MXPA98010574A - Synthetic plan promoter - Google Patents

Abstract

The present invention relates to synthetic elements for increasing the expression of genes in plant cells. These include a promoter with a "TATA to start" sequence containing 64% or more GC content and an upstream element incorporating several OCS binding motifs and novel flanking sequences.

Description

SYNTHETIC PROMOTER OF THE HEART OF A PLANT AND REGULATOR ELEMENT CURRENT UP FIELD OF THE INVENTION This invention relates generally to the field of molecular biology of plants and in particular to an upstream element and synthetic promoter sequences and their combined arrangement within a promoter region, so as to increase expression of a gene. The invention allows the enhanced or amplified expression of the desired structural genes in monocotyledonous and dicotyledonous plants.

BACKGROUND OF THE INVENTION The expression of a gene encompasses a number of steps ranging from the DNA standard to the protein product or final protein. The control and regulation of the expression of a gene can occur through numerous mechanisms. The start of transcription of a gene is generally through the predominant control of gene expression. Transcriptional controls (or promoters) are generally relegated to relatively short sequences embedded in the flanking region or upstream 5 'of the transcribed gene. There are DNA sequences which affect the expression of the gene in response to environmental stimuli, availability of REF. 29109 nutrients or adverse conditions including thermal shocks, anaerobiosis or the presence of heavy metals. There are also DNA sequences which control the expression of the gene during development or in a specific tissue or organ. The promoters contain the signals for RNA polymerase to begin transcription, so that protein synthesis can occur. The binding of DNA to nuclear proteins that specifically interact with those known promoter DNA sequences promotes the formation of the transcriptional complex and eventually initiates the process of gene expression. One of the most common sequence motifs present in the promoters of genes transcribed by the APJST polymerase II (polll) eukaryotic system is the WTATA element "which resides upstream of the start of transcription." Eukaryotic promoters are complex and they are comprised of components which include a TATA block in the consensus sequence at approximately 35 base pairs 5 'relative to the transcription start site or top or top site which is defined as a + 1. The TATA motif is the site where the TATA (TB?) binding protein as part of the complex of several polypeptides (TFIID complex) binds and interacts productively (directly or indirectly) with factors linked to other elements of the promoter sequence. TFIID in turn recruits the RNA polymerase II complex to place it for the start of transcription, generally 25 to 30 base pairs downstream of the TATA element and promotes elongation, thereby producing RNA molecules. The sequences around the start of the transcription (designated INR) of some polll genes seem to provide an alternative binding site for factors that also recruit members of the TFIID complex and thus "acLive" the transcription.These INR sequences are particularly relevant in promoters lacking functional TATA elements that provide the binding sites of the heart promoter or plant nucleus for eventual transcription It has been proposed that promoters containing a TATA motif and functional INR are the most efficient in activity (Zenzie-Gregory et al, 1992. J. Biol. Chem. 267: 2823-2830) In the majority of cases, sequence elements other than the TATA motif are required for accurate transcription. upstream of the TATA motif and a subset may have homology to the CCAAT consensus sequence.

It has been found that other DNA sequences raise the total level of expression of nearby genes. One of the ccmúnes elements that have been described reside far upstream from the start site and appear to exhibit independent position and orientation characteristics. The upstream elements have been designated as amplifiers. One of the least common elements by virtue of their specificities are the sequences that interact with specific DNA binding factors. These sequence motifs are collectively known as upstream elements which usually depend on their position and orientation. Many upstream elements have been identified in a number of plant promoters based initially on the function and the second on sequence homologies. These upstream promoter elements fluctuate widely in the type of control: from environmental responses such as temperature, humidity, hurting, etc., developmental signals, (germination, maturation of the seed, flowering, etc.) to spatial information (tissue specificity). These elements also seem to exhibit odularity since they can be exchanged with other elements while maintaining their characteristic control over gene expression. The promoters are usually 5 'or upstream relative to the start of the coding region of the corresponding gene, and the entire region containing all the auxiliary elements that affect the regulation or absolute levels of transcription may be comprised of less than 100 pairs of bases or as much as 1 pair of kilobases. Numerous promoters have been described in the literature, which are active in plant cells. These include the promoters of nopaline synthase (NOS) and octopine synthase (OCS) (which are found in tumor-inducing plasmids of Agrobacterium tumefaciens). Also included are the 19S and 35S promoters of cauliflower mosaic virus (CaMV), the photoinducible promoter of the small subunit of ribulose carboxylase biphosphate (ssRUBICSO, a polypeptide very abundant in plants), and the promoter of sucrose synthase. Promoters for use in plants are also described in EP-A-0 459 643; WO-A-95 14098; and EP-A-0 342 926. All of those promoters have been used to create various types of DNA cructs, which have been expressed in plants. (See, for example, PCT publication WO84 / 02913 by Rogers et al.).

