medium, optionally supplemented with mammalian serum (such as fetal bovine serum) or trace elements and growth maintenance supplements (such as feeder cells, such as normal mouse peritoneal effusion cells, splenocytes, bone marrow macrophages, 2-aminoethanol, insulin, transferrin, low density lipoproteins, oleic acid, and the like). Propagation of bacterial cells or yeast cells as host cells is likewise carried out in suitable media known in the art, for example in LB, NZCYM, NZM, Terrific broth, SOB, SOC, 2XYT, or M9 minimal medium for bacteria and YPD, YEPD, minimal medium, or complete minimal Dropout medium for yeast.

In vitro production can provide relatively pure antibody preparations and can be scaled up to produce large quantities of the desired antibody. Techniques for bacterial cell, yeast, or mammalian cell culture are known in the art and include homogeneous suspension culture (e.g., airlift reactors or continuous stirred reactors), immobilized or entrapped cell culture (e.g., hollow fibers, microcapsules, agarose beads, or ceramic columns).

A large amount of the desired antibody can also be obtained by in vivo proliferation of mammalian cells. For this purpose, hybridoma cells producing the desired antibody are injected into a histocompatible mammal, causing the growth of an antibody-producing tumor. Optionally, the animal is primed with a hydrocarbon, especially a mineral oil such as pristane (tetramethylpentadecane), prior to injection. After 1-3 weeks, antibodies are isolated from the body fluids of those mammals. For example, hybridoma cells obtained by fusion of suitable myeloma cells with antibody-producing splenocytes from Balb/c mice, or transfected cells derived from the hybridoma cell line Sp2/O and producing the desired antibody, are injected intraperitoneally into Balb/c mice (which are optionally pretreated with pristane), and ascites are collected from the animals 1-3 weeks later.

The cell culture supernatant is screened for the desired antibody, preferably by immunofluorescence staining of β -diox II expressing cells, by immunoblotting, by an enzyme immunoassay (such as a sandwich assay or dot assay), or a radioimmunoassay.

For isolation of the antibody, the immunoglobulins in the culture supernatant or ascites can be concentrated, for example by ammonium sulfate precipitation, dialysis in hygroscopic materials such as polyethylene glycol, filtration through selective membranes, etc. If necessary and/or desired, the antibody may be purified by conventional chromatography, for example, gel filtration, ion exchange chromatography, DEAE-cellulose chromatography, and/or immunoaffinity chromatography, such as affinity chromatography using β -diox II protein or protein A.

The invention also relates to hybridoma cells secreting the monoclonal antibodies of the invention. Preferred hybridoma cells of the invention are genetically stable, secrete monoclonal antibodies of the invention with the desired specificity, and can be activated by deep-frozen cultures by thawing and recloning.

The invention also relates to a method for preparing a hybridoma cell line secreting monoclonal antibodies directed against beta-diox II, characterized in that a suitable mammal (e.g. a Balb/c mouse) is immunized with purified beta-diox II protein, an antigen carrier containing purified beta-diox II, or cells carrying beta-diox II, antibody-producing cells of the immunized animal are fused to cells of a suitable myeloma cell line, the hybrid cells obtained in the fusion are cloned, and cell clones secreting the desired antibody are selected. For example, splenocytes from Balb/c mice immunized with cells bearing β -diox II are fused with cells of the myeloma cell line PAI or the myeloma cell line Sp2/O-Agl4, the resulting hybrid cells are screened for secretion of the desired antibody, and positive hybridoma cells are cloned.

Preferably for the preparation of hybridoma cell lines, characterized by several (e.g. 4-6) subcutaneous and/or intraperitoneal injections 10 over several months (e.g. 2-4 months)⁷-10⁸Balb/c mice are immunized with cells of human tumor origin and expressing beta-diox II with a suitable adjuvant, splenocytes are collected from the immunized mice 2-4 days after the last injection, and the cells of the myeloma cell line PAI are fused in the presence of a fusion-promoting agent, preferably polyethylene glycol. Preferably, myeloma cells are fused to a 3-20 fold excess of splenocytes from immunized mice in a solution containing about 30-50% polyethylene glycol having a molecular weight of about 4000. After fusion, cells were expanded in the appropriate medium described above, with the option of adding at regular intervalsSexual media (e.g., HAT media) to prevent overgrowth of normal myeloma cells relative to desired hybridoma cells.

The invention also relates to recombinant DNA comprising an insert encoding a heavy chain variable domain and/or a light chain variable domain of an antibody directed against a β -diox II protein. These DNAs include, by definition, coding single-stranded DNA, double-stranded DNA consisting of the coding DNA and its complementary DNA, or these complementary (single-stranded) DNAs themselves.

In addition, the DNA encoding the heavy chain variable domain and/or the light chain variable domain of an antibody against β -diox II may be enzymatically or chemically synthesized DNA having the authentic DNA sequence encoding the heavy chain variable domain and/or the light chain variable domain or a mutant thereof. Mutants of authentic DNA are DNA encoding the heavy and/or light chain variable domains of the above-described antibodies, in which one or more amino acids are deleted or substituted with one or more other amino acids. Preferably, the modification is located outside the CDRs of the heavy chain variable domain and/or the light chain variable domain of the antibody. Such mutant DNA may also be silent mutants in which one or more nucleotides are replaced by other nucleotides and the new codon encodes the same amino acid. Such mutant sequences are also degenerate sequences. Degenerate sequences are degenerate within the meaning of the genetic code, i.e.in which an unlimited number of nucleotides are replaced by other nucleotides, without changing the amino acid sequence originally encoded. These degenerate sequences are useful because they have different restriction sites and/or specific codon frequencies preferred by the particular host (in particular E.coli) in order to obtain optimal expression of the murine variable domain of the heavy chain and/or the murine variable domain of the light chain.

The term "mutant" is intended to include DNA mutants obtained by in vitro mutagenesis of authentic DNA according to methods known in the art.

To assemble the complete tetrameric immunoglobulin molecule and express the chimeric antibody, recombinant DNA inserts encoding the heavy and light chain variable domains are fused to the corresponding DNA encoding the heavy and light chain constant domains, and then transferred to an appropriate host cell, for example after incorporation into a hybrid vector.

In the case of a diagnostic composition, it is preferred that the antibody is provided with means for detecting the antibody, which may be enzymatic, fluorescent, radioisotope, or other means. Antibodies and detection means may be provided for simultaneous, simultaneous but separate, or sequential use in a diagnostic kit intended for use in diagnosis.

For example, the invention provides diagnostic methods for pathologies characterized by an increase or decrease in β -diox II levels in a given subject or individual. For example, a test sample is obtained and contacted with an agent capable of specifically binding to β -diox II or a nucleotide sequence capable of binding to a nucleic acid molecule encoding β -diox II under suitable conditions that allow for specific binding between the agent or the nucleotide sequence and the β -diox II target amino acid or nucleic acid sequence. Subsequently, the amount of specific binding in the test sample can be compared to the amount of specific binding in a control sample, wherein a pathology associated with a pathway induced by β -diox II can be diagnosed from an increase or decrease in the amount of specific binding in the test sample relative to the control sample.

The invention also provides methods of increasing or decreasing the amount of beta-diox II in a cell or tissue, which can modulate the level of vitamin a or other retinoids. For example, the amount of β -diox II in a given target cell or tissue can be increased by introducing into the cell or tissue a nucleic acid molecule comprising a nucleic acid sequence encoding β -diox II or a functional fragment thereof. An increase in the amount of β -diox II in cells or tissues can induce or promote vitamin a accumulation, which would be beneficial not only for humans, but also for animals and feed that are frequently given vitamin preparations to improve nutritional quality.

Preservation of biological materials

Coli cells carrying the gene coding for beta-carotene dioxygenase derived from Drosophila melanogaster have been deposited under the Budapest treaty in Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) in Germany with the identification reference "beta-diox", number DSM 13304.

The following examples are illustrative, but not limiting, of the invention.

ExamplesPlasmid constructsConstruction of beta-carotene-accumulating Escherichia coli strains

The vector pFDY297 was used to construct a plasmid carrying the β -carotene biosynthesis gene from Erwinia herbicola (Erwinia herbicoloa). pFDY297 is a derivative of pACYC177 (bp 486-3130) having bp 1-485 into which pBluescriptSK has been introduced. To clone the beta-carotene biosynthesis genes from Erwinia herbicola, appropriate endonuclease restriction sites were introduced at both ends of the PCR product. The crtE gene was first inserted into pFDY 297. Using primers: 5'-GCGTCGACCGCGGTCTACGGTTAACTG-3' (SEQ ID NO: 3) and 5'-GGGGTACCCTTGAACCCAAAAGGGCGG-3' (SEQ ID NO: 4) and the Expand PCR System, an amplification PCR System (Boehringer, Mannheim, Germany), by PCR amplification of CrtE from pBL376(Hundle BS et al, mol. Gen. Genet., 245: 406. sup. 416, 1994), which plasmid encodes the entire carotenoid biosynthesis gene cluster from Erwinia herbicola. The PCR product was digested with KpnI and SalI and ligated into the appropriate site of pFDY297 to yield plasmid pCRTE. Using primers: 5'-GCTCTAGACGTCTGGCGACGGCCCGCCA-3' (SEQ ID NO: 5) and 5'-GCGTCGACACCTACAGGCGATCCTGCG-3' (SEQ ID NO: 6) and Expand PCRSystem (Boehringer, Mannheim, Germany), the genes crtB, crtI, and crtY were amplified from pBL376 by PCR. The PCR product was digested with XbaI and SalI and ligated into the appropriate site of pCRTE to generate plasmid pORANGE. After transformation of the plasmid into E.coli JM109, the resulting strain was able to synthesize beta-carotene. Cloning of beta-diox from Drosophila melanogaster

We isolated total RNA from the heads of adult drosophila by manual dissection. Reverse transcription was performed using oligo (T) -adaptor primer 5'-GACCACGCGTATCGATGTCGACTTTTTTTTTTTT TTTTTT-3' (SEQ ID NO: 7) and Superscript reverse transcriptase (Gibco, Germany). For cloning of the full-length cDNA, PCR was performed using specific upstream primer 5'-GCAGCCGGTGTCTTCAAGA G-3' (SEQ ID NO: 8) derived from the published EST fragment (accession No. AI063857) and 3 ' anchored primer 5'-GACCACGCGTATCGATGTCG A-3' (SEQ ID NO: 9) and the ExpandPCR System, amplification PCR System (Boehringer, Mannheim, Germany). After 0.8% agarose gel electrophoresis, the resulting PCR product was isolated, directly ligated into the vector pBAD-TOPO (Invitrogen, the Netherlands) and transformed into a strain of E.coli that accumulates β -carotene. Using this cloning strategy, Drosophila cDNA was translationally fused to the short open reading frame of the vector and under the control of a positively regulated promoter inducible by L-arabinose. The bacteria were plated on LB agar containing ampicillin (100. mu.g/ml), kanamycin (50. mu.g/ml), and L-arabinose (0.2%). Positive colonies were identified by fading from yellow to almost white. To analyze the resulting plasmid p β diox and confirm its structure, both strands were sequenced completely. Expression, purification, and enzymatic Activity of beta-diox-gex

For expression of β -diox, cDNA was amplified using Gex upstream primer 5'-GGAATTCGCAGCCGGTGTCTTCAAGAG-3' (SEQ ID NO: 10) and Gex downstream primer 5'-CCTCGAGGTAGTCTTCCCATATAAGG-3' (SEQ ID NO: 11) and the Expand PCR System, amplification PCR System (Boehringer, Mannheim, Germany). Appropriate restriction sites were introduced at both ends of the PCR product by oligonucleotide primers. After restriction digestion with EcoRI and NcoI, the PCR product was cloned into the appropriate site of the expression vector pGEX-4T-1(Pharmacia, Frieberg, Germany). The resulting plasmid, p β diox-gex, was transformed into E.coli strain JM 109. Expression of the fusion protein β -diox-gex in E.coli and subsequent purification on glutathione sepharose 4B (Pharmacia, Frieburgh, Germany) was performed as described in the manufacturer's protocol. Determination of beta-diox enzyme Activity

The purified protein was incubated in 300. mu.l of a buffer containing 50mM Tricine/NaOH (pH7.6) and 100mM NaCl and 0.05% Triton X100. The reaction was started by adding 5. mu.l of beta-carotene (80. mu.M in ethanol). Adding FeSO respectively₄And L-antibodyAscorbic acid to final concentrations of 5. mu.M and 10 mM. After 2 hours at 30 ℃ incubation 100. mu.l of 2M NH was added₂The reaction was stopped with OH (pH6.8) and 200. mu.l of methanol. Extraction and HPLC analysis were performed as described above. Determination of mRNA levels in different parts of the body by RT-PCR

Total RNA was isolated from adult Drosophila (male and female). The head, chest, and abdomen were obtained by manual dissection (legs and wings had been removed beforehand). To measure the amount of steady state mRNA of β -diox, RT-PCR was performed as described (von Lintig J et al, Plant J., 12: 625-634, 1997). Using oligo (dT)₁₇) Reverse transcription was performed with primers and Superscript reverse transcriptase (Gibco, Germany). PCR was performed using β -diox II upstream 5'-CTGCAAACGGACCGACCACGT-3' (SEQ ID NO: 12) and downstream 5 ' -GCAAATCTATCGAAGATCGAG-3(SEQ ID NO: 13) primers and Taq polymerase (Pharmacia, Frieburg, Germany). The mRNA level of the constitutively expressed ribosomal protein rp49 was investigated as an internal control using the intron-spanning upstream primer 5'-GACTTCATCCGCCACCAGTC-3' (SEQ ID NO: 14) and downstream primer 5'-CACCAGGAACTTCTTGAATCCG-3' (SEQ ID NO: 15). PCR was performed as two separate primer assays for β -diox and rp49, and all four primers were combined in one assay. Extraction of beta-carotene and retinoids from E.coli and HPLC analysis

Coli strains were cultured in LB medium in 50ml flasks under safety Red light until OD of the culture₆₀₀To reach 1. The expression of beta-diox was induced for 6 or 16 hours by addition of L-arabinose (0.2% w/v). The bacteria were then harvested by centrifugation. The precipitate was extracted by the following scheme: A. the pellet was resuspended in 200. mu.l of 6M formaldehyde and incubated at 30 ℃ for 2 minutes, then 2ml of dichloromethane were added. Carotene and retinoids were extracted 3 times with 4ml n-hexane. The collected organic phases were evaporated and dissolved in HPLC solvent. B. Resuspend pellet in 2ml 1M NH₂OH (dissolved in 50% methanol) and incubated at 30 ℃ for 10 min. Extracted 3 times with petroleum ether. Collecting the organic phase in N₂Dried under reflux and dissolved in HPLC solvent. After equipping a multi-diode-array (model 166, Beckman) and System Gold noveau software (Beckman,U.S. A) was performed on Hypersil 3 μm (Knaur, Germany) on System Gold (Beckman). HPLC solvent A (n-hexane/ethanol 99.75: 0.25) was used for retinal and solvent B (n-hexane/ethanol 99.5: 0.5) was used for retinal oxime. The reference all-trans, 13-cis, and 9-cis retinaldehyde were purchased from Sigma (germany); 11-cis retinal was isolated from dark-adapted bovine eyes. Respectively by NaBH₄By reduction with NH or₂Reaction of OH to obtain the corresponding retinol and oxime. To quantify the molar mass, the peak integrals were measured with a defined amount of reference. Preparation of Total RNA from different tissues of mice