The two promoters that have been widely used in plant cell transformatiare those of the genes encoding alcohol dehydrogenase, Adhl and AdhlI. Both genes are induced after the t of anaerobiosis. The maize Adhl has been cloned and sequenced as the AdhlI has been. The formation of a Adhl, Adh-CAT chimeric gene comprising the Adhl promoter is linked to the coding sequences of chloramphenicol acetyltransferase (CAT) and the 3 'signal of nopaline synthase (NOS) which causes the expression of CAT be approximately 4 times higher at low oxygen concentratithan under control conditi The sequence elements necessary for the induction of chimeric Adh-CAT have also been identified. The existence of an anaerobic regulatory element (ARE) between positi-140 and -99 of the Adhl promoter of the maize composed of at least two sequence elements at positi-133 to -124 and positi-113 to 99 both of which it has been found necessary and sufficient for the expression of the activity of the Adh-CAT gene at low levels of oxygen. The Adh promoter, however, responds to anaerobiosis and is not a citutive promoter that drastically limits its effectiveness. Another promoter commonly used is the 35S promoter of the Cauliflower Mosaic Virus. The 35S promoter (CaMV) is a promoter of dicotyledonous viruses that, however, directs the expression of the genes introduced in the protoplasm of dicotyledand monocotyled The 35S promoter is a very strong promoter and is cderable because of its widespread use due to the high level of expression of traits or characteristics in transgenic plants. The CaMV35S promoter, however, has also shown relatively low activity in several graminaceous plants of agricultural importance such as wheat. Although all of these promoters give high expression in dicots, none give the high levels of expression in monocotyled There is a need for synthetic promoters and other elements that induce expression in cells of the protoplasts of transformed monocots. The main objective of this invention is to provide synthetic regulatory elements that promote the expression of induced genes in plant cells and plant tissues. Another objective of this invention is to provide a recombinant promoter molecule that provides highly reliable levels of expression of the genes introduced into target cells.

Yet another objective of this invention is to provide upstream amplifying elements that can further stimulate the activity of any promoter. Yet another objective of this invention is to provide plants, plant cells and plant tissues containing either or both of the recombinant promoter or upstream element of the invention. Yet another object of the invention is to provide vehicles for the transformation of plant cells including viral or plasmid vectors and expression cassettes incorporating the synthetic upstream promoter of the invention. Yet another objective of the invention is to provide bacillus cells comprising such vectors for the maintenance, reproduction and transformation of the plant. The other objects of the invention will be apparent from the following description of the invention.

BRIEF DESCRIPTION OF THE INVENTION The invention provides a synthetic promoter molecule of the core or core and the upstream element that will allow those skilled in the art to obtain highly reliable levels of expression of structural genes introduced into target cells. The heart or core promoter comprises a TATA block and a start of transcription. further, the 'TATA to start' region is 64% or more GC rich One modality is shown in SEQ ID N0: 1. A novel upstream element, SEQ ID NO: 2 is also described which helps to enhance transcription. The synthetic promoter of the heart or core can be used with combinations of sequences of the amplifying elements and / or upstream of the 5 'flanking regions of the structural genes expressible by the plant. Similarly the upstream element can be used in combination with several promoter sequences of the heart or nucleus of several plants In a preferred embodiment the heart or core promoter and the upstream element are used together to obtain a tenfold expression of a marker gene introduced into transgenic monocotyledonous plants obtained with the promoter of corn ubiquitin 1. The pro-motor of the heart or nucleus comprises a TATA motif and a TATA region for the start of the t "GC-rich" transcription (64% or more of GC content than in animal promoters is traditionally characteristic) • The sequence was placed 5 'of a structural gene and will promote non-specific constitutive tissue expression in plant cells transgenic The invention also comprises an expression cassette comprising (the upstream element) the synthetic promoter of the heart or core of a plant, a structural gene, the expression of which is desired in plant cells, and a polyadenylation or high signal. The expression cassette can be encompassed by the plasmid or viral vectors for the transformation of plant protoplast cells. The invention also encompasses transformed bacteria cells for the maintenance and reproduction of the vector, as well as transformed monocot or dicotyledonous cells and finally transgenic plants. In another embodiment, the invention encompasses an upstream element that can be used in combination with the synthetic promoter or with other promoters known in the art. The upstream element comprises at least 3 OCS binding motifs (TGACG) with a novel intervening sequence. One embodiment is described in SEQ ID NO: 2 and a promoter sequence of the heart or nucleus was placed 5 'to increase the transcription levels of the product of the resulting gene. Thus, the invention comprises an expression cassette comprising the upstream synthetic element of the invention, 5 'to the inducible promoter of the plant which is 5' to a structural gene. This expression cassette can be incorporated into vectors and plasmids as described at the beginning. In a preferred embodiment the upstream synthetic element was used in combination with the synthetic promoter sequence of the heart or nucleus to achieve non-specific tissue constitutive expression of the gene product, which is ten times larger than that of the ubil promoter of the corn .

DESCRIPTION OF THE FIGURES Figure 1 is a description of an arrangement of peak nucleotide bases of a heart or core promoter containing the consensus sequences of the TATA motifs and INR present in plant promoters. A designates +1 of the transcribed region. Figure 2 is a description of the Sequence Heart Promoter or Syn II Nucleus with an example of a plant promoter and both are aligned at the main start of transcription (bold). The TATA motif is underlined. The CaMV 35S promoter is shown with percentages of GC containing the sequences shown in parentheses.