For experiments, 7-week-old BALB/c mice (male and female) were sacrificed and different tissues (colon, small intestine, stomach, spleen, brain, liver, heart, kidney, lung, and testis) were dissected by hand and immediately frozen in liquid nitrogen. 50-100mg of each tissue was homogenized in liquid nitrogen in a mortar with a pestle, and total RNA was isolated using the RNeasy Kit, RNA easy Kit (Qiagen, Hilden, Germany). The concentration of the isolated total RNA was determined spectrophotometrically. Cloning of cDNA encoding beta-diox homologous protein from mice

To clone the full-length cDNA encoding the putative mouse β -carotene dioxygenase, RACE-PCR was performed using the 5 '/3' RACE kit (Roche Molecular Biochemicals, Mannheim, Germany). Reverse transcription was performed using 500ng of total RNA isolated from liver, oligo dT anchor primer, and Superscript reverse transcriptase (Life Technologies, Inc.). PCR was performed using the anchor primer and the specific upstream primer 5'-ATGGAGATAATATTTGGCCAG-3' (SEQ ID NO: 22) for beta, beta-carotene-15, 15 'dioxygenase (beta-diox I) or the specific upstream primer 5' -ATGTTGGGACCGAAGCAAAGC-3(SEQ ID NO: 24) for beta-diox II and the Expand PCR System, i.e., amplification PCR System (Roche molecular biochemicals). The PCR products were ligated into the vector pBAD-TOPO (Invitrogen, Netherlands) to yield plasmids pDIox I and pDIox II. Tissue-specific expression of beta-carotene dioxygenase in mice

RT-PCR was performed using total RNA isolated from different tissues (100ng) as described (von Lintig J, Welsch R, Bonk M, Giuliano G, Batschauera, and Kleinig H, Plant J., 12: 625-634, 1997). The following primer sets were used. β -dioxI: an upstream primer 5'-ATGGAGATAATATTTGGCCAG-3' (SEQ ID NO: 22) and a downstream primer 5'-AACTCAGACACCACGATTC-3' (SEQ ID NO: 23); β -diox II: an upstream primer 5'-ATGTTGGGACCGAAGCAAAGC-3' (SEQ ID NO: 24) and a downstream primer 5'-TGTGCTCATGTAGTAATCACC-3' (SEQ ID NO: 25). The mRNA for β -actin was analyzed using upstream primer 5'-CCAACCGTGAAAAGATGACCC-3' (SEQ ID NO: 26) and downstream primer 5'-CAGCAATGCCTGGGTACATGG-3' (SEQ ID NO: 27) as controls for the integrity of each RNA sample. In vitro assay for enzymatic Activity

The plasmid pDIOx II was transformed into E.coli strain XL1-blue (Stratagene Co.) for heterologous expression of the β -diox II polypeptide. The bacteria were cultured at 28 ℃ until A₆₀₀Up to 1.0. L- (+) -arabinose was then added to a final concentration of 0.8% (w/v) and the bacteria were incubated for a further 3 hours. After harvesting the bacteria, they were disrupted in a buffer containing 50mM Tricine/KOH (pH7.6), 100mM NaCl, and 1mM dithiothreitol using a French press. The crude extract was centrifuged at 20,000Xg for 20 minutes. The supernatant was dialyzed against the same buffer at 4 ℃ for 1 hour. The enzyme activity in the crude extract (100. mu.g total protein) was determined as described (Nagao A, During A, Hoshino C, Terao J, Olson JA, Arch. biochem. Biophys., 328: 57-63, 1996) by adding beta-carotene in Tween 40 micelles (final concentration in the assay 300. mu.M beta-carotene and 0.2% Tween 40). Lipophilic compounds were then extracted and analyzed by HPLC as (von Lintig J and Vogt K, J.biol.chem., 275: 11915-11920, 2000). HPLC analysis of E.coli strains expressing two different classes of beta-carotene dioxygenases, accumulating beta-carotene and lycopene from mice

Plasmids pDiox I and pDiox II were transformed into appropriate strains of escherichia coli. Culture conditions and analysis of carotene and its cleavage products were as previously described (von Lintig J and VogtK, J.biol.chem., 275: 11915-11920, 2000). LC-MS and GC-MS Mass Spectrometry of cleavage products

Coli strains were grown overnight and isolatedThe bacteria were harvested from the hearts. For solid phase extraction, SPME syringes (100 μm PDMS, Supelco, Deisenhofen, Germany) were incubated in the supernatant for 15 min. The compound adsorbed to the solid phase was then directly subjected to GC-MS (GC: Hewlett-Packard 6890; MS: Hewlett-Packard 5973(70eV), Waldbronn, Germany) with a temperature program which was increased from the initial 100 ℃ to 300 ℃ at 6 ℃/min. DB-1(30m x 0.25mm x 0.25 μm film thickness, J)&W, folcom, canada) as a column and helium as a carrier gas. For the LC-MS analysis, bacterial pellets were extracted in the presence of hydroxylamine as previously described (von Lintig J and Vogt K, J.biol.chem., 275: 11915-11920, 2000). LC/MS was performed on an HP1100 HPLC module system (Hewlett packard. waldbronn, germany) coupled to a Micromass (manchester, uk) VG platform II quadrupole mass spectrometer equipped with an APcI interface (atmospheric chemical ionization). uV absorption was monitored using a Diode Array Detector (DAD). MS parameters (APcI)⁺Type) is as follows: the source temperature is 120 ℃; APcI probe temperature 350 ℃; crown 3.2 kV; high voltage lens 0.5 kV; the cone voltage is 30V. The system is operated in full scan mode (m/z 250-1000). Data were acquired and processed using MassLynx 3.2 software. For peak separation, a Bischoff (Leonberg, Germany) Nucleosil RP-C18 column (5 μm, 250X4.6mm) was used and maintained at 25 ℃. The mobile phase consists of an acetonitrile/methanol 85: 15(v/v) mixture (A) and isopropanol (B); gradient% a (min) 100(8) -70(10) -70(25) -100(28) -100 (32); flow rate 1 ml/min; the injection volume was 20. mu.l. Sequence comparison and phylogenetic tree analysis

The results generated using the vector NTI Suite 6.0(InforMax, Oxford, UK) are shown in FIG. 15.

Using chemicals: beta-ionone (Roth, Carlsrue, Germany), 12 '-beta-apocarotene aldehyde (BASF, Lodvichg, Germany), and 8' -beta-apocarotene aldehyde (Sigma, Deisenhofen, Germany). Results

The insect EST library was searched for a homologue of vp14, a plant carotenoid cleavage enzyme, and the published EST fragment from Drosophila melanogaster was found (accession number AI 063857). To clone the full-length cDNA and directly test beta-HuRaphanus sativus dioxygenase I Activity Escherichia coli capable of synthesizing and accumulating beta-carotene was constructed by introducing a set of beta-carotene biosynthesis genes from Erwinia herbicola (Hundle BS et al, S.a.). The method enables the detection of the formation of retinoids by the fading of the colony from yellow (beta-carotene) to almost white (retinoids) and provides a rapid and efficient in vitro test system for the identification of beta-carotene dioxygenase I activity. For this purpose, total RNA was isolated from Drosophila heads and cDNA was synthesized. Using specific oligonucleotides derived from EST fragments and dT₁₇The anchored oligonucleotides were subjected to RACE-PCR. The obtained PCR product was directly cloned into an expression vector pBAD-TOPO and transformed into the E.coli strain. After plating the cells on LB medium containing 0.2% L-arabinose to induce expression of putative β -carotene dioxygenase I, several almost white colonies were found and further analyzed (fig. 2). The overnight incubation was performed under safe red light to minimize photo-oxidation induced isomerization and non-specific cleavage of beta-carotene. Beta-carotene and retinoids were extracted and analyzed by HPLC. The control strain transformed with the vector alone lacked the ability to cleave beta-carotene and no trace of retinoids were detected. But bacteria expressing drosophila cDNA contained significant amounts of retinoids in addition to β -carotene (fig. 3 a). Retinoids were identified by residence time (retentivity time) and co-chromatography with authentic standards and by their absorption spectra (fig. 4). The predominant retinal isomer is the all-trans form, with only about 20% of the 13-cis form. Depending on the incubation time of the bacteria after induction, it is also possible to detect significant amounts of all-trans and 13-cis retinol and esters of these retinol isomers. The retinoids found were consistent with their isomeric composition of the β -carotene precursor, which was identified by a separate HPLC system. Extraction was also carried out in the presence of hydroxylamine in order to confirm the formation of retinal and to improve the yield of retinoids and to isolate their isomers. Figure 3b shows that this treatment resulted in the formation of all-trans and 13-cis retinal oximes with a corresponding blue shift in their absorption spectra. Analysis ofIt was demonstrated that in addition to retinal, significant amounts of retinol and retinyl ester were formed in E.coli (Table 1). The question was whether E.coli is also able to produce retinoic acid from retinal. To analyze retinoic acid formation, cells were lysed and the extracts analyzed on an HPLC system using established protocols (Thaller C and Eichele G, Nature, 327: 625-62814, 1987). The results revealed that under these conditions, significant amounts of retinal and retinol could be detected in E.coli, but no retinoic acid was formed.

TABLE 1

Escherichia coli^(-)Strain escherichia coli⁽⁺⁾Bacterial strains

All-trans retinal n.d. 4.7

13-cis-retinal n.d. 1.5

All-trans retinol n.d. 8.0

13-cis-Retinol n.d. 2.4

n.d. 1.8

Retinoids of the sigma type-18.4

Beta-carotene 56.021.4

n.d.: no detectable E.coli was obtained from the bacterial culture (16 hours at 28 ℃ C.)⁽⁺⁾Strain and Escherichia coli^(-)Molar amounts of beta-carotene and retinoids in the bacterial strain (pmol/mg dry weight).

Taken together, these results demonstrate that the cloned cDNA encodes beta-carotene dioxygenase, the correspondingIt was named as β -diox I. Since the only retinoid, i.e., C, was found in the E.coli test system₂₀The compound, and therefore presumably, catalyzes the central cleavage of β -carotene, resulting in the formation of two molecules of retinal.

To further analyze the enzymatic properties of β -diox I, the cDNA was cloned into the expression vector pGEX-4T-1 and expressed as a fusion protein. To exclude the possibility that fusion to the N-terminus of glutathione-S-transferase would abolish enzymatic activity, the construct (. beta. -diox-gex) was transformed into a strain of E.coli which synthesizes β -carotene. Using the test methods described above, it was shown that the extent of retinoid formation was the same as that of unfused β -diox I (data not shown). After expression of beta-diox-gex in E.coli, the protein was purified by affinity chromatography. Purification was achieved without the addition of detergent, indicating that the fusion protein was soluble and not tightly associated with the membrane. To test the enzyme activity in vitro, 1. mu.g of purified protein was incubated in an assay containing 0.05% Triton X100 in the presence of beta-carotene for 2 hours. To analyze the products formed, the reaction was stopped by adding hydroxylamine/methanol and the products were analyzed by HPLC after extraction. Analysis revealed the formation of retinal (figure 5). Addition of FeSO in the assay₄Ascorbic acid resulted in increased formation of cleavage products (fig. 5A), while the addition of EDTA inhibited the conversion of β -carotene to retinal (fig. 5C). These results indicate that the enzymatic activity of the dioxygenase is iron dependent, as reported in several in vitro systems of animal origin. In conclusion, the activity of the β -diox II enzyme identified so far in E.coli can likewise be measured in vitro on purified proteins and lead to the formation of the same product.

Sequence analysis revealed that the cDNA encoded a 620 amino acid protein (SEQ ID NO: 2), with a calculated molecular weight of 69.9kDa (FIG. 6). The deduced amino acid sequence shares sequence homology with plant-like retinoids dioxygenase vpl4, with a lignin stilbene synthase from Pseudomonas paucimobilis (Pseudomonas paucimobilis), and with several proteins of the cyanobacterium genus Synechocystis (Synechocystis) of which the function is unknown. However, the highest sequence homology was found with RPE65 (a protein from vertebrate retinal pigment epithelium, first described in bovine eyes). RPE65 showed 36.7% overall sequence identity with β -diox I. The alignment of the beta-diox I deduced amino acid sequence, RPE65, to vp14 using program Map showed a unique pattern of conserved regions (fig. 7). Insect proteins have long extensions near the C-terminus compared to RPE65 and vp 14. The N-terminal extension of the plant protein vp14 relative to its animal homologue is most likely due to the target sequence for plastid import. Sequence homology of β -diox II with bacterial and plant dioxygenases suggests that we are investigating a novel dioxygenase enzyme present in bacteria, plants, and animals.

The expression pattern of beta-diox I mRNA was investigated by RT-PCR. As shown in fig. 8, mRNA was exclusively restricted to the head, whereas β -diox ImRNA was not detectable in the chest and abdomen by this method. Although Drosophila uses 3-hydroxy retinal for vision, it has been shown that in addition to 3-hydroxy retinoids (zeaxanthin and lutein), beta-carotene may also be a suitable precursor. In addition, it has been demonstrated that fruit flies are capable of hydroxylating retinal at position 3 of the β -ionone ring and forming the very visible enantiomer (3S) -3-hydroxy retinal, which is the unique chromophore of ring-split Cyclorrhaph fruit flies. These results demonstrate that in drosophila, β -carotene cleavage and further metabolism of retinoids as well as the visual cycle are located in the same part of the body. Cloning of cDNA encoding novel Carotene dioxygenase (beta-diox II)

To clone the cDNA encoding the putative β -carotene dioxygenase, we searched the mouse EST database and found two EST fragments with significant peptide sequence similarity to the β -diox I identified so far by drosophila. One EST fragment (AWO44715) encodes mouse β -diox I (Redmond TM, Gentleman S, Duncan T, Yu S, Wiggert B, GanttE, and Cunningham FX jr., j.biol.chem., online, 2000), and the other (AW611061) has significant similarity to drosophila, chicken, and mouse β -diox I and mouse RPE 65. However, it is not identical and thus is a hitherto unknown new representative of this class of dioxygenases. To obtain full-length cDNA, we designed upstream primers deduced from EST fragments.We then performed RACE-PCR on total RNA preparations derived from the liver of 7-week-old BALB/c male mice. The PCR product was cloned into the vector pBAD-TOPO and subjected to sequence analysis. The cDNA (SEQ ID NO: 16) encodes a protein of 532 amino acids. Sequence comparison revealed that the deduced amino acid sequence (SEQ ID NO: 17) shares 39% sequence identity with mouse β, β -15, 15' -dioxygenase (β -dioxI) (FIG. 10). Several highly conserved amino acid sequences were found and may be involved in binding the cofactor Fe²⁺Six conserved histidines, indicating that the encoded proteins belong to the same class of enzymes. Thus, in addition to β -diox I and RPE65, a3 rd polyene chain dioxygenase, β -diox II, is present in mice. Novel carotenes dioxygenases catalyze the asymmetric cleavage of beta-carotene leading to the formation of beta-10' -apocarotenal and beta-ionone

To functionally identify β -diox II, we expressed it as a recombinant protein in e.coli and performed in vitro tests for enzyme activity under the conditions described for β -diox I (Nagao a, During a, HoshinoC, Terao J, Olson JA, arch, biochem, biophysis, 328: 57-63, 1996). HPLC analysis revealed no formation of retinoids from β -carotene. But one compound with a residence time of 4.6 minutes could be detected (fig. 11A). When hydroxylamine was present during the extraction, the residence time of the compound was changed from 4.6 minutes to 16 minutes, indicating that the compound had an aldehyde group, whereby the corresponding oxime could be formed (fig. 11B). The putative β -carotene cleavage products catalyzed by the novel β -carotene dioxygenase increased linearly over incubation times as long as 2 hours. The UV/VIS absorption spectrum of the compound was similar to that of beta-apocarotene aldehyde or beta-apocarotene aldoxime (FIG. 11C). However, they are not identical to 8 '-apocarotene aldehyde/oxime and 12' - β -apocarotene aldehyde/oxime, according to spectral comparison of the stock solutions of our laboratory controls. The UV/VIS spectra of these compounds are similar to those found in the literature (Barua AB and Olson JA, J.Nutr., 130: 1996-2001, 2000) for β -10 '-apocarotene aldehyde (424nm) and β -10' -apocarotene aldoxime (435 nm). The turnover rate and thus the amount of cleavage products formed is relatively low in vitroAs was observed earlier in beta-diox. To obtain large quantities of this material for further chemical analysis, we decided to use an E.coli test system that has been used successfully for identifying Drosophila beta-diox I. Mouse beta 0-diox I was expressed as a control. In the case of the formation of retinoids from β 1-carotene, the test system offers the advantage of being able to visualize β 2-carotene cleavage by a color change of the bacteria from yellow to almost white. Coli expressing mouse β 3-diox I turned white, while in β 4-diox II, this significant color change was not seen, indicating that the enzyme catalyzes the formation of β 5-apocarotene aldehyde in e. In the E.coli strain expressing mouse beta-diox II, the beta-carotene content was significantly reduced (22.8pmol/mg dry weight versus 60.9pmol/mg dry weight for the control strain). To identify these compounds, the compounds were extracted and analyzed by HPLC as described above. Two classes of species were identified with absorption maxima at 424nm and 386nm, respectively (FIGS. 13B and C). The presence of compounds having the same spectrum but different residence times may be due to the stereoisomeric composition of the product formed and/or to the cis and trans configuration of the oxime formed. This result was obtained as early as after analysis of the Drosophila's beta-diox I. Depending on the induction time, first a putative β -10 '-apocarotene aldehyde could be detected and then a putative β -10' -apocarotene alcohol could be detected, indicating the conversion of the aldehyde to the corresponding alcohol in E.coli (data not shown). The conversion of retinal to retinol in E.coli has been previously discovered by expression of beta-diox I from Drosophila or mice, as described herein (FIG. 13A). To positively identify the putative β -10 '-apocarotene aldehyde formed, we converted it to the corresponding β -10' -apocarotene aldoxime and performed LC-MS analysis on it. Because it is in APcI⁺Operating the system in mode, the excimer ions typically behave as [ M + H ]]⁺A signal. By using a catalyst in the presence of M/z 392[ M + H ]]⁺This is the excimer ion of the base peak of the spectrum to identify 10' -beta-apocarotene aldoxime. Even number [ M + H ]]⁺The mass spectral signal clearly demonstrates the presence of nitrogen in the compound, from which the conversion of the hydroxyl group to the corresponding oxime can be determined.No fragmentation of polyene chains (production of characteristic daughter ions) was observed. In addition, the characteristic UV spectra showing maxima at 405nm (shoulder), 424nm and 446nm fit the 10' - β -apocarotene aldoxime chromophore system and are consistent with the previously reported spectral data (Barua AB and 0lson JA, J.Nutr., 130: 1996-2001, 2000).

Thus, beta-10' -apocarotene aldehyde is formed from beta-carotene. But the second compound, β -ionone, which should result from oxidative cleavage of the 9 ', 10' double bond of β -carotene, was not detected by HPLc. This may be due to its volatility and/or due to its partitioning into the medium. Therefore, we analyzed the bacterial growth medium by GC-MS after solid phase extraction of lipophilic compounds. In the medium of this E.coli strain, in addition to a large amount of indole, a significant amount of beta-ionone could be detected, which could not be found in the E.coli control strain. Taken together, these analyses demonstrate that β -diox II catalyzes the asymmetric cleavage of β -carotene at the 9 ', 10 ' carbon double bond, resulting in the formation of β -10 ' -apocarotene aldehyde and β -ionone. Therefore, we refer to this enzyme as β - β -carotene-9 ', 10' -dioxygenase (β -diox II). It should be noted, however, that β -diox ii from other sources not identified herein may attack other double bonds. Thus, the activity of β -diox II, i.e., the asymmetric cleavage of β -carotene, is not limited to the 9 ', 10' carbon double bonds disclosed above.

To test whether this enzyme catalyzes the oxidative cleavage of carotenes other than beta-carotene, we transformed it into a strain of E.coli that was able to synthesize and accumulate lycopene (FIG. 12). The experiments were performed as described above. In this strain, a significant amount of putative apolycopene aldehyde could be detected. This can be shown by converting the aldehyde to the corresponding oxime (data not shown). Thus, this novel carotene dioxygenase also catalyzes the oxidative cleavage of lycopene in the E.coli test system, resulting in the formation of apolycopene aldehyde, which is temporarily identified by their UV/VIS spectra. Cloning of cDNAs encoding novel carotene dioxygenases from human and zebrafish

To confirm the presence of this second class of dioxygenases in other metazoan, we searched the database for EST fragments with sequence identity. We found EST fragments from human and zebrafish. Then we cloned the full-length cDNA and sequenced. A556 amino acid protein (SEQ ID NO: 21) was encoded by cDNA (SEQ ID NO: 20) cloned from total RNA derived from human liver. And cDNA (SEQ ID NO: 18) isolated from zebrafish encodes a 549 amino acid protein (SEQ ID NO: 19). The deduced amino acid sequence shares 72 and 49% sequence identity with mouse beta-diox II. We performed phylogenetic tree calculations based on the sequence distance method and used a neighbor algorithm to deduce the amino acid sequence with metazoan polyene dioxygenase and plant VP 14. As shown in fig. 15, three groups of polyene chain dioxygenases, two different types of β -carotene dioxygenases (I and II) and RPE65, were found in vertebrates. In Drosophila and Caenorhabditis seleegans, only one class of dioxygenases (I) is found throughout the genome. C. elegans dioxygenase catalyzes the symmetric cleavage of β -carotene to form retinal, as judged by the e. Sequence analysis revealed that the three vertebrate polyenyldioxygenases are likely to be from a common ancestor. Thus, the presence of other genes encoding such enzymes, β -diox and RPE65, is clearly associated with vertebrate carotene/retinoid metabolism. Tissue-specific expression of novel carotene dioxygenases

We analyzed total RNA from several tissues of 7-week-old BALB/c mice (male and female) and assessed the steady state mRNA levels of both carotenoid dioxygenases by RT-PCR. RT-PCR products of two carotenoid dioxygenase mRNAs can be detected in the small intestine, liver, kidney, and testis. mRNA for the novel carotene dioxygenase was also present in the spleen and brain, and low abundance steady-state mRNA levels of both carotenoid dioxygenases could be detected in the lung and heart (fig. 16). The integrity of the RNA preparation was confirmed by analysis of β -actin mRNA. By omitting the reverse transcriptase enzyme in the assay, it can be shown that the RT-PCR product is derived from mRNA rather than DNA contamination. By multiple tissue mRNA blots analyzed with human cDNA riboprobe, we can find the 2.2kb messenger of the novel carotene dioxygenase in heart and liver, while the 2.4kb transcript of β -diox II was observed mainly in kidney (data not shown). Discussion reflecting the results above

In accordance with the present invention, Drosophila beta-diox I is the first beta-carotene dioxygenase identified at the molecular level. In the course of the experiments that led to the principles of the present invention, two alternative pathways starting from beta-carotene as substrate could be demonstrated, characterized by different enzymatic activities with homologous beta-diox I and II gene types. The information disclosed herein provides a key to open a broad field of further studies of carotenoid/retinoid metabolism in animals.

Beta-diox I encodes a 620 amino acid protein with a calculated molecular weight of 69.9 kDa. Sequence comparison revealed that β -diox I belongs to a novel dioxygenase which has been found so far only in bacteria and plants. The enzymatic activity of β -diox I, which is responsible for cleavage of 9-cis-neoxanthin in the ABA biosynthetic pathway, can be measured under the same conditions reported for plant carotenoid cleavage enzyme vp 14. In animals, dependence of β -carotene dioxygenase activity on iron has been reported. Addition of FeSO to the assay₄Ascorbic acid resulted in increased enzyme activity, while EDTA was added to significantly reduce retinal formation. The enzyme activity can be measured without adding cofactors such as thiol reagents or electron acceptors. This indicates that beta-diox II is Fe dependent²⁺Furthermore, no other co-factors are required for enzyme activity, as reported for plant vp 14. Since beta-carotene is insoluble in an aqueous environment, the enzyme activity test was performed in the presence of 0.05% Triton X100. In vivo, beta-carotene is not freely diffusible, but must bind to lipophilic structures such as membranes or binding proteins. Thus, the question is whether β -diox binds to membranes and interacts with its lipophilic substrate. The β -diox fusion protein can be purified without the addition of detergents, which are directed to its soluble state rather than to membrane-bound topology. But the glutathione-S-transferase portion of the fusion protein may also contribute to its solubility. Since the visual chromophore of Drosophila is 3-hydroxy retinal, we testedWhether β -diox I is able to form this hydroxylated retinoid directly using zeaxanthin as substrate, but under the conditions we apply, the enzyme fails to catalyze this reaction. In addition, we expressed β -diox I in E.coli strains that accumulated zeaxanthin, but could only detect the formation of non-hydroxylated retinoids. In this E.coli strain, a significant amount of beta-carotene, the direct precursor of zeaxanthin, was found, which can serve as a substrate for beta-diox I. An explanation for this result may lie in the fact that drosophila are able to hydroxylate retinal at position 3 of the β -ionone ring. In summary, we were able to show that β -diox I catalyzes the symmetric cleavage of β -carotene.

The β -diox I gene is located on chromosome 3 of Drosophila melanogaster 87F. The drosophila mutant, ninaB, has been localized to this region by cytological methods (FlyBase mapseed 87). In all types of photoreceptors, the mutant phenotype has reduced rhodopsin content. But the mutant phenotype could be rescued by dietary addition of retinal, whereas even higher doses of beta-carotene did not. Both the effectiveness of the visual pigment chromophore and the transcriptional regulation of the visual pigment protein module (opsin) by retinoic acid are dependent on β -diox II enzyme activity. Thus, it appears likely that the niaB phenotype is caused by a mutation in β -diox I.

The highest sequence homology was found for β -diox I with RPE65 (a multilateral cooperation first described in bull's eye). Thus, the question was whether RPE65 is a vertebrate counterpart of β -diox I. Although the exact function of RPE65 is not known, it has been proposed to play a role in vitamin a metabolism, and it has recently been found that mutations in this gene are responsible for the early onset of a severe form of retinal dystrophy in humans. In the eyes of mice with disrupted RPE65 gene, accumulation of all-trans vitamin A occurred. It was concluded that RPE65 is involved in the all-trans to 11-cis isomerization of vitamin a in the visual cycle of mammals. However, isomerization of all-trans retinol to 11-cis retinol was not affected after removal of RPE65 from the RPE-membrane fraction. To our knowledge, beta-carotene dioxygenase activity has never been reported in RPE, nor has significant amounts of its substrate beta-carotene been measured in vertebrate eyes. We expressed RPE65 cloned from bovine RPE by RT-PCR in the test system, but failed to detect the formation of retinoids or eccentric cleavage products such as apocarotene aldehyde. Thus, the exact function of RPE65 remains to be studied further, and we speculate that other yet undiscovered members of this family with different tissue specificities (small intestine, liver) are responsible for vertebrate β -carotene dioxygenase activity. Sequence homology of β -diox I with RPE65 and plant and bacterial dioxygenases suggests that we are studying novel dioxygenases that catalyze the cleavage of conjugated carbon double bonds. This type of reaction involves cleavage of carotenoids, as well as a variety of other compounds. The large intestine bacillus test system provides a powerful tool for identifying novel genes involved in retinoid formation and for screening for potential agonists or antagonists of the enzymes of the invention. In addition, the retinoids producing E.coli strains can be successfully used for the further step of identifying the carotene/retinoids metabolism.

In accordance with another aspect of the present invention, we report the cloning, identification, and tissue-specific expression of a second novel carotene dioxygenase enzyme derived from mouse, human, and zebrafish and which catalyzes the asymmetric cleavage of β -carotene. The formation of beta-apocarotene aldehyde from beta-carotene was shown by expressing the enzyme in a strain of E.coli that synthesizes beta-carotene. The cleavage products formed can be identified by absorption spectroscopy, conversion of the aldehyde to the corresponding oxime, and by LC-MS or GC-MS as being β -10' -apocarotene aldehyde and β -ionone. In vitro, the enzyme catalyzes the same reaction as in the E.coli test system. Thus, the enzymes identified catalyze the oxidative cleavage of the 9 ', 10' double bonds in the polyene backbone of its substrate, beta-carotene.