Figure 3 is a DNA sequence of the upstream element Rsyn7. The TGACG motifs are indicated in bold type. Figure 4 is a plasmid map of one embodiment of the invention comprising the Syn II Heart or Nucleus promoter and the Rsyn7 elements directing a GUS-containing construct. Figure 5 describes various schemes of synthetic promoters according to the present invention tested on transient and stable transformants. Figure 6 is a description of the transient assay data using the plasmids incorporating the promoter sequences of the invention. Figure 7 (A). It is the activity of Rsyn7 :: GUS (6086) in corn plants TO. Figure 7 (B) is a scheme of corn plants in VT stage with indicated tissue sample sites. Figure 8 describes the activity of the GUS in root segments of a segregating population of transgenic IT maize seedlings containing the construct Rsyn7 :: GUS (6086) or the UBI: GUS (3953). Figure 9 describes the expression of GUS of three synthetic promoters in corn TO gene transgenic plants that include the promoter sequences of the invention as a comparison.

DETAILED DESCRIPTION OF THE INVENTION In the following description numerous terms were used extensively. The following definitions are provided to eliminate ambiguities about what was intended or - the scope of its use in the specification and claims for fat. litar the understanding of the invention. A structural gene is a DNA sequence that was transcribed into messenger RNA (mRNA), which is then translated into an amino acid sequence characteristic of a specific polypeptide. A promoter is a DNA sequence that directs the transcription of a structural gene. Typically, a promoter is located in the 5 'region of a gene, near the transcriptional start site of a structural gene. A minimal promoter or core promoter or core contains the nucleotide sequences essential for proper functioning, including the TATA block and initiation of transcription. By this definition, a promoter of the heart or nucleus may or may not have detectable activity in the absence of specific sequences that may increase activity or confer tissue-specific activity. For example, the heart or core promoter of maize consists of approximately 37 nucleotides 5 'from the start site of transcription of the SGB6 gene, while the heart or nucleus 35S promoter of the Cauliflower Mosaic Virus (CaMV) consists of of about 33 nucleotides 5 'from the start site of the 35S genome transcript. ADH refers generally to the alcohol dehydrogenase gene expressible in plants and specifically to the maize alcohol dehydrogenase gene. The ADH 1 promoter refers to the DNA fragment spanning the region between the nucleotide positions of about -1094 to about -106 of the maize dehydrogenase-1 gene, or the homologous fragment that is functionally equivalent. The sequence was numbered with the transcription start site designated as +1 according to the correction published by Ellis et al., (1987) supra. TATA to start "will mean the sequence between the TATA motif and the start of transcription." A synthetic DNA is an artificially created DNA sequence that does not occur naturally, and an organism or an ancestor of that organism must be introduced to control or to be expressed.

The OCS element refers to the identified TGACG motif of the octopine synthase gene, histone genes, enzyme genes for agropin biosynthesis, the manpir-a synthase gene, the CaMV 35S gene, the histone H3 gene and the nopaline synthase gene. As used herein the term includes any sequence capable of binding to the ASF-1 factor identified in U.S. Patent No. 4,990,607 to Katagiri, the disclosure of which is incorporated herein by reference. An amplifier is a regulatory element of DNA that can increase the efficiency of transcription regardless of the distance or orientation of the amplifier in relation to the site of initiation of transcription. The term "expression" refers to the biosynthesis of a genetic product. In the case of a structural gene, transcription involves the structural gene in the mRNA and therefore the translation of the mRNA into one or more polypeptides. A cloning vector is a DNA molecule such as a plasmid, cosmid or bacterial phage that has the ability to reproduce autonomously in a host cell. Cloning vectors typically contain one or a small number of restriction endonuclease recognition sites in which external DNA sequences can be inserted in a determinable manner without loss of the essential biological function of the vector, as well as a marker gene that is suitable for use in the identification and selection of cells transformed with the cloning vector. Marker genes typically include genes that provide resistance to tetracycline, resistance to hygromycin, or resistance to ampicillin. An expression vector is a DNA molecule that comprises the gene that is expressed in a host cell. Typically the expression of the gene is placed under the control of certain regulatory elements including promoters, specific regulatory elements of a tissue, and amplifiers. It is said that such a gene is "operably linked" to the regulatory elements. A recombinant host can be any prokaryotic or eukaryotic cells containing a cloning vector or an expression vector. This term also includes those prokaryotic or eukaryotic cells that have been genetically engineered to contain the cloned genes in the chromosome or genome of the host cell. A transgenic plant is a plant that has one or more plant cells that contain an expression vector.

It should be understood that there may be minor sequence variations within the sequence or fragments used or described in this application. These variations can be determined by standard techniques to enable those skilled in the art to manipulate and give utility to the functional units of the promoter elements necessary to direct the initiation of transcription in the structural gene followed by a transcription termination signal. (and perhaps polyadenylation) expressible in the plant. The tissue of the plant includes differentiated and undifferentiated tissues or plants, including but not limited to roots, stems, buds, leaves, pollen, seeds, tumor tissue and various cell and crop forms such as single cells, protoplasts, embryos and callus tissue. The tissue of the plant can be in the plant or in an organ, tissue cultures or cells. The promoter of the invention as seen in SEQ ID NO: 1 and / or SEQ ID NO: 2, can be used to obtain high expression levels of structural genes. Similarly, the upstream element of the invention (SEQ ID NO: 2) can be used in combination with other promoters or the promoter of the invention to enhance transcription levels in genetically modified plants. The production of a genetically modified plant tissue expressing a structural gene under the control of the regulatory elements of the invention combines the teachings of the present disclosure with a variety of techniques and expedients known in the art. In most cases there are alternative records for each stage of the total process. The choice of files depends on variables such as the system of the plasmid vector chosen for the cloning and introduction of the recombinant DNA molecule, the plant species to be modified, the particular structural gene, the promoter elements and upstream elements used. Those skilled in the art are capable of selecting and using the appropriate alternatives to achieve functionality. Culture conditions for expressing desired structural genes and cultured cells are known in the art. Numerous monocotyledonous and dicotyledonous plant species are also known in the art to be transformable and regenerable so that all plants contain and express the desired genes under the regulatory control of the promoter molecules and the upstream elements of the invention can be obtained. As is known to those skilled in the art, expression in transformed plants can be tissue specific and / or specific for certain stages of development. The selection of the truncated promoter and the selection of the structural gene are other parameters that can be optimized to achieve the expression of the desired plant as is known to those skilled in the art and as taught herein. The selection of an appropriate expression vector will depend on the method of introducing the expression vector into the host cells. Typically an expression vector contains (1) prokaryotic DNA elements that code for a bacterial origin of reproduction and a marker of resistance to an antibiotic to provide growth and selection of the expression vector in a bacterial cell; (2) DNA elements that control the initiation of transcription such as a promoter; (3) DNA elements that control the processing of transcripts such as the termination of transcription / polyadenylation; and (4) a reporter gene that is operably linked to the DNA elements to control the initiation of transcription. Useful reporter genes include ß-glucuronidase, β-galactosidase, chloramphenicol, acetyl transferase, luciferase, green fluorescent protein (GFP) and the like. Preferably, the reporter gene is any of the β-glucuronidase, (GUS), GFP or luciferase. General descriptions of expression vectors and plant reporter genes can be found in Gruber et al., 'Vectors for Plant Transformation, in Methods in Plant Molecular Biology & Biotechnology "in Glinch et al., (Eds. Pp. 89-119, CRC Press, 1993) In addition, GUS expression vectors and GUS gene cassettes are available from Clonetech Laboratories, Inc., Palo Alto , California, while luciferase expression vectors and luciferase gene cassettes are available from Promega Corp. (Madison, Wis.) Expression vectors containing genomic or synthetic fragments can be introduced into protoplasts or into intact tissues. or isolated cells Preferably the expression vectors are introduced into intact tissue The general methods for growing plant tissues are provided for example in Maki et al., "Procedures for Introducing Foreign DNA into Plants" in Methods in Plant Molecular Biology & Biotechnology, Glich et al., (Eds. Pp. 67-88 CRC Press, 1993); and by Phillips et al., "Cell-Tissue Culture and In-Vitro Manipulation" in Corn & Corn Improvement, 3rd Edition Sprague et al., (Eds. Pp. 345-387) American Society of Agrono and Inc. et al. 1988. Methods for introducing expression vectors into plant tissues include direct infection or cocultivation of plant cells with Agrobacterium. Turne Faciens, Horsch et al., Science, 227: 1229 (1985). The descriptions of vector systems and methods for gene transfer mediated by Agrobacterium are provided in Gruber et al. supra. Preferably, the expression vectors are introduced into corn or other plant tissues using the direct gene transfer method such as microprojectile-mediated release, DNA injection, electroporation and the like. More preferably, the expression vectors are introduced into plant tissues using the release by means of microprojectiles with a ballistic device. The vectors of the invention can only be used for the expression of structural genes but can also be used for the cloning of exon traps, or methods for trapping promoters to detect the differential expression of genes in tissue varieties, K. Lindsey et al. ., 1993"Tagging Genomic Sequences That Direct Transgene Expression by Activation of a Promoter Trap in Plants", Transgenic Research 2: 33-47. D. Auch & Reth, et al., "Exon Trap Cloning: Using Elastomeric PCR Rapidly Detect and Clone Exons from Genomic DNA Fragments" Nuclic Acids Research, Vol. 18, No. 22, p. 6743. This promoter of the invention was based in part on the discovery that the "TATA to start" region rich in GC in a plant acts as a promoter of the heart or specific core of the very strong tissue that induces constitutive expression in plant cells. The TATA element of the plant promoters of the polIII genes generally have the sequence TATA (A / T) A (A / T) A, SEQ ID NO: 3 while the consensus of the start of transcription consists of the sequence 5 '. ... TYYTCAT (A / C) AA ... 3 '. SEQ ID NO: 3, wherein A designates the starting base for transcription. The typical plant promoter sequence is described in Figure 1. It has been shown that the sequences involved in the TATA element and the initiation of transcription play a significant role in the efficiency of transcriptional activation. It has been shown that the TATA binding protein interacts as a minor groove in the junction of the double helix of the TATA motif by bending this one towards the side of the larger slot (Kim, et al., 1993, Nature, 365: 512-520). Thus, the downstream sequences of the TATA motif that impact this discovery will affect the efficiency of stable transcriptional complex formation and ultimately expression. Studies of the "TATA to start" regions of plant promoters show a significantly higher level of AT sequences leading to potential compression of the minor groove (Yaurawaj et al., Biologícal Anstracts Vol. 47, Issue 8, Ref. 144712"Consensus Sequences for Plant Mini to Producers" Annual Meeting of the American Society of Plant Physiologists, July 29-August 2, 1995, Plant Physilogy 108 [2 Supp.] 1994, 114). In general, animal promoters show a 'TATA to start' sequence rich in GC leading to greater groove compression, suggesting that the transcriptional complexes of the heart or core promoter of average plants and animals recognize and interact with a somewhat different TATA to start the structure with the corresponding sequence difference.A very surprisingly the applicant has found that GC-rich synthetic animal-type promoters work very well in plants.While not wishing to join any theory, it is It is possible that the AT-rich TATA motif present in a GC-rich sequence may present itself more prominently to the TATA-binding complex by means of a fine demarcation of the TATA sequence from GC-rich to AT-rich. The 'joined' TATA motif could interact more closely with the TATA binding complex. This could improve the efficiency of the start of transcription, diverting the binding balance towards a more stabilized form, while the 'unbound' version, that is, having more AT rich sequence instead of flanking the TATA motif, could Potentially slip or deviate downward and effectively reduce binding efficiency Few data related to this region of plant promoters are available except for crude deletions and some point fluctuations.The obvious design of a synthetic heart promoter or core for the Expression of a plant could include the 'TATA to start' sequence rich in AT. However, based on the 'bound' mechanism, it was postulated by the mechanism of the invention that a more efficient core or heart promoter could be present in a TATA motif included in a GC-rich sequence. promoter of the heart or nucleus Syn II, SEQ ID NO: 1 of the invention with examples of promoters of the heart or core of the plant aligned with the major onset of transcription Another example of a 35S plant promoter of CaMV (FIG. SEQ ID NO: 4) is shown with the percent of GC-rich sequences shown on the right side in parentheses.The heart sequence or Syn II core does not show any significant sequence homology with the sequences in the public database. Synthetic promoter sequence from the Heart o Nucleus Syn II shows a sequence 'TATA to start' rich in GC in a 64% different to the sequence rich in GC in 40% of the total present in the promoters of traditional plants (CaMV35S for example) .The heart or core promoter Natural and isolated UBI that potentiates very high levels of activity shows a 'TATA to start' sequence rich in GC by 64% more similar to animal promoters. Such examples provided the impetus for designing a sequence of 'TATA to start' very rich in GC for efficient transcription as opposed to the current dogma of the promoters of the heart or core of the plant.In this way the invention comprises a synthetic promoter sequence of the plant. heart or core of the synthetic plant comprising a TATA motif and a 'TATA to start' region that is 64% richer in GC. In a preferred embodiment the promoter can include target sites of restriction endonucleotides to facilitate cloning. In a more preferred embodiment the sequence is that of SEQ ID NO: l. As will be appreciated by those skilled in the art, several base transversions within SEQ ID NO: 1 may occur which will maintain the percent GC content and which are intended to be within the scope of this invention. SEQ ID NO: 10. For example, guanines could be replaced with cytosines and vice versa without affecting the total efficacy of promoter, as long as the percentage of GC content is maintained.