In addition to the overall sequence identity with β -diox I discussed above, there are also unique conserved patterns of histidine residues that may be involved in the cofactor Fe²⁺In combination with (1). Thus, three distinct representatives of the family of polyene dioxygenases, including RPE65, are found in vertebrates. RPE65 proteinThe biochemical function of (a) remains to be elucidated and we show that asymmetric cleavage occurs in addition to symmetric cleavage of β -carotene, conclusively solving the debate on the significance of this reaction. Analysis of tissue-specific expression shows that mRNA for both classes of enzymes is found in several tissues (e.g., small intestine and liver). These findings confirm, at the molecular level, the biochemical result of the presence of both symmetric and asymmetric cleavage of β -carotene in the same tissue. The expression pattern in mice and humans is not consistent. This may be due to inter-species differences in carotene metabolism or differences reflecting age and nutritional status of the individual under study, and there may be another factor that may explain the conflicting results obtained in several studies. In early studies with tissue homogenates, a variety of β -apocarotene aldehydes of varying chain length resulting from asymmetric cleavage of β -carotene could be found. Thus, several authors use the term random cleavage for this reaction. Here we show that the enzyme β -diox II does not catalyze this side reaction, but is specific for the 9 ', 10' double bond. The formation of beta-apocarotenal, which is different from 10' -beta-apocarotenal, found in vitro, may be caused by further metabolism of the primary cleavage products or by other carotene dioxygenases not yet known. However, it is difficult to obtain the in vitro activity of metazoan polyene chain dioxygenase, and the formation of beta-apocarotene aldehyde from beta-carotene by non-enzymatic degradation in an aqueous environment has been reported (Henry LK, Pupatisri-Nienaber NL, Jaren-Galan M, van Breemen RB, Catignani GL, and Schwartz SJ, J.Agric.food chem., 48: 5008-

The molecular identification of the cDNA coding for this novel carotene dioxygenase (. beta. -diox II) has led to problems of physiological relevance in the metabolism of carotenes in vertebrates. It has been shown in rats and chickens that β -apocarotenal can be a biologically active precursor for RA formation. After absorption of these compounds, the corresponding acids are first formed and then shortened to produce retinoic acid. The same study also showed that only a small fraction of beta-apocarotenal is attacked by beta-diox to produce retinal. This possibility may be important to consider the co-expression of both classes of dioxygenases in several tissues as described herein. Several tissues were also found to be able to synthesize RA, and retinaldehyde, the primary product of symmetrically cleaving β -carotene, was found to be not an intermediate. By analyzing the formation of RA from β -apocarotenal, a mechanism similar to that of fatty acid β -oxidation was proposed. In these studies, the formation of RA from β -apocarotenal was ensured by administration of a potent inhibitor of citral-retinal dehydrogenase, which catalyzes the oxidation of retinal to RA. Thus, asymmetric cleavage reactions are likely to represent the first step in the formation of alternative pathways for RA, and may contribute to RA homeostasis in the body, certain tissues, or cells. The second product resulting from the asymmetric cleavage of beta-ionone is known as a plant aroma compound. Such short chain compounds are volatile, and their putative physiological role in animals remains to be investigated.

In drosophila, vitamin a is formed exclusively by a symmetrical cleavage reaction. In vertebrates, two different classes of carotene dioxygenases β -diox I and β -diox II are found, as well as RPE65 protein. Sequence comparison showed that the vertebrate dioxygenase was from a common ancestor. In contrast to drosophila, RA plays an important role in development and cell differentiation in vertebrates. Thus, the presence of different β -carotene dioxygenases may be associated with the occurrence of RA effects. By in situ hybridization of zebrafish embryos, highly stable mRNA levels of the zebrafish homologue of β -diox were found prior to gastrulation. The zebrafish homologue of beta-carotene-9 ', 10' -dioxygenase was detectable only after organogenesis. High steady-state mRNA levels of β -diox I found early in development have been reported for mice (Redmond TM, Gentleman S, duncan t, Yu S, Wiggert B, Gantt E, and Cunningham FX jr., j.biol.chem., online, 2000). This suggests that the formation of retinoids from β -carotene catalyzed by symmetric oxidative cleavage reactions may contribute to retinoid homeostasis in the embryo. Thus, in addition to the parent pre-formed vitamin a, de novo biosynthesis from provitamins appears to be an important source of retinoids in the developmental process. However, asymmetric cleavage reactions may contribute to RA formation in certain tissues during late stages of development. In this regard, the expression of β -diox II in the brain and lung may be relevant. RA plays an important role in the cellular differentiation process of the nervous system. In the ferret model, under certain conditions, such as exposure to cigarette smoke, toxicity of β -carotene to the lungs was reported. In this regard, it is argued that asymmetric cleavage of β -carotene may be involved in these toxic effects (see review of Russell RM, am.J. Clin.Nutr., 71: 878-884, 2000). In addition, RA has been found to be formed from β -carotene in vitro in the testis, small intestine, liver, kidney, and lung. Here we show that mRNAs encoding two different types of carotene dioxygenases are found in all these tissues. This suggests that, in addition to the small intestine and liver, several tissues may promote their own RA homeostasis by endogenously forming retinoids from β -carotene, a feature in retinoids homeostasis that has heretofore been underestimated and unappreciated.

This enzyme is capable of catalyzing the oxidative cleavage of lycopene as judged in the E.coli test system. This suggests that the polyene chain backbone of carotene plays an important role in substrate specificity, while the ionone ring structure of β -carotene appears to be of little relevance. This result was also obtained after analysis of mouse β -diox I. The beneficial effects of lycopene on human health have been reported. Lycopene accumulates primarily in the liver, but also in the intestine, prostate, and testis (i.e., tissues expressing mRNA of both β -diox I and β -diox II). The cleavage of lycopene and the formation of apolycopene aldehyde are indicative of a putative role in vertebrate physiology. In vertebrates, there are several nuclear receptors, such as orphan receptors, for which the ligand is unknown. In addition to possibly being a putative precursor for RA formation in the case of beta-carotene cleavage, it is speculated that compounds formed by the asymmetric cleavage reaction of beta-carotene and/or lycopene might also represent putative ligands for these receptors.

In summary, the data presented herein molecularly identifies the enzyme that catalyzes the asymmetric cleavage of β -carotene, β -carotene-9 ', 10' -dioxygenase. Thus, in addition to the symmetric cleavage of β -carotene, a second enzyme activity is present in vertebrates. The molecular identification of enzymes involved in the cleavage of beta-carotene would open new avenues for studying the effects of metabolites derived from carotene on animal physiology and human health.

In recent years, there has been great progress in understanding the retinoid receptors and their ligands and their various roles in development and cell differentiation. By virtue of the findings of the present invention, the influence of the cleavage reaction on tissue distribution, the isomeric specificity of retinoids, and the regulation of vitamin a uptake may soon be further elucidated.

In addition, the identification of cDNAs encoding β -carotene dioxygenases I and II has a great influence on the applications in medicine, pharmacy, and biotechnology. In medicine, cloning of the corresponding gene from a human or mammal enables more detailed physiological characterization of the carotene/retinoid metabolism in mammals and will affect a variety of effects caused by vitamin a and its derivatives, thus providing several therapeutic applications.

Vitamin a deficiency is known to be a serious problem. cDNA equipped with the necessary regulatory sequences can be used for expression in non-retinoid containing organisms such as most plants, most bacteria, and fungi. Thus, vitamin a production can be achieved according to the invention in crops and microorganisms used in food technology or, more generally, in organisms which hitherto have no retinoids but are capable of synthesizing provitamin a (β -carotene).

Obviously, many modifications and variations of the present invention are possible in light of the above teachings. It is, therefore, to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described herein.

Sequence listing <110> greenovation Pflanzenbiotechnoholoae GmbH <120> dioxygenase catalyzing the cleavage of beta-carotene <130> dioxygenase catalyzing the cleavage of beta-carotene <140> <141> <150>00105822.1<151>2000-03-20<150>99125895.5<151>1999-12-24<160>27<170> PatentIn version 2.1< 210>1<211>2037<212> DNA <213> Drosophila melanogaster (Drosophila melanogaster) <220> <221> CDS <222> (1) · (1860) <400>1atg gca gcc ggt gtc ttc aag agt ttt atg cgc gac ttc ttt gcg gtg 48Met Ala Ala Gly Val Phe Lys Ser Phe Met Arg Asp Phe Phe Ala Val 151015 aa tac gat gag cag cga aat gat ccg caa gcg gaa cga ctg gat ggc 96Lys Tyr Asp Glu Gln Arg Asn Asp Pro Gln Ala Glu Arg Leu Asp Gly.

20 25 30aac gga cga ctg tat ccc aac tgc tcg tcg gat gtg tgg ctg cga tcc 144Asn Gly Arg Leu Tyr Pro Asn Cys Ser Ser Asp Val Trp Leu Arg Ser

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85 90 95ctg ctg cac cga ttt gcc att cgg aat gga cgc gtc acc tac cag aat 336Leu Leu His Arg Phe Ala Ile Arg Asn Gly Arg Val Thr Tyr Gln Asn

100 105 110cgc ttc gtg gac acg gaa aca ctg cga aag aat cgc tct gcc cag cgg 384Arg phe Val Asp Thr Glu Thr Leu Arg Lys Asn Arg Ser Ala Gln Arg

115 120 125att gtg gtc acg gag ttt ggc aca gct gct gtt ccg gat ccc tgt cac 432Ile Val Val Thr Glu Phe Gly Thr Ala Ala Val Pro Asp Pro Cys His

130 135 140tcg atc ttc gat aga ttt gcg gcc att ttt cga ccg gat agt gga acg 480Ser Ile Phe Asp Arg Phe Ala Ala Ile Phe Arg Pro Asp Ser Gly Thr145 150 155 160gat aac tcg atg att tcc ara tat cct ttc ggg gat cag tat tac aca 528Asp Ash Ser Met Ile Ser Ile Tyr Pro Phe Gly Asp Gln Tyr Tyr Thr

165 170 175ttt acg gag acg cct ttt atg cat aga ata aat ccc tgc act ttg gcc 576Phe Thr Glu Thr Pro Phe Met His Arg Ile Ash Pro Cys Thr Leu Ala

180 185 190acc gaa gca cga atc tgc acc acc gac ttc gtg ggc gtg gtg aac cac 624Thr Glu Ala Arg Ile Cys Thr Thr Asp Phe Val Gly Val Val Asn His

195 200 205aca tcg cat ccg cat gtt ctt ccc agt ggc act gtc tac aac ctg ggc 672Thr Ser His Pro His Val Leu Pro Ser Gly Thr Val Tyr Asn Leu Gly

210 215 220acc aca atg acc aga tct gga ccg gca tac act ata crc agt ttc ccg 720Thr Thr Met Thr Arg Ser Giy Pro Ala Tyr Thr Ile Leu Ser Phe Pro225 230 235 240cac ggc gag cag atg ttc gag gat gct cat gtg gtg gcc aca ctg ccg 768His Gly Glu Gln Met Phe Glu Asp Ala His Val Val Ala Thr Leu Pro

245 250 255tgc cgc tgg aaa ctg cat ccc ggt tat atg cac acc ttc ggc tta acg 816Cys Arg Trp Lys Leu His Pro Gly Tyr Met His Thr Phe Gly Leu Thr

260 265 270gat cac tac ttt gtg att gtg gag cag ccg ttg tcc gtt tcg ctt acg 864Asp His Tyr Phe Val Ile Val Glu Gln Pro Leu Ser Val Ser Leu Thr

275 280 285gag tat atc aaa gcc cag cra ggt gga cag aat tta tcg gcg tgt crc 912Glu Tyr Ile Lys Ala Gln Leu Gly Gly Gln Asn Leu Ser Ala Cys Leu

290 295 300aag tgg ttc gag gat cga ccg aca cra ttt cac ctt ata gat cgg gtt 960Lys Trp Phe Glu Asp Arg Pro Thr Leu Phe His Leu Ile Asp Arg Val305 310 315 320tcc ggc aaa ctg gtg cag acc tac gaa tcg gaa gcc ttc ttc tac ctg 1008Ser Gly Lys Leu Val Gln Thr Tyr Glu Ser Glu Ala Phe Phe Tyr Leu

325 330 335cac arc atc aac tgc ttt gaa cgg gat ggc cac gtg gtg gtg gac att 1056His Ile Ile Asn Cys Phe Glu Arg Asp Gly His Val Val Val Asp Ile

340 345 350tgc agc tac agg aat ccc gag atg atc aac tgc atg tat ctg gag gcc 1104Cys Ser Tyr Arg Asn Pro Glu Met Ile Asn Cys Met Tyr Leu Glu Ala

355 360 365att gcc aat atg caa acg aat ccc aat tat gct acc crc ttt cgt gga 1152Ile Ala Asn Met Gln Thr Asn Pro Asn Tyr Ala Thr Leu Phe Arg Gly

370 375 380cgt ccc ttg aga ttc gtc ctg ccc ttg ggc aca att cct ccg gca agc 1200Arg Pro Leu Arg Phe Val Leu Pro Leu Gly Thr Ile Pro Pro Ala Ser385 390 395 400atc gcc aag cgg gga ctg gtc aag tcc ttc tcc crt gct gga cta agt 1248Ile Ala Lys Arg Gly Leu Val Lys Ser Phe Ser Leu Ala Gly Leu Ser

405 410 415gct ccg cag gtt tct cgc acc atg aag cac tcg gtc tcg caa tat gcg 1296Ala Pro Gln Val Ser Arg Thr Met Lys His Ser Val Ser Gln Tyr Ala

420 425 430gat ata acc tac atg ccc acc aat gga aag caa gcc act gct gga gag 1344Asp Ile Thr Tyr Met Pro Thr Asn Gly Lys Gln Ala Thr Ala Gly Glu

435 440 445gaa agc ccc aag cga gat gcc aaa cgt ggc cgc tat gag gag gag aat 1392Glu Ser Pro Lys Arg Asp Ala Lys Arg Gly Arg Tyr Glu Glu Glu Asn

450 455 460ctt gtc aat ctg gtt acc atg gag ggc agt caa gcg gag gcg ttt cag 1440Leu Val Asn Leu Val Thr Met Glu Gly Ser Gln Ala Glu Ala Phe Gln465 470 475 480ggc acc aat ggc arc caa ctg cgt ccg gaa atg ctg tgt gat tgg ggc 1488Gly Thr Asn Gly Ile Gln Leu Arg Pro Glu Met Leu Cys Asp Trp Gly

485 490 495tgt gaa aca cct agg atc tat tat gaa cgg tat atg ggc aag aac tac 1536Cys Glu Thr Pro Arg Ile Tyr Tyr Glu Arg Tyr Met Gly Lys Asn Tyr

500 505 510cga tac ttc tac gcg att agc tcc gat gtg gat gca gtg aat ccg ggc 1584Arg Tyr Phe Tyr Ala Ile Ser Ser Asp Val Asp Ala Val Asn Pro Gly

515 520 525acc ctc atc aag gtg gat gtg tgg aat aag agc tgt cta acc tgg tgc 1632Thr Leu Ile Lys Val Asp Val Trp Asn Lys Ser Cys Leu Thr Trp Cys

530 535 540gag gag aat gtc tat ccc agt gag ccc att ttt gtg cct tcg ccg gat 1680Glu Glu Ash Val Tyr Pro Ser Glu Pro Ile Phe Val Pro Ser Pro Asp545 550 555 560ccg aaa tcc gag gac gat ggc gtt atc ctg gcc tcc atg gtg ctg ggc 1728Pro Lys Set Glu Asp Asp Gly Val Ile Leu Ala Ser Met Val Leu Gly

565 570 575ggt ctc aac gat cgc tat gtg ggc cra att gtg cra tgt gcc aaa acg 1776Gly Leu Ash Asp Arg Tyr Val Gly Leu Ile Val Leu Cys Ala Lys Thr

580 585 590atg acc gag ctg ggc cgt tgt gat ttc cat acc aat gga ccc gtg ccc 1824Met Thr Glu Leu Gly Arg Cys Asp Phe His Thr Asn Gly Pro Val Pro

595 600 605aag tgt ctc cat gga tgg ttt gca ccc aat gcc att tagatacgga 1870Lys Cys Leu His Gly Trp Phe Ala Pro Ash Ala Ile

610615620 actccttata tgggaagact acttagctta ggagataggg taaagcatat gcccagtatt 1930acgtttagat ttagactaga gcatttaatc ttagaactta gaattttgga ttcaagacat 1990tcgcaataaa ctcctgccac ttgcgctgga acaaaaaaaa aaaaaaa 2037<210>2<211>620<212> PRT <213> Drosophila melanogaster <400>2Met Ala Ala Gly Val Phe Lys Ser Phe Met Arg Asp Phe Phe Ala Val 151015 Lys Tyr Asp Glu Gln Arg Asn Asp Pro Gln Ala Glu Arg Leu Asp Gly

20 25 30Asn Gly Arg Leu Tyr Pro Asn Cys Ser Ser Asp Val Trp Leu Arg Ser

35 40 45Cys Glu Arg Glu lie Val Asp Pro lie Glu Gly His His Ser Gly His

50 55 60Ile Pro Lys Trp Ile Cys Gly Ser Leu Leu Arg Asn Gly Pro Gly Ser65 70 75 80Trp Lys Val Gly Asp Met Thr Phe Gly His Leu Phe Asp Cys Ser Ala

85 90 95Leu Leu His Arg Phe Ala Ile Arg Asn Gly Arg Val Thr Tyr Gln Asn

100 105 110Arg Phe Val Asp Thr Glu Thr Leu Arg Lys Asn Arg Ser Ala Gln Arg

115 120 125Ile Val Val Thr Glu Phe Gly Thr Ala Ala Val Pro Asp Pro Cys His

130 135 140Ser Ile Phe Asp Arg Phe Ala Ala Ile Phe Arg Pro Asp Ser Gly Thr145 150 155 160Asp Asn Ser Met Ile Ser Ile Tyr Pro Phe Gly Asp Gln Tyr Tyr Thr

165 170 175Phe Thr Glu Thr Pro Phe Met His Arg Ile Asn Pro Cys Thr Leu Ala

180 185 190Thr Glu Ala Arg Ile Cys Thr Thr Asp Phe Val Gly Val Val Asn His

195 200 205Thr Ser His Pro His Val Leu Pro Ser Gly Thr Val Tyr Asn Leu Gly

210 215 220Thr Thr Met Thr Arg Ser Gly Pro Ala Tyr Thr Ile Leu Ser Phe Pro225 230 235 240His Gly Glu Gln Met Phe Glu Asp Ala His Val Val Ala Thr Leu Pro

245 250 255Cys Arg Trp Lys Leu His Pro Gly Tyr Met His Thr Phe Gly Leu Thr

260 265 270Asp His Tyr Phe Val Ile Val Glu Gln Pro Leu Ser Val Ser Leu Thr

275 280 285Glu Tyr Ile Lys Ala Gln Leu Gly Gly Gln Asn Leu Ser Ala Cys Leu

290 295 300Lys Trp Phe Glu Asp Arg Pro Thr Leu Phe His Leu Ile Asp Arg Val305 310 315 320Ser Gly Lys Leu Val Gln Thr Tyr Glu Ser Glu Ala Phe Phe Tyr Leu

325 330 335His Ile Ile Asn Cys Phe Glu Arg Asp Gly His Val Val Val Asp Ile

340 345 350Cys Ser Tyr Arg Asn Pro Glu Met Ile Asn Cys Met Tyr Leu Glu Ala

355 360 365Ile Ala Asn Met Gln Thr Asn Pro Asn Tyr Ala Thr Leu Phe Arg Gly

370 375 380Arg Pro Leu Arg Phe Val Leu Pro Leu Gly Thr Ile Pro Pro Ala Ser385 390 395 400Ile Ala Lys Arg Gly Leu Val Lys Ser Phe Ser Leu Ala Gly Leu Ser

405 410 415Ala Pro Gln Val Ser Arg Thr Met Lys His Ser Val Ser Gln Tyr Ala

420 425 430Asp Ile Thr Tyr Met Pro Thr Asn Gly Lys Gln Ala Thr Ala Gly Glu

435 440 445Glu Ser Pro Lys Arg Asp Ala Lys Arg Gly Arg Tyr Glu Glu Glu Asn

450 455 460Leu Val Asn Leu Val Thr Met Glu Gly Ser Gln Ala Glu Ala Phe Gln465 470 475 480Gly Thr Asn Gly Ile Gln Leu Arg Pro Glu Met Leu Cys Asp Trp Gly

485 490 495Cys Glu Thr Pro Arg Ile Tyr Tyr Glu Arg Tyr Met Gly Lys Asn Tyr

500 505 510Arg Tyr Phe Tyr Ala Ile Ser Ser Asp Val Asp Ala Val Asn Pro Gly

515 520 525Thr Leu Ile Lys Val Asp Val Trp Asn Lys Ser Cys Leu Thr Trp Cys

530 535 540Glu Glu Asn Val Tyr Pro Ser Glu Pro Ile Phe Val Pro Ser Pro Asp545 550 555 560Pro Lys Ser Glu Asp Asp Gly Val Ile Leu Ala Ser Met Val Leu Gly

565 570 575Gly Leu Asn Asp Arg Tyr Val Gly Leu Ile Val Leu Cys Ala Lys Thr

580 585 590Met Thr Glu Leu Gly Arg Cys Asp Phe His Thr Asn Gly Pro Val Pro

595600605 Lys Cys Leu His Gly Trp Phe Ala Pro Asn A1a Ile 610615620 <210>3<211>27<212> DNA <213> Artificial sequence <220> <223> description of the artificial sequence: description of CrtE upstream primer <400>3gcgtcgaccg cggtctacgg ttaactg 27<210>4<211>27<212> DNA <213> Artificial sequence <220> <223> derived from Erwinia herbicola: description of CrtE downstream primer <400>4ggggtaccct tgaacccaaa agggcgg 27<210>5<211>28<212> DNA <213> Artificial sequence <220> <223> Artificial sequence derived from Erwinia herbicola: description of crtI upstream primer <400>5gctctagacg tctggcgacg gcccgcca 28<210>6<211>27<212> DNA <213> Artificial sequence <220> <223> derived from Erwinia herbicola: description of CrtI downstream primer <400>6gcgtcgacac ctacaggcga tcctgcg 27<210>7<211>40<212> DNA <213> Artificial sequence <220> <223> Artificial sequence derived from Erwinia herbicola: description of oligo (T) -adaptor primer <400>7gaccacgcgt atcgatgtcg actttttttt tttttttttt 40<210>8<211>20<212> DNA <213> Artificial sequence <220> <223> Artificial sequence: description of specific upstream primers <400>8gcagccggtg tcttcaagag 20<210>9<211>21<212> DNA <213> Artificial sequence <220> <223> derived from EST (accession number AI 063857): description of the Anchor primer <400>9gaccacgcgt atcgatgtcg a 21<210>10<211>27<212> DNA <213> Artificial sequence <220> <223> Artificial sequence: description of Gex upstream primer <400>10ggaattcgca gccggtgtct tcaagag 27<210>11<211>26<212> DNA <213> Artificial sequence <220> <223> derived from Drosophila melanogaster: description of Gex downstream primer <400>11cctcgaggta gtcttcccat ataagg 26< 26 >12<211>21<212> DNA <213> Artificial sequence <220> <223> derived from Drosophila melanogaster: description of beta-diox RT-PCR upstream primer <400>12ctgcaaacgg accgaccacg t 21<21 > 210>13<211>21<212> DNA <213> Artificial sequence <220> <223> derived from Drosophila melanogaster: description of the beta-diox RT-PcR downstream primer <400>13gcaaatctat cgaagatcga g 21<21 >14<211>20<212> DNA <213> Artificial sequence <220> <223> derived from Drosophila melanogaster: description of the rp49 RT-PCR upstream primer <400>14gacttcatcc gccaccagtc 20<210>15<211>22<212> DNA <213> Artificial sequence <220> <223> derived from ribosomal protein rp 49: rp49 RT-PCR downstream primer <400>15caccaggaac ttcttgaatc cg 22<210>16<211>1855<212> DNA <213> rattus muscovi (Mus musculus) <220> <221> CDS <222> (1) } (1596) <400>16atg ttg gga ccg aag caa agc ctg cca tgc att gcc cca ctg ctg acc 48Met Leu Gly Pro Lys Gln Ser Leu Pro Cys Ile Ala Pro Leu Leu Thr 151015 acg gcg gag gag act ctg agt gct gtc tct gct cgg gtc cga gga cat 96Thr Ala Glu Glu Thr Leu Ser Ala Val Ser Ala Arg Val Arg Gly His derived from ribosomal protein rp49

20 25 30att cct gaa tgg ctt aat ggt tat cta crt cga gtt gga cct ggg aag 144Ile Pro Glu Trp Leu Asn Gly Tyr Leu Leu Arg Val Gly Pro Gly Lys

35 40 45ttt gaa ttt ggg aag gat aga tac aat cat tgg ttt gat gga atg gcg 192Phe Glu Phe Gly Lys Asp Arg Tyr Asn His Trp Phe Asp Gly Met Ala

50 55 60ttg ctt cac cag ttc cga atg gag agg ggc aca gtg aca tac aag agc 240Leu Leu His Gln Phe Arg Met Glu Arg Gly Thr Val Thr Tyr Lys Ser65 70 75 80aag ttt cta cag agt gac aca tat aag gcc aac agt gct gga ggt aga 288Lys Phe Leu Gln Ser Asp Thr Tyr Lys Ala Asn Ser Ala Gly Gly Arg

85 90 95att gtg atc tca gaa ttt ggc acg ctg gcc ctt cct gac cca tgc aag 336Ile Val Ile Ser Glu Phe Gly Thr Leu Ala Leu Pro Asp Pro Cys Lys

100 105 110agc atc ttt gaa cgt ttc atg tca agg ttt gag cca cct act atg act 384Ser Ile Phe Glu Arg Phe Met Ser Arg Phe Glu Pro Pro Thr Met Thr

115 120 125gac aac acc aac gtc aac ttt gtg cag tac aaa ggt gat tac tac atg 432Asp Asn Thr Asn Val Asn Phe Val Gln Tyr Lys Gly Asp Tyr Tyr Met

130 135 140agc aca gag act aat ttt atg aat aag gtg gac att gag atg ctg gaa 480Ser Thr Glu Thr Asn Phe Met Asn Lys Val Asp Ile Glu Met Leu Glu145 150 155 160agg aca gaa aag gtg gac tgg agc aaa ttc att gct gtg aat gga gcc 528Arg Thr Glu Lys Val Asp Trp Ser Lys Phe Ile Ala Val Asn Gly Ala

165 170 175act gca cat cct cat tac gac cca gat ggg aca gca tac aac atg ggg 576Thr Ala His Pro His Tyr Asp Pro Asp Gly Thr Ala Tyr Asn Met Gly

180 185 190aac agc tat ggg cca aga ggt tct tgc tat aat att att cgt gtt cct 624Asn Ser Tyr Gly Pro Arg Gly Ser Cys Tyr Asn Ile Ile Arg Val Pro

195 200 205cca aaa aag aaa gag ccc ggg gag acg att cac gga gca cag gtg cta 672Pro Lys Lys Lys Glu Pro Gly Glu Thr Ile His Gly Ala Gln Val Leu

210 215 220tgt tcc att gcc tcc act gag aaa atg aag cct tct tac tac cat agc 720Cys Ser Ile Ala Ser Thr Glu Lys Met Lys Pro Ser Tyr Tyr His Ser225 230 235 240ttt gga atg aca aaa aac tac ata atc ttt gtc gaa cag cct gta aag 768Phe Gly Met Thr Lys Asn Tyr Ile Ile Phe Val Glu Gln Pro Val Lys

245 250 255atg aag ctg tgg aaa ara arc act tct aaa atc cgg gga aag ccc ttt 816Met Lys Leu Trp Lys Ile Ile Thr Ser Lys Ile Arg Gly Lys Pro Phe

260 265 270gct gat ggg ara agc tgg gag ccc cag tat aac acg cgg ttt cat gtg 864Ala Asp Gly Ile Ser Trp Glu Pro Gln Tyr Asn Thr Arg Phe His Val

275 280 285gtg gat aaa cac act gga cag ctt ctc cca gga atg tac tac agc atg 912Val Asp Lys His Thr Gly Gln Leu Leu Pro Gly Met Tyr Tyr Ser Met

290 295 300cct ttt ctt acc tat cat caa atc aat gcc ttt gag gac cag ggc tgt 960Pro Phe Leu Thr Tyr His Gln Ile Asn Ala Phe Glu Asp Gln Gly Cys305 310 315 320att gtg att gat ctg tgc tgc cag gat gat ggg aga agc cta gac ctt 1008Ile Val Ile Asp Leu Cys Cys Gln Asp Asp Gly Arg Ser Leu Asp Leu

325 330 335tac caa cta cag aat ctc agg aaa gct gga gag ggg ctt gat cag gtc 1056Tyr Gln Leu Gln Asn Leu Arg Lys Ala Gly Glu Gly Leu Asp Gln Val

340 345 350tat gag tta aag gca aag tct ttc cct cga aga ttt gtc ttg ccc tta 1104Tyr Glu Leu Lys Ala Lys Ser Phe Pro Arg Arg Phe Val Leu Pro Leu

355 360 365gat gtt agt gtg gat gct gct gaa gga aag aac ctc agc cca ctg tcc 1152Asp Val Ser Val Asp Ala Ala Glu Gly Lys Asn Leu Ser Pro Leu Ser

370 375 380tat tct tca gcc agc gct gtg aaa cag ggt gat gga gag atc tgg tgc 1200Tyr Ser Ser Ala Ser Ala Val Lys Gln Gly Asp Gly Glu Ile Trp Cys385 390 395 400tct cct gaa aat cta cac cac gaa gac ctg gaa gag gaa ggg ggg att 1248Ser Pro Glu Asn Leu His His Glu Asp Leu Glu Glu Glu Gly Gly Ile

405 410 415gaa ttc cct cag arc aac tat ggc cga ttc aat ggc aaa aag tat agt 1296Glu Phe Pro Gln Ile Asn Tyr Gly Arg Phe Asn Gly Lys Lys Tyr Ser

420 425 430ttc ttc tat ggc tgc ggt ttt cga cat ttg gtg ggg gat tct ctg att 1344Phe Phe Tyr Gly Cys Gly Phe Arg His Leu Val Gly Asp Ser Leu Ile

435 440 445aag gtt gac gtg acg aac aag aca cra agg gtt tgg aga gaa gaa ggc 1392Lys Val Asp Val Thr Asn Lys Thr Leu Arg Val Trp Arg Glu Glu Gly

450 455 460ttt tat ccc tcg gag ccc gtt ttt gtt ccg gtg cca gga gca gat gag 1440Phe Tyr Pro Ser Glu Pro Val Phe Val Pro Val Pro Gly Ala Asp Glu465 470 475 480gaa gac agt ggg gtt ata ctc tct gtg gtg atc act ccc aac cag agt 1488Glu Asp Ser Gly Val Ile Leu Ser Val Val Ile Thr Pro Asn Gln Ser

485 490 495gaa agc aac ttc ctc ctt gtc ttg gat gcc aag agc ttc aca gag ctg 1536Glu Ser Asn Phe Leu Leu Val Leu Asp Ala Lys Ser Phe Thr Glu Leu

500 505 510ggg cga gcg gaa gta ccc gtg cag atg cct tac ggg ttc cat ggc acc 1584Gly Arg Ala Glu Val Pro Val Gln Met Pro Tyr Gly Phe His Gly Thr