In another embodiment, the invention comprises an upstream synthetic element placed 5 'to any natural or synthetic promoter for use in plants, particularly expression. genetics in corn From the activity of numerous promoters, basic elements (binding sites) have been defined. These include, for example, AT-rich regions of cardiac shock promoters, and elements of the ASF-1 (AS-1) binding site present in octopine synthase (SCO) and promoters of the Cauliflower Mosaic Virus. AS-1 is one of the best known upstream elements and its binding sequence (OCS element) is present in many plant constitutive promoters such as CaMV35S, A. tumefaciens, wheat histone NOS and OCS promoters. The OCS element was isolated for the first time as an amplifier element in the OCS gene promoter where it was identified as a palindromic sequence of 16 base pairs (Ellis et al-, 1987 MBOJ 6: 11-16), but it has been reduced to its essential characteristics as a TGACG motif. See U.S. Patent No. 4,990,607 to Katagiri previously incorporated herein by reference. The upstream element of the invention has a 71% homology with the amplifier element described in U.S. Patent No. 5,023,179 to Lam et al. The two sequences are very different in their flanking sequences surrounding the TGACG motif, regions which have been shown to have an impact on the amplification levels of the transcript. The amplifying or enhancing activity of the transcription of the OCS element was correlated with the in-vitro binding of a transcriptional factor. Similar elements were also identified in the promoter regions of six other cDNA genes involved in the synthesis of opine and three viral plant promoters including the 35S promoter of CaMV (Bouchez et al., 1989) supra. These elements showed binding to the transcription factor OCS in vi tro and increased transcription in plant cells. In tobacco, a TGA1 DNA binding factor was shown to specifically interact with the AS-1 element alone or in conjunction with other promoter elements. Katagiri et al. 1989, Nature 340: 727-730. This factor also showed to be expressed preferably in the root of tobacco plants. The promoters of the heart or nucleus with one or two copies of the upstream element OCS tend to enhance the expression of the gene while 4 or more repeats of this element produce more or less constitutive but relatively low activity to interact with the 35S promoters.