515 520 525ttt gtg cct arc tgacggcaga ggcgcaagga aggctaggat cgggcttcga 1636Phe Val Pro Ile

530tgagcacact ctgaggaaaa gagaaaatgg tggatctcac tcaaaagctg ttgtagtttg 1696gacctgaccc tgacccctaa ggaatcatag acccgactcc cgtgggctca tcgaccctga 1756cccccaacgt gctgatagat cctgaccacc acgggatcat atttaaattc ttgttcccag 1816cttgtggcaa tacttttttt tttttttgta gcagtggta 1855<210>17<211>532<212> PRT <213> mus musculus <400>17Met Leu Gly Pro Lys Gln Ser Leu Pro Cys Ile Ala Pro Leu Leu Thr 151015 Thr Ala Glu Glu Thr Leu Ser Ala Val Ser Ala Arg Val Arg Gly His

20 25 30Ile Pro Glu Trp Leu Asn Gly Tyr Leu Leu Arg Val Gly Pro Gly Lys

35 40 45Phe Glu Phe Gly Lys Asp Arg Tyr Asn His Trp Phe Asp Gly Met Ala

50 55 60Leu Leu His Gln Phe Arg Met Glu Arg Gly Thr Val Thr Tyr Lys Ser65 70 75 80Lys Phe Leu Gln Ser Asp Thr Tyr Lys Ala Asn Ser Ala Gly Gly Arg

85 90 95Ile Val Ile Ser Glu Phe Gly Thr Leu Ala Leu Pro Asp Pro Cys Lys

100 105 110Ser Ile Phe Glu Arg Phe Met Ser Arg Phe Glu Pro Pro Thr Met Thr

115 120 125Asp Asn Thr Asn Val Asn Phe Val Gln Tyr Lys Gly Asp Tyr Tyr Met

130 135 140Ser Thr Glu Thr Asn Phe Met Asn Lys Val Asp Ile Glu Met Leu Glu145 150 155 160Arg Thr Glu Lys Val Asp Trp Ser Lys Phe Ile Ala Val Asn Gly Ala

165 170 175Thr Ala His Pro His Tyr Asp Pro Asp Gly Thr Ala Tyr Asn Met Gly

180 185 190Asn Ser Tyr Gly Pro Arg Gly Ser Cys Tyr Asn Ile Ile Arg Val Pro

195 200 205Pro Lys Lys Lys Glu Pro Gly Glu Thr Ile His Gly Ala Gln Val Leu

210 215 220Cys Ser Ile Ala Ser Thr Glu Lys Met Lys Pro Ser Tyr Tyr His Ser225 230 235 240Phe Gly Met Thr Lys Asn Tyr Ile Ile Phe Val Glu Gln Pro Val Lys

245 250 255Met Lys Leu Trp Lys Ile Ile Thr Ser Lys Ile Arg Gly Lys Pro Phe

260 265 270Ala Asp Gly Ile Ser Trp Glu Pro Gln Tyr Asn Thr Arg Phe His Val

275 280 285Val Asp Lys His Thr Gly Gln Leu Leu Pro Gly Met Tyr Tyr Ser Met

290 295 300Pro Phe Leu Thr Tyr His Gln Ile Asn Ala Phe Glu Asp Gln Gly Cys305 310 315 320Ile Val Ile Asp Leu Cys Cys Gln Asp Asp Gly Arg Ser Leu Asp Leu

325 330 335Tyr Gln Leu Gln Asn Leu Arg Lys Ala Gly Glu Gly Leu Asp Gln Val

340 345 350Tyr Glu Leu Lys Ala Lys Ser Phe Pro Arg Arg Phe Val Leu Pro Leu

355 360 365Asp Val Ser Val Asp Ala Ala Glu Gly Lys Asn Leu Ser Pro Leu Ser

370 375 380Tyr Ser Ser Ala Ser Ala Val Lys Gln Gly Asp Gly Glu Ile Trp Cys385 390 395 400Ser Pro Glu Asn Leu His His Glu Asp Leu Glu Glu Glu Gly Gly Ile

405 410 415Glu Phe Pro Gln Ile Asn Tyr Gly Arg Phe Asn Gly Lys Lys Tyr Ser

420 425 430Phe Phe Tyr Gly Cys Gly Phe Arg His Leu Val Gly Asp Ser Leu Ile

435 440 445Lys Val Asp Val Thr Asn Lys Thr Leu Arg Val Trp Arg Glu Glu Gly

450 455 460Phe Tyr Pro Ser Glu Pro Val Phe Val Pro Val Pro Gly Ala Asp Glu465 470 475 480Glu Asp Ser Gly Val Ile Leu Ser Val Val Ile Thr Pro Asn Gln Ser

485 490 495Glu Ser Asn Phe Leu Leu Val Leu Asp Ala Lys Ser Phe Thr Glu Leu

500 505 510Gly Arg Ala Glu Val Pro Val Gln Met Pro Tyr Gly Phe His Gly Thr

515 520 525Phe Val Pro Ile

530<210>18<211>2134<212>DNA<213>Danio rerio<220><221>CDS<222>(29)..(1675)<400>18aagatagcaa tccataacac ctaaagtc atg tct aca tct gca aat gat caa 52

Met Ser Thr Ser Ala Asn Asp Gln

1 5atg tat aaa gtg cca gct aac aaa aaa cgt cca tct gcc agc ggc ctg 100Met Tyr Lys Val Pro Ala Asn Lys Lys Arg Pro Ser Ala Ser Gly Leu

10 15 20gag ttc atc ggt cct crt gtc agc tct gtt gag gag arc ccg gat ccc 148Glu Phe Ile Gly Pro Leu Val Ser Ser Val Glu Glu Ile Pro Asp Pro25 30 35 40atc act aca ctc att aaa ggt caa att ccc tcc tgg atc aac ggc agc 196Ile Thr Thr Leu Ile Lys Gly Gln Ile Pro Ser Trp Ile Asn Gly Ser

45 50 55ttc ctt aga aat gga cct gga aaa ttt gag ttt ggt gaa agc aaa ttc 244Phe Leu Arg Asn Gly Pro Gly Lys Phe Glu Phe Gly Gln Ser Lys Phe

60 65 70acc cac tgg ttt gac ggt atg gct ttg atg cat cgt ttc aac att aag 292Thr His Trp Phe Asp Gly Met Ala Leu Met His Arg Phe Asn Ile Lys

75 80 85gat ggc cag gtg acc tac agc agc cga ttt ttg caa agt gat tct tat 340Asp Gly Gln Val Thr Tyr Ser Ser Arg Phe Leu Gln Ser Asp Ser Tyr

90 95 100gtg cag aac tca gag aaa aac cga att gtg gtt tct gaa ttt ggt acc 388Val Gln Asn Ser Glu Lys Asn Arg Ile Val Val Ser Glu Phe Gly Thr105 110 115 120ctg gca aca cct gac cca tgc aag aac atc ttc gcc cgc ttc ttt tca 436Leu Ala Thr Pro Asp Pro Cys Lys Asn Ile Phe Ala Arg Phe Phe Ser

125 130 135cgc ttt cag atc cca aaa aca act gat aat gca gga gtg aac ttt gtt 484Arg Phe Gln Ile Pro Lys Thr Thr Asp Asn Ala Gly Val Asn Phe Val

140 145 150aag tac aag gga gat ttc tac gta agc aca gag acc aac ttc atg cgc 532Lys Tyr Lys Gly Asp Phe Tyr Val Ser Thr Glu Thr Asn Phe Met Arg

155 160 165aaa att gac cct gtg agc cta gaa acc aaa gaa aag gtg gat tgg tcc 580Lys Ile Asp Pro Val Ser Leu Glu Thr Lys Glu Lys Val Asp Trp Ser

170 175 180aaa ttt att gca gtc agt gca gcc aca gct cat cca cat tat gat cgg 628Lys Phe Ile Ala Val Ser Ala Ala Thr Ala His Pro His Tyr Asp Arg185 190 195 200gaa gga gca act tac aac atg gga aac rca tat ggc cga aaa ggc ttc 676Glu Gly Ala Thr Tyr Asn Met Gly Asn Ser Tyr Gly Arg Lys Gly Phe

205 210 215ttc tac cat ara crc aga gta cca cca ggt gaa aaa cag gac gat gat 724Phe Tyr His Ile Leu Arg Val Pro Pro Gly Glu Lys Gln Asp Asp Asp

220 225 230gct gat ctg tct ggc gct gaa att crt tgc tcg att cct gct gct gac 772Ala Asp Leu Ser Gly Ala Glu Ile Leu Cys Ser Ile Pro Ala Ala Asp

235 240 245ccc aga aaa cca rca tac tac cac agt ttt gtc atg rca gag aat tac 820Pro Arg Lys Pro Ser Tyr Tyr His Ser Phe Val Met Ser Glu Asn Tyr

250 255 260ara gtc ttt att gag cag ccg arc aag ctg gac ctg ctg aag ttc atg 868Ile Val Phe Ile Glu Gln Pro Ile Lys Leu Asp Leu Leu Lys Phe Met265 270 275 280ctg tac aga att gct gga aag agc ttt cat aag gtc atg tcc tgg aac 916Leu Tyr Arg Ile Ala Gly Lys Ser Phe His Lys Val Met Ser Trp Asn

285 290 295ccg gaa cta gac aca atc ttt cat gtg gca gac cga cac aca ggc cag 964Pro Glu Leu Asp Thr Ile Phe His Val Ala Asp Arg His Thr Gly Gln

300 305 310ctc ctc aac aca aaa tac tac agc agt gcc atg ttc gcc ctg cac cag 1012Leu Leu Asn Thr Lys Tyr Tyr Ser Ser Ala Met Phe Ala Leu His Gln

315 320 325att aat gca tat gaa gag aat gga tat ctg att atg gac atg tgc tgc 1060Ile Asn Ala Tyr Glu Glu Asn Gly Tyr Leu Ile Met Asp Met Cys Cys

330 335 340gga gat gat ggc aat gtg att ggt gaa ttc aca ctg gag aat cta cag 1108Gly Asp Asp Gly Asn Val Ile Gly Glu Phe Thr Leu Glu Asn Leu Gln345 350 355 360tcg acc ggg gaa gat ctc gac aag ttt ttc aat rca ctg tgt aca aac 1156Ser Thr Gly Glu Asp Leu Asp Lys Phe Phe Asn Ser Leu Cys Thr Asn

365 370 375tta cca cgc cga tat gta ctg cct ctg gag gtg aag gag gat gaa ccc 1204Leu Pro Arg Arg Tyr Val Leu Pro Leu Glu Val Lys Glu Asp Glu Pro

380 385 390aat gac caa aac ctc atc aat ttg cca tac acc acc gct agc gct gtg 1252Asn Asp Gln Asn Leu Ile Asn Leu Pro Tyr Thr Thr Ala Ser Ala Val

395 400 405aaa act caa act ggg gtg ttc ctc tac cat gag gat ctc tac aat gat 1300Lys Thr Gln Thr Gly Val Phe Leu Tyr His Glu Asp Leu Tyr Asn Asp

410 415 420gac ctg ttg cag tac ggt ggt crt gag ttt cca cag ara aac tac gct 1348Asp Leu Leu Gln Tyr Gly Gly Leu Glu Phe Pro Gln Ile Asn Tyr Ala425 430 435 440aac tac aac gct cgt cct tat cgg tat ttc tat gcc tgt ggc ttt ggt 1396Asn Tyr Asn Ala Arg Pro Tyr Arg Tyr Phe Tyr Ala Cys Gly Phe Gly

445 450 455cat gtg ttt ggt gac tct ctg ctt aag atg gat ttg gag gga aag aag 1444His Val Phe Gly Asp Set Leu Leu Lys Met Asp Leu Glu Gly Lys Lys

460 465 470ctg aag gtg tgg cgc cat gct ggt ttg ttc ccc rca gaa cca gtg ttt 1492Leu Lys Val Trp Arg His Ala Gly Leu Phe Pro Ser Glu Pro Val Phe

475 480 485att cca gca cct gat gct cag gat gag gat gat ggc gtg gtc atg tct 1540Ile Pro Ala Pro Asp Ala Gln Asp Glu Asp Asp Gly Val Val Met Ser

490 495 500gtg atc att aca cct aga gag aaa aag agc agt ttc cra ctt gtc ctt 1588Val Ile Ile Thr Pro Arg Glu Lys Lys Ser Ser Phe Leu Leu Val Leu505 510 515 520gat gcc aag acg ttc aca gag ctc gga cga gca gaa gtt cca gtg gac 1636Asp Ala Lys Thr Phe Thr Glu Leu Gly Arg Ala Glu Val Pro Val Asp

525 530 535atc cca tac ggc act cat gga ctc ttc aat gag aag agc taaacagaaa 1685Ile Pro Tyr Gly Thr His Gly Leu Phe Asn Glu Lys Ser

540 545atctatcatt aaaatatcta atcaaacaat ttcactcatt ttgataattt ccatctaaac 1745agggaagagt tttttgtaat ggagtagtgt tttttgtatt atgcctgatt ttccttggct 1805gattgtgatt tagtattggt acagtatatt tgggtgaagg atctgttata atagggcttt 1865tacttatgct ttttcgaata agttaagcat gatgttaatc tattgtattt atatattctc 1925tacagcattt tttgttattc aagtgcatat tttattcatg tatattttat acttactttt 1985atatacattt taatagtttt acttttttta aatatacaaa ttaattacat ctgtgaaatt 2045tgtgagaccc tcgcctgcaa acccagctca gtggattagc catgtaattc ttttttaata 2105aatgttgtgc cttaaaaaaa aaaaaaaaa 2134<210>19<211>549<212>PRT<213>Danio rerio<400>19Met Ser Thr Ser Ala Asn Asp Gln Met Tyr Lys Val Pro Ala Asn Lys1 5 10 15Lys Arg Pro Ser Ala Ser Gly Leu Glu Phe Ile Gly Pro Leu Val Ser

20 25 30Ser Val Glu Glu Ile Pro Asp Pro Ile Thr Thr Leu Ile Lys Gly Gln

35 40 45Ile Pro Ser Trp Ile Asn Gly Ser Phe Leu Arg Asn Gly Pro Gly Lys

50 55 60Phe Glu Phe Gly Glu Ser Lys Phe Thr His Trp Phe Asp Gly Met Ala65 70 75 80Leu Met His Arg Phe Asn Ile Lys Asp Gly Gln Val Thr Tyr Ser Ser

85 90 95Arg Phe Leu Gln Ser Asp Ser Tyr Val Gln Asn Ser Glu Lys Asn Arg

100 105 110Ile Val Val Ser Glu Phe Gly Thr Leu Ala Thr Pro Asp Pro Cys Lys

115 120 125Asn Ile Phe Ala Arg Phe Phe Ser Arg Phe Gln lle Pro Lys Thr Thr

130 135 140Asp Asn Ala Gly Val Asn Phe Val Lys Tyr Lys Gly Asp Phe Tyr Val145 150 155 160Ser Thr Glu Thr Asn Phe Met Arg Lys Ile Asp Pro Val Ser Leu Glu

165 170 175Thr Lys Glu Lys Val Asp Trp Ser Lys Phe Ile Ala Val Ser Ala Ala

180 185 190Thr Ala His Pro His Tyr Asp Arg Glu Gly Ala Thr Tyr Asn Met Gly

195 200 205Asn Ser Tyr Gly Arg Lys Gly Phe Phe Tyr His Ile Leu Arg Val Pro

210 215 220Pro Gly Glu Lys Gln Asp Asp Asp Ala Asp Leu Ser Gly Ala Glu Ile225 230 235 240Leu Cys Ser Ile Pro Ala Ala Asp Pro Arg Lys Pro Ser Tyr Tyr His

245 250 255Ser Phe Val Met Ser Glu Asn Tyr Ile Val Phe Ile Glu Gln Pro Ile

260 265 270Lys Leu Asp Leu Leu Lys Phe Met Leu Tyr Arg Ile Ala Gly Lys Ser

275 280 285Phe His Lys Val Met Ser Trp Asn Pro Glu Leu Asp Thr Ile Phe His

290 295 300Val Ala Asp Arg His Thr Gly Gln Leu Leu Asn Thr Lys Tyr Tyr Ser305 310 315 320Ser Ala Met Phe Ala Leu His Gln Ile Asn Ala Tyr Glu Glu Asn Gly

325 330 335Tyr Leu Ile Met Asp Met Cys Cys Gly Asp Asp Gly Asn Val Ile Gly

340 345 350Glu Phe Thr Leu Glu Asn Leu Gln Ser Thr Gly Glu Asp Leu Asp Lys

355 360 365Phe Phe Asn Ser Leu Cys Thr Asn Leu Pro Arg Arg Tyr Val Leu Pro

370 375 380Leu Glu Val Lys Glu Asp Glu Pro Asn Asp Gln Asn Leu Ile Asn Leu385 390 395 400Pro Tyr Thr Thr Ala Ser Ala Val Lys Thr Gln Thr Gly Val Phe Leu

405 410 415Tyr His Glu Asp Leu Tyr Asn Asp Asp Leu Leu Gln Tyr Gly Gly Leu

420 425 430Glu Phe Pro Gln Ile Asn Tyr Ala Asn Tyr Asn Ala Arg Pro Tyr Arg

435 440 445Tyr Phe Tyr Ala Cys Gly Phe Gly His Val Phe Gly Asp Ser Leu Leu

450 455 460Lys Met Asp Leu Glu Gly Lys Lys Leu Lys Val Trp Arg His Ala Gly465 470 475 480Leu Phe Pro Ser Glu Pro Val Phe Ile Pro Ala Pro Asp Ala Gln Asp

485 490 495Glu Asp Asp Gly Val Val Met Ser Val Ile Ile Thr Pro Arg Glu Lys

500 505 510Lys Ser Ser Phe Leu Leu Val Leu Asp Ala Lys Thr Phe Thr Glu Leu

515 520 525Gly Arg Ala Glu Val Pro Val Asp Ile Pro Tyr Gly Thr His Gly Leu

530535540 Phe Asn Glu Lys Ser545<210>20<211>1934<212> DNA <213> human (Homo sapiens) <220> <221> CDS <222> (1). (1668) <400>20atg gtg tac cgg crc cca gtt ttc aaa agg tac atg gga aat act cct 48Met Val Tyr Arg Leu Pro Val Phe Lys Arg Tyr Met Gly Asn Thr Pro 151015 cag aaa aaa gcc gtc ttt ggg cag tgt cgg ggt ctg cca tgt gtt gca 96Gln Lys Lys Ala Val Phe Gly Gln Cys Arg Gly Leu Pro Cys Val Ala

20 25 30ccg ctg ctg acc aca gtg gaa gag gct cca cgg ggc atc tct gct cga 144Pro Leu Leu Thr Thr Val Glu Glu Ala Pro Arg Gly Ile Ser Ala Arg

35 40 45gtc tgg gga cat ttt cct aag tgg ctc aat ggc tct cra ctt cga att 192Val Trp Gly His Phe Pro Lys Trp Leu Asn Gly Ser Leu Leu Arg Ile

50 55 60gga cct ggg aaa ttc gag ttt ggg aag gat aag tac aat cat tgg ttt 240Gly Pro Gly Lys Phe Glu Phe Gly Lys Asp Lys Tyr Asn His Trp Phe65 70 75 80gat ggg atg gcg ctg crt cac cag ttc aga atg gca aag ggc aca gtg 288Asp Gly Met Ala Leu Leu His Gln Phe Arg Met Ala Lys Gly Thr Val

85 90 95aca tac agg agc aag ttt cta cag agt gat aca tat aag gcc aac agt 336Thr Tyr Arg Ser Lys Phe Leu Gln Ser Asp Thr Tyr Lys Ala Asn Ser

100 105 110gct aaa aac cga att gtg atc rca gaa ttt ggc aca ctg gct crc ccg 384Ala Lys Asn Arg Ile Val Ile Ser Glu Phe Gly Thr Leu Ala Leu Pro

115 120 125gat cca tgc aag aat gtt ttt gaa cgt ttc atg tcc agg ttt gag ctg 432Asp Pro Cys Lys Asn Val Phe Glu Arg Phe Met Ser Arg Phe Glu Leu

130 135 140cct ggt aaa gct gca gcc atg act gac gat act aat gtc aac tat gtg 480Pro Gly Lys Ala Ala Ala Met Thr Asp Asp Thr Asn Val Asn Tyr Val145 150 155 160cgg tac aag ggt gat tac tac crc tgc acc gag acc aac ttt atg aat 528Arg Tyr Lys Gly Asp Tyr Tyr Leu Cys Thr Glu Thr Asn Phe Met Asn

165 170 175aaa gtg gac att gaa act ctg gaa aaa aca gaa aag gta gat tgg agc 576Lys Val Asp Ile Glu Thr Leu Glu Lys Thr Glu Lys Val Asp Trp Ser

180 185 190aaa ttt att gct gtg aat gga gca act gca cat cct cat tat gac ccg 624Lys Phe Ile Ala Val Asn Gly Ala Thr Ala His Pro His Tyr Asp Pro

195 200 205gat gga aca gca tac aat atg ggg aac tcc ttt ggg cca tat ggt ttc 672Asp Gly Thr Ala Tyr Asn Met Gly Asn Ser Phe Gly Pro Tyr Gly Phe

210 215 220tcc tat aag gtt att cgg gtt cct cca gag gag gtg gac ctt ggg gag 720Ser Tyr Lys Val Ile Arg Val Pro Pro Glu Glu Val Asp Leu Gly Glu225 230 235 240aca arc cat gga gtc cag gtg ata tgt tct att gct tct aca gag aaa 768Thr Ile His Gly Val Gln Val Ile Cys Ser Ile Ala Ser Thr Glu Lys

245 250 255ggg aaa cct tct tac tac cat agc ttt gga atg aca agg aac tat ata 816Gly Lys Pro Ser Tyr Tyr His Ser Phe Gly Met Thr Arg Asn Tyr Ile

260 265 270att ttc att gaa caa cct cra aag atg aac ctg tgg aaa att gcc act 864Ile Phe Ile Glu Gln Pro Leu Lys Met Asn Leu Trp Lys Ile Ala Thr

275 280 285tct aaa att cgg gga aag gcc ttt tca gat ggg ata agc tgg gaa ccc 912Ser Lys Ile Arg Gly Lys Ala Phe Ser Asp Gly Ile Ser Trp Glu Pro

290 295 300cag tgt aat acg cgg ttt cat gtg gtg gaa aaa cgc act gga cag ctc 960Gln Cys Asn Thr Arg Phe His Val Val Glu Lys Arg Thr Gly Gln Leu305 310 315 320ctt cca ggg aga tac tac agc aaa cct ttt gtt aca ttt cat caa atc 1008Leu Pro Gly Arg Tyr Tyr Ser Lys Pro Phe Val Thr Phe His Gln Ile

325 330 335aat gcc ttt gag gac cag ggc tgt gtt ara att gat ttg tgc tgt caa 1056Asn Ala Phe Glu Asp Gln Gly Cys Val Ile Ile Asp Leu Cys Cys Gln

340 345 350gat aat gga aga acc cta gaa gtt tac cag tta cag aat ctc agg aag 1104Asp Asn Gly Arg Thr Leu Glu Val Tyr Gln Leu Gln Asn Leu Arg Lys

355 360 365gct ggg gaa ggg ctt gat cag gtc cat aat tca gca gcc aaa tct ttc 1152Ala Gly Glu Gly Leu Asp Gln Val His Asn Ser Ala Ala Lys Ser Phe

370 375 380cct cga agg ttt gtt ttg cct tta aat gtc agt ttg aat gcc cct gag 1200Pro Arg Arg Phe Val Leu Pro Leu Asn Val Ser Leu Asn Ala Pro Glu385 390 395 400gga gac aac ctg agt cca ttg tcc tat act tca gcc agt gct gtg aaa 1248Gly Asp Asn Leu Ser Pro Leu Ser Tyr Thr Ser Ala Ser Ala Val Lys

405 410 415cag gct gat gga acg arc tgc tgc tct cat gaa aat cra cat cag gag 1296Gln Ala Asp Gly Thr Ile Cys Cys Ser His Glu Asn Leu His Gln Glu

420 425 430gac cta gaa aag gaa gga ggc att gaa ttt cct cag atc tac tat gat 1344Asp Leu Glu Lys Glu Gly Gly Ile Glu Phe Pro Gln Ile Tyr Tyr Asp

435 440 445cga ttc agt ggc aaa aag tat cat ttc ttt tat ggc tgt ggc ttt cgg 1392Arg Phe Ser Gly Lys Lys Tyr His Phe Phe Tyr Gly Cys Gly Phe Arg

450 455 460cat tta gtg ggg gat tct ctg atc aag gtt gat gtg gtg aat aag aca 1440His Leu Val Gly Asp Ser Leu Ile Lys Val Asp Val Val Asn Lys Thr465 470 475 480ctg aag gtt tgg aga gaa gat ggc ttt tat ccc rca gaa cct gtt ttt 1488Leu Lys Val Trp Arg Glu Asp Gly Phe Tyr Pro Ser Glu Pro Val Phe

485 490 495gtt cca gca cca gga acc aat gaa gaa gat ggt ggg gtt att ctt tct 1536Val Pro Ala Pro Gly Thr Asn Glu Glu Asp Gly Gly Val Ile Leu Ser

500 505 510gtg gtg arc act ccc aac cag aat gaa agc aat ttt ctc cta gtt ttg 1584Val Val Ile Thr Pro Asn Gln Asn Glu Ser Asn Phe Leu Leu Val Leu

515 520 525gat gcc aag aac ttt gaa gag ctg ggc cga gca gag gta cct gtg cag 1632Asp Ala Lys Asn Phe Glu Glu Leu Gly Arg Ala Glu Val Pro Val Gln

530535540 atg cct tat ggg ttc cat ggt acc ttc ata ccc atc tgatgggaca 1678Met Pro Tyr Gly Phe His Gly Thr Phe Ile Pro Ile 545550555 accacaaggt ctggaaacta ggtttaaaat aagtgtgcac ttggacataa agactggaga 1738aataaacact gaggactcca aaaggggggc aaggaggaag aggggcaggg gttaaaaagc 1798tacctattga atactatgtt ccctatttgg gtgatgggtt cgttagaagt ccaaacctca 1858gcagcacaca atatactcat gtaacaagcc tgcacatgta ccccagaatt taaaataaaa 1918tttttttttt tttttt 1934<2l0>2l <211>556<212> PRT <213> human <400>21Met Val Tyr Arg Leu Pro Val Phe Lys Arg Tyr Met Gly Asn Thr Pro 151015 Gln Lys Lys Ala Val Phe Gly Gln Cys Arg Gly Leu Pro Cys Val Ala

20 25 30Pro Leu Leu Thr Thr Val Glu Glu Ala Pro Arg Gly Ile Ser Ala Arg

35 40 45Val Trp Gly His Phe pro Lys Trp Leu Asn Gly Ser Leu Leu Arg Ile

50 55 60Gly Pro Gly Lys Phe Glu Phe Gly Lys Asp Lys Tyr Asn His Trp Phe65 70 75 80Asp Gly Met Ala Leu Leu His Gln Phe Arg Met Ala Lys Gly Thr Val

85 90 95Thr Tyr Arg Ser Lys Phe Leu Gln Ser Asp Thr Tyr Lys Ala Asn Ser

100 105 110Ala Lys Asn Arg Ile Val Ile Ser Glu Phe Gly Thr Leu Ala Leu Pro

115 120 125Asp Pro Cys Lys Asn Val Phe Glu Arg Phe Met Ser Arg Phe Glu Leu

130 135 140Pro Gly Lys Ala Ala Ala Met Thr Asp Asp Thr Asn Val Asn Tyr Val145 150 155 160Arg Tyr Lys Gly Asp Tyr Tyr Leu Cys Thr Glu Thr Asn Phe Met Asn

165 170 175Lys Val Asp Ile Glu Thr Leu Glu Lys Thr Glu Lys Val Asp Trp Ser

180 185 190Lys Phe Ile Ala Val Asn Gly Ala Thr Ala His Pro His Tyr Asp Pro

195 200 205Asp Gly Thr Ala Tyr Asn Met Gly Asn Ser Phe Gly Pro Tyr Gly Phe

210 215 220Ser Tyr Lys Val Ile Arg Val Pro Pro Glu Glu Val Asp Leu Gly Glu225 230 235 240Thr Ile His Gly Val Gln Val Ile Cys Ser Ile Ala Ser Thr Glu Lys

245 250 255Gly Lys Pro Ser Tyr Tyr His Ser Phe Gly Met Thr Arg Asn Tyr Ile

260 265 270Ile Phe Ile Glu Gln Pro Leu Lys Met Asn Leu Trp Lys Ile Ala Thr

275 280 285Ser Lys Ile Arg Gly Lys Ala Phe Ser Asp Gly Ile Ser Trp Glu Pro

290 295 300Gln Cys Asn Thr Arg Phe His Val Val Glu Lys Arg Thr Gly Gln Leu305 310 315 320Leu Pro Gly Arg Tyr Tyr Ser Lys Pro Phe Val Thr Phe His Gln Ile

325 330 335Asn Ala Phe Glu Asp Gln Gly Cys Val Ile Ile Asp Leu Cys Cys Gln

340 345 350Asp Asn Gly Arg Thr Leu Glu Val Tyr Gln Leu Gln Asn Leu Arg Lys

355 360 365Ala Gly Glu Gly Leu Asp Gln Val His Asn Ser Ala Ala Lys Ser Phe

370 375 380Pro Arg Arg Phe Val Leu Pro Leu Asn Val Ser Leu Asn Ala Pro Glu385 390 395 400Gly Asp Asn Leu Ser Pro Leu Ser Tyr Thr Ser Ala Ser Ala Val Lys