Thus the invention incorporates an upstream synthetic element which can be used with the promoter of the core or core of the invention or other core or core promoters to increase the expression of the gene. The element incorporates three motifs similar to the OCS and novel intervening sequences which enhance the expression of a gene. Figure 3, SEQ ID NO: 2 shows the complete sequence of a modality (Rsyn7) of the upstream synthetic element which incorporates at least three motifs similar to the OSC, SEQ ID NO: 5, TGACG, which are indicated in bold type. Sequences that weaken many elements such as the motif of SEQ ID NO: 5 TGACG have shown to have profound impacts on the binding affinities of DNA binding factors and thus play as important a role as the motifs'. central ones. (Burrows et al., 1992, Plant Molecular Biology 19: 665-675, Shindler et al., 1992, Plant Cell 4: 1309-1319, Foster et al., 1994, FASEBJ 8: 192-200). The novel sequences that weaken the TGACG motifs in the Rsyn7 promoter have been determined and a clear increase in transcriptional activity with several promoters has been determined, particularly when used with the Syn II Heart or Nucleus promoter.

The upstream element Rsyn7 has been cloned upstream of the GUS Heart or Nucleus promoter directing a GUS construct and haplopening the GUS activity levels in transgenic corn plants approximately ten times more than the ubiquitin promoter, the corn promoter. strongest to date. The following examples are for purposes of illustration only and are intended not to limit in any way the scope or application of the present invention. Those skilled in the art will appreciate that many permutations can be achieved and in fact are intended to be within the scope of the invention. All references and citations through the specification are hereby incorporated by reference expressly.

EXAMPLE 1 Plasmids were designed using the multiple cloning site of pBlueScriptIIKS + from Stratagene. (To facilitate the cloning of the different combination of elements). The cligonucleotides containing the sequence of the elements were synthesized with restriction endonuclease sites at the ends. In this way the elements could be added or removed and replaced as necessary. GUS and Luciferase were used for the reporter genes. For transient assays, plasmid DNA was introduced into intact 3 day old corn seedbeds by particle bombardment. After 16 hours of incubation at 25 ° C in the dark, expression was assayed by measuring the enzymatic activity of the GUS in root and shoot extracts of each seedbed to determine if any preferred tissue expression could be demonstrated. The activity of the GUS was measured using a light test kit for the GUS from Tropix (47 Wiggins Avenue, Bedford, MA 01730). Constructs were introduced that give high levels of expression in a cell line to produce stable transformants. Those stable transformants (TO) were tested by PCR to determine the presence of the GUS gene by MUG assay (4-methylumbelliferyl-glucuronide) to quantify the level of activity of the GUS protein that is produced. When the plants were ready to be transferred to the greenhouse, they were tested histochemically with X-gluc to determine where the GUS product began to be synthesized. Plants that showed preferred expression levels grew in the greenhouse until stage V6.

EXAMPLE 2 Construction of Plasmids Containing the Heart or Syn II Promoter Standard molecular biology techniques were carried out according to Maniatis et al., (1982) Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. All plasmids used in the invention can be prepared according to the specification guidelines by one skilled in the art without undue experimentation using materials already available in the art. 6 TCGACACTGC AGCTCTAGGG ATGGTAGCGC AGGGTCGCGTA GGACGTATT TATAGCCGCT CGAGTG-3: oligos N306 SEQ ID NO were synthesized 'and N307 SEQ ID NO: 7 GATCCACTCG AGCGGCTATA AATACGTACC TACGACCCT GCGCTACCAT CCCTAGAGCT GCAGTG-3' according to the guidelines in an automated DNA synthesizer (such As the DNA Synthesizer of Applied Biosystems Inc. (Model 380B), these automated synthesizers are commercially available.The oligos were then ligated to the BamHI fragment of the pBlueScriptlIKSt plasmid comprising the β-glucuronidase gene interrupted by the intronic region of intron 1 Maize ADH1 A map of a plasmid incorporating both the core promoters or Syn II core and the upstream element is depicted in Figure 4. Various other modalities are shown in other plasmids described in Figure 5. The numbers of the plasmids they are shown to the right of each diagram of the promoter with the corresponding legend placed below the diagrams The upper diagram shows the complete transplant transcriptional unit with the following diagrams focusing on the salient differences between the promoters of the Heart or nucleus of the 35S and the Syn II. The legend shows the number and nature of the different subelements of the promoter, the sequence if it is relatively short, the source of the element and the position in relation to the transcription site. The heart or nucleus promoter sequence consists of 35 base pairs with enzymatic sites upstream of a TATA block and a start of transcription with 10 to 15 base pairs downstream. The upstream elements (binding sites of Gal 4, Rsyn, AT-GBL etc.) were fused to the heart or nucleus sequence with the ADH1 intron and different marker genes (LUC or GUS) and proved to be functional in transient assays ( Figure 6) and plants stably transformed with Rsyn (Figure 7).