405 410 415Gln Ala Asp Gly Thr Ile Cys Cys Ser His Glu Asn Leu His Gln Glu

420 425 430Asp Leu Glu Lys Glu Gly Gly Ile Glu Phe Pro Gln Ile Tyr Tyr Asp

435 440 445Arg Phe Ser Gly Lys Lys Tyr His Phe Phe Tyr Gly Cys Gly Phe Arg

450 455 460His Leu Val Gly Asp Ser Leu Ile Lys Val Asp Val Val Asn Lys Thr465 470 475 480Leu Lys Val Trp Arg Glu Asp Gly Phe Tyr Pro Ser Glu Pro Val Phe

485 490 495Val Pro Ala Pro Gly Thr Asn Glu Glu Asp Gly Gly Val Ile Leu Ser

500 505 510Val Val Ile Thr Pro Asn Gln Asn Glu Ser Asn Phe Leu Leu Val Leu

515 520 525Asp Ala Lys Asn Phe Glu Glu Leu Gly Arg Ala Glu Val Pro Val Gln

530535540 Met Pro Tyr Gly Phe His Gly Thr Phe Ile Pro Ile 545550555 <210>22<211>21<212> DNA <213> Artificial sequence <220> <223> description of the artificial sequence: description of β -diox I RT-PCR upstream primer <400>22atggagataa tatttggcca g 21<21 > 210>23<211>19<212> DNA <213> Artificial sequence <220> <223> Artificial sequence: description of the beta-diox I RT-PCR downstream primer <400>23aactcagaca ccacgattc 19<210>24<211>21<212> DNA <213> Artificial sequence <220> <223> Artificial sequence: description of β -diox II RT-PCR upstream primer <400>24atgttgggac cgaagcaaag c 21<210>25<211>21<212> DNA <213> Artificial sequence <220> <223> Artificial sequence: description of the beta-diox II RT-PCR downstream primer <400>25tgtgctcatg tagtaatcac c 21<210>26<211>21<212> DNA <213> Artificial sequence <220> <223> Artificial sequence: description of β -actin RT-PCR upstream primer <400>26ccaaccgtga aaagatgacc c 21<210>27<211>21<212> DNA <213> artificial sequence <220> <223> artificial sequence: beta-actin RT-PCR downstream primer <400>27cagcaatgcc tgggtacatg g 21

Claims (38)

1. An isolated beta-carotene dioxygenase (beta-diox II) polypeptide or functional fragment thereof having the biological activity of specifically cleaving beta-carotene and lycopene to form beta-apocarotene aldehyde and beta-ionone, respectively, and apolycopene aldehyde.

2. A β -diox II polypeptide or functional fragment thereof according to claim 1 comprising one or more amino acid sequences selected from the group consisting of: SEQ ID NO: 17 at positions 39-47, 96-118, 361-368 and 466-487, and SEQ ID NO: 19, positions 55-63, 112-134, 378-385 and 482-503 of SEQ ID NO: 21 at positions 59-67, 116-138, 385-392, and 490-511.

3. A β -diox II polypeptide according to claim 1 or 2 or a functional fragment thereof having an amino acid sequence substantially identical to that of SEQ ID NO: 17. 19, or 21, is at least 45% identical to the amino acid sequence set forth in seq id no.

4. A β -diox II polypeptide according to claim 1 or 2 or a functional fragment thereof having an amino acid sequence substantially identical to that of SEQ ID NO: 17. 19, or 21, is at least 60% identical to the amino acid sequence set forth in seq id no.

5. A β -diox II polypeptide according to claim 1 or 2 or a functional fragment thereof having an amino acid sequence substantially identical to that of SEQ ID NO: 17. 19, or 21, which is at least 75% identical to the amino acid sequence set forth in seq id no.

6. A β -diox II polypeptide according to claim 1 or 2 or a functional fragment thereof having an amino acid sequence substantially identical to that of SEQ ID NO: 17. 19, or 21, which is at least 90% identical to the amino acid sequence set forth in seq id no.

7. A β -diox II polypeptide according to claim 1 or 2 or a functional fragment thereof having the amino acid sequence of SEQ ID NO: 17. 19, or 21, or a portion thereof.

8. A β -diox II polypeptide or functional fragment thereof according to claim 1 or 2 having an amino acid sequence encoded by a DNA sequence selected from the group consisting of:

(1) SEQ ID NO: 16 and/or SEQ ID NO: 18 and/or SEQ ID NO: 20 or a complementary strand thereof; and

(2) SEQ ID NO: 16at the 115-141 position, the 286-354 position, the 1081-1104 position and the 1396-1461 position or complementary strands thereof; and

(3) SEQ ID NO: 18 at positions 191-217, 362-430, 1160-1183 and 1472-1537 or complementary strands thereof; and

(4) SEQ ID NO: 20at the 175-201 position, the 346-414 position, the 1153-1176 position and the 1468-1533 position or complementary strands thereof; and

(5) a DNA sequence or a functional fragment thereof which hybridizes under high stringency conditions with the DNA sequence or the complementary strand thereof defined in (1), (2), (3), and (4); and

(6) DNA sequences which hybridize to the DNA sequences defined in (1), (2), (3), (4) and (5) if the degeneracy of the non-genetic code is not present.

9. A DNA molecule comprising a DNA sequence encoding a β -diox II polypeptide according to any one of claims 1 to 8, or a functional fragment thereof.

10. A DNA molecule comprising a DNA sequence for ensuring the expression of a β -diox II polypeptide or a functional fragment thereof having the biological activity of specifically cleaving β -carotene and lycopene to form β -apocarotene aldehyde and β -ionone and apolycopene aldehyde, respectively, or for determining the presence of a nucleic acid characteristic of said polypeptide or functional fragment thereof, said nucleic acid being selected from the group consisting of:

(1) SEQ ID NO: 16 and/or SEQ ID NO: 18 and/or SEQ ID NO: 20 or a complementary strand thereof; and

(2) SEQ ID NO: 16at the 115-141 position, the 286-354 position, the 1081-1104 position and the 1396-1461 position or complementary strands thereof; and

(3) SEQ ID NO: 18 at positions 191-217, 362-430, 1160-1183 and 1472-1537 or complementary strands thereof; and

(4) SEQ ID NO: 20at the 175-201 position, the 346-414 position, the 1153-1176 position and the 1468-1533 position or complementary strands thereof; and

(5) a DNA sequence or a functional fragment thereof which hybridizes under high stringency conditions with the DNA sequence or the complementary strand thereof defined in (1), (2), (3), and (4); and

(6) DNA sequences which hybridize to the DNA sequences defined in (1), (2), (3), (4) and (5) if the degeneracy of the non-genetic code is not present.

11. The DNA molecule according to claim 9 or 10, comprising a DNA sequence which is a cDNA, genomic, or artificial DNA sequence.

12. The DNA molecule according to any one of claims 9 to 11, comprising at least one selectable marker gene or cDNA operably linked to a constitutive, inducible or tissue-specific promoter sequence allowing its expression in bacterial, fungal, including yeast, insect, animal or plant cells, seeds, tissues or whole organisms.

13. The DNA molecule according to any one of claims 9 to 12, wherein the coding nucleotide sequence is fused to a suitable plastid transit peptide coding sequence, both preferably expressed under the control of a tissue-specific or constitutive promoter.

14. A plasmid or vector system comprising one or more DNA molecules according to any one of claims 9 to 13.

15. A method for producing a β -diox II polypeptide comprising the steps of:

(1) expressing in a suitable host a polypeptide encoded by a DNA according to any one of claims 9 to 14 and

(2) isolating said beta-diox II polypeptide.

16. A protein product obtained by the method of claim 15.

17. A prokaryotic or eukaryotic host cell, seed, tissue or whole organism transformed or transfected with a DNA molecule according to any of claims 9 to 13 or with a plasmid or vector system according to claim 14 in a manner enabling said host cell, seed, tissue or whole organism to express a polypeptide or functional fragment thereof having the biological activity of specifically cleaving β -carotene and lycopene to form β -apocarotene aldehyde and β -ionone and apolycopene aldehyde, respectively, and/or having the ability to specifically bind antibodies prepared against said polypeptide or functional fragment thereof.

18. A prokaryotic or eukaryotic host cell, seed, tissue, or whole organism according to claim 17 selected from the group consisting of: bacteria, fungi include yeast, insect, animal, and plant cells, seeds, tissues, or whole organisms.

19. Prokaryotic host cell or whole organism according to claim 18, which is a bacterium selected from the group consisting of: protobacteria, including members of the alpha, beta, gamma, delta, and epsilon subgenera; gram-positive bacteria including Actinomycetes (Actinomycetes), Firmicutes (Firmicutes), clostridia (Clostridium) and its relatives, yellow bacteria, cyanobacteria, green sulfur bacteria, green non-sulfur bacteria, and archaea.

20. Prokaryotic host cell or whole organism according to claim 19, belonging to a genus of protobacteria selected from the group consisting of: agrobacterium, Rhodobacter, ammonia-oxidizing bacteria such as Nitrosomonas, Enterobacteriaceae, Myxobacteria such as Myxococcus, preferably Agrobacterium aureus, Rhodobacter capsulatus, species of Nitrosomonas sp ENI-11, Escherichia coli, and Myxococcus xanthus.

21. Prokaryotic host cell or whole organism according to claim 19, belonging to a gram-positive bacterium selected from the group consisting of: actinomycetes (Actinomycetes) and Firmicutes (Firmicutes) include the genera Clostridium (Clostridium) and relatives thereof, such as Bacillus (Bacillus) and Lactococcus (Lactococcus), preferably Bacillus subtilis and Lactococcus lactis.

22. Prokaryotic host cell or whole organism according to claim 19, belonging to the group of yellow bacteria selected from the group consisting of: bacteroides (Bacteroides), Cellophaga (Cytophaga), and Flavobacterium (Flavobacterium), preferably Flavobacterium such as Flavobacterium ATCC 21588.

23. Prokaryotic host cell or whole organism according to claim 19, belonging to a cyanobacterium selected from the group consisting of: chlorococcales, including Synechocystis (Synechocystis) and Synechococcus (Synechococcus), preferably Synechocystis species and Synechococcus species PS 717.

24. Prokaryotic host cell or whole organism according to claim 19, belonging to the group of green sulfur bacteria or green non-sulfur bacteria selected from the group of: respectively of the genus Chlorella (Chlorobium) or of the family Chloroflexaceae (Chloroflexaceae), such as the genus Chloroflexa (Chloroflexus), preferably respectively of the species Chlorobium limicola (Chlorosulfatophilum) and Chloroflexa aurantium (Chloroflexa aurantiacus).

25. Prokaryotic host cell or whole organism according to claim 19, belonging to an archaebacterium selected from the group consisting of: halobacteriaceae (Halobacteriaceae) such as Halobacterium (Halobacterium), preferably Halobacterium salinum.

26. Eukaryotic host cell or whole organism according to claim 18, which is a fungus selected from the group comprising yeast: ascomycota (Ascomycota) includes Saccharomyces species (Saccharomyces) such as Pichia and Saccharomyces, and Anamorphic Ascomycota (Anamorphic Ascomycota) includes Aspergillus, preferably Saccharomyces cerevisiae and Aspergillus niger.

27. The eukaryotic host cell according to claim 18, which is an insect cell selected from the group consisting of: SF9, SF21, Trychplusiani, and MB 21.

28. The eukaryotic host cell according to claim 18, which is an animal cell selected from the group consisting of: baby Hamster Kidney (BHK) cells, Chinese Hamster Ovary (CHO) cells, Human Embryonic Kidney (HEK) cells, and COS cells, with NIH 3T3 and 293 cells being most preferred.

29. The eukaryotic host cell, seed, tissue, or whole organism according to claim 18, which is a plant cell, seed, tissue, or whole organism selected from the group consisting of: eukaryotic algae, embryonated plants include the phylum Bryophyta (Bryophyta), the phylum Pteridophyta (Pteridophyta), and the phylum angiosperma (spectatophyta) such as the subphylum Gymnospermae (Gymnospermae) and the subphylum Angiospermae (Angiospermae), the latter including magnoliopsia, rospsida, and Liliopsida ("monocots").

30. A eukaryotic host cell, seed, tissue, or whole organism according to claim 29 selected from the group consisting of: cereal seeds, preferably rice, wheat, barley, oats, amaranth, flax, triticale, rye, and corn; oilseeds, preferably canola seeds, cotton seeds, soybeans, safflower, sunflower, coconut, and palm; other edible seeds or seeds containing edible parts selected from the group consisting of: pumpkin, sesame, poppy, grape, mung bean, peanut, pea, bean, radish, alfalfa, cocoa, coffee, hemp; tree nuts, preferably walnuts, almonds, pecans, and chickpeas; potatoes, carrots, sweet potatoes, sugar beets, tomatoes, peppers, cassava, willows, oaks, elms, maples, apples, and bananas.

31. A method of transforming a bacterial, fungal, yeast, insect, animal, or plant cell, seed, tissue, or whole organism to produce a transformant capable of expressing a beta-carotene dioxygenase (beta-diox II) polypeptide or functional fragment thereof, said polypeptide or functional fragment thereof having the biological activity of specifically cleaving beta-carotene and lycopene to form beta-apocarotene aldehyde and beta-ionone and apolycopene aldehyde, respectively, and/or having the ability to specifically bind to antibodies raised against said polypeptide or functional fragment thereof, the method comprises transforming said bacteria, fungi including yeast, insect, animal or plant cells, seeds, tissues or whole organisms with a DNA molecule according to any one of claims 9 to 13 or with a plasmid or vector system according to claim 14.

32. Transformed bacteria, fungi including yeast, insect, animal, or plant cells, seeds, tissues, or whole organisms provided or regenerated from a transformant produced according to claim 31.

33. The transformed plant cell, seed, tissue, or whole organism according to claim 32, selected from the group consisting of: eukaryotic algae, embryonated plants include the phylum Bryophyta (Bryophyta), the phylum Pteridophyta (Pteridophyta), and the phylum Spermatophyta (stertophyta) such as the subphylum Gymnospermae (Gymnospermae) and the subphylum Angiospermae (Angiospermae), the latter including magnoliopsia, rospsida, and Liliopsida ("monocots").

34. The transformed plant cell, seed, tissue, or whole organism according to claim 33, selected from the group consisting of: cereal seeds, preferably rice, wheat, barley, oats, amaranth, flax, triticale, rye, and corn; oilseeds, preferably canola seeds, cotton seeds, soybeans, safflower, sunflower, coconut, and palm; other edible seeds or seeds containing edible parts selected from the group consisting of: pumpkin, sesame, poppy, grape, mung bean, peanut, pea, bean, radish, alfalfa, cocoa, coffee, hemp; tree nuts, preferably walnuts, almonds, pecans, and chickpeas; potatoes, carrots, sweet potatoes, sugar beets, tomatoes, peppers, cassava, willows, oaks, elms, maples, apples, and bananas.

35. An antibody specifically immunoreactive with the polypeptide of any one of claims 1-8 and 16.

36. Use of a DNA molecule according to any one of claims 9 to 13 for diagnostic and/or therapeutic purposes.

37. Use of a polypeptide according to any one of claims 1-8 and 16 for therapeutic purposes.

38. Use of an antibody according to claim 35 for the isolation and/or quantification of a polypeptide according to any one of claims 1-8 and 16 and for diagnostic and/or therapeutic purposes.