Example 3 Construction of the Upstream Element Rs? N7 Fused to the Heart Promoter or Syn II Nucleus that results in the P5903 and P6086 Plasmids The oligos to construct the promoter subelement Rsyn7 N1965: (SEQ ID NO: 8) GATCCTATGA CGTATGGTAT GACGTGTTGTT CAAGATGATG ACTT AAASN TACCTATGAC GTATGGTATG ACGTGTGTCG ACTGATGACT TA and (SEQ ID NO: 9) GATCTAAGTC ATCAGTCGAC ACACGTCATA CCATACGTCA TAGGTAGGTT TGAAGTCATC ATCCTTGAACA CACGTCATAC CATACGTCA TAG were synthesized as described earlier and cloned into the BainH1 site of plasmid pBlueScriptIIKS +. The oligos were annealed or matured and cloned into a P3398 plasmid upstream of the heart sequence or Syn II core and resulted in several versions of the original Rsyn7 sequence due to spontaneous deletions. The Rsyn7-2 version involved the deletion of a single base, which resulted in a 3X reiterative TGACG motif upstream of the heart promoter or Syn II core (Rsyn7 :: LUC, P5903). The sequence encoding the LUC was replaced by the sequence encoding the GUS to produce the Rsyn7 construct P6086. P6086 was introduced later in the transgenic maize resulting in high levels of constitutive activity in four of the six active events examined (Figure 7). The progeny of TO plants of various transformation events were examined and the activity of the GUS fluctuated from the 400 PPM (micrograms of GUS / GFW enzyme in 7-day seedling root tissue), Figure 8. Those TO and TI plants generally they produced a GUS 4X-10X activity greater than the plants that contain the ubiquitin reporter gene:: GUS. Thus, starting from the above, it can be seen that the invention achieves at least all of its objectives.

EXAMPLE 4 Transformation and Expression with Heart Promoter or Syn II Nucleus and / or Upstream Element Rsyn7 Using the transient bombardment assays, the promoter sequence of the heart or Syn II core was compared against the heart or nucleus sequence 35S either alone or in set with numerous activation elements. Figure 6 is a description of the transient assay data using plasmids incorporating the promoter sequences of the invention and shows transient GUS or LUC activity in three-day maize roots or BMS calluses bombarded with chimeric promoter constructs:: GUS or LUC. The -33 CaMV35S in the promoter versions of the Heart or Núcle Syn II of the synthetic promoter constructs :: GUS (or LUC) were bombarded in three-day maize roots (or BMS callus cultured according to what was described above) and they were tested to determine the activity of the enzyme twenty hours after the bombings. The data shown are the crude enzyme units from a compilation of at least three experiments and have not been normalized in any way due to the inherent variability of the transient assays. Control plasmids 1654 and 3537 are the LUC constructs tested on corn bms callus. There is a difference of approximately 4 to 20 times in the transient activity between the versions of the Heart or Nucleus of 35S and Syn II. The Y axis is in logarithmic scale. Both core or core promoters were directed to a construct containing GUS (Figures 4 and 5) and generated a basal level of activity (Figure 6). However, when the activating elements were placed upstream of the TATA motif, the Heart or Syn II Nucleus generally provided higher levels of activity (2-4 times better) in corn cells than when the activating elements were placed upstream of the heart. or 35S nucleus (Figure 6).

The sequence of the Heart or Nucleus Syn II has been shown to enhance the activity in plants transformed in a stable manner. In addition, with certain activating sequences upstream, the activity levels of the TATA element in stably transformed corn plants reached levels ten times higher than the corn ubiquitin constructs which produce extremely high levels of activity. Figures 7 and 8 show the GUS activity levels of tissues isolated from TO plants in the VT stage and corn tissue from IT seedlings, respectively. These data demonstrate that this sequence of the heart or nucleus can participate in the potentiation of very high levels of activity as a functional partner of the active chimeric promoters. Figure 7 shows the activity of Rsyn:: GUS (6086) in TO corn plants. Plants in the VT stage with post-pollinated ears of 3 to 8 days were dissected and tested to determine GUS activity. Figure 7A describes the expression of GUS in the designated tissues. Figure 7B describes a scheme of corn plants with the indicated measurement sites. The plants of the TO events that demonstrated a range of activities with the Rsyn7 promoter were tested. Again the logarithmic scale must be noted. The activity interval for the UBI :: GUS plants was indicated on the right side of the graph as a comparison. These data demonstrate that the Rsyn7 promoter can potentiate activity up to ten times above the levels of the ubiquitin promoter which still shows little preference for tissues making the Rsyn7 suitable as a strong constitutive promoter. Figure 8 describes the GUS activity in root segments of a segregating population of transgenic IT seedlings of corn containing the construct Rsyn7 :: GUS (6086) or UBI :: GUS (3953). 1 cm root segments of transgenic maize seedlings were dissected from six to seven days, weighed and tested for GUS using light equipment for GUS. The activity was represented as parts per million of fresh weight. The activity of the root in several IT plants containing the Rsyn7 :: GUS promoter show higher activity than many of the activity levels produced by the UBI promoter. These are consistent with the data of a transgenic TO plant. Activity levels in young leaves containing Rsyn7 :: GUS are also much higher than the activity levels of young leaves containing UBI :: GUS (data not shown). The Heart or Syn II Nucleus sequence showed to work well with a variety of upstream elements including GAL 4 binding sites, Rsyn7 elements, GBL elements, etc. Figure 9 shows the GUS expression of three synthetic promoters in corn TO gene transgenic plants. The dissected tissues (See Figure 7B) of transgenic plants TO from the VT stage harboring the constructs of Rsyn7 (Rsyn), Atsyn or the core promoter Syn II (syn-core) promoter constructs:: GUS were quantitatively assayed to determine the activity of the GUS. Each circle represents an average of the tissue activity of transgenic corn plants of a single transformation event. The TGACG motif corresponds to the sequence of Rsyn7 and the motif "t-com" refers to the composite element similar to the consensus AT, Atcom, by W. Gurley, et al., 1993. In: Control of Plant Gene Expression, ed. by Desh Pal Verma, CRC press, Boca Raton, FL, pp. 103-123 Syn-heart refers to the promoter sequence of the Heart or Syn II Nucleus that contains the TATA element and the start of transcription.

LIST OF SEQUENCES GENERAL INFORMATION: (i) APPLICANT: Pioneer Hi-Bred International, Inc. (ii) TITLE OF THE INVENTION: Synthetic Promoter of the Heart of a Plant and Upstream Regulating Element (iii) SEQUENCE NUMBER: 10 (iv) ADDRESS FOR CORRESPONDENCE: (A) ADDRESS: Pioneer Hi-Bred International, Inc.

(B) STREET: 7100 N. W. 62nd Avenue, Dar in Building (C) CITY: Johnston (D) STATE: Iowa (E) COUNTRY: United States (F) ZIP CODE: 50131 (v) COMPUTER LEGIBLE FORM: (A) TYPE OF MEDIA: Flexible disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: Patentln Relay # 1.0, Version # 1.30 (vi) DATA OF THE CURRENT APPLICATION: (A) APPLICATION NUMBER: (B) DATE OF SUBMISSION: (C) CLASSIFICATION: (viii) INFORMATION FROM THE MANDATORY / AGENT: (A) NAME: JERVIS, Herbert H .; SWEENEY, Patricia A .; BOBROWICZ, Donna, RAN David. (B) REGISTRATION NUMBER: (ix) INFORMATION FOR TELECOMMUNICATION: (A) TELEPHONE: 515-334-4468 (B) TELEFAX: 515-334-6883 (2) INFORMATION FOR SEQ ID NO: l: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 72 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (iii) HYPOTHETIC: NO (iv) ANTICIPATION: NO (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: l: GGATCCACTC GñGCGGCTAT AñATACGTAC CTñCGCACGC TGCGCTñCCA TCCCGAGCAC 60 TGCAGTGTCG AC 72 (2) INFORMATION FOR SEQ ID NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 96 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (iii) HYPOTHETICAL: NO (iv) ANTICIPATION: NO (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2 GGATGCTATG CGTATGGTAG GACGTGTGTT CAAGAGGAGG ACTTCA? ACC TACCTATGAC 60 GTATGGTATG ACGTGTGTCG ACTGATGACT TAGATC 96 (2) INFORMATION FOR SEQ ID NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 18 base pairs (B) TYPE; nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (iii) HYPOTHETICAL: NO (iv) ANTICIPATION: NO (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3: TMAWftWA-TY YTCA2M? A 18 (2) INFORMATION FOR SEQ ID NO: 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 40 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTICIPATION: NO (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4 CCTCTATATA AGCAAGTTCA TTTCATTTGG AGAGG ACG 40 (2) INFORMATION FOR SEQ ID NO: 5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 5 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SUIT: NO (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 5: TGAGC (2) INFORMATION FOR SEQ ID NO: 6: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 66 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (iii) HYPOTHETICAL: NO (iv) ANTICIPATION: NO (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6: TCGACACTGC AGCTCTAGGG ATGGTAGCGC AGGGTGCGTA GGTACGTATT TATAGCCGCT 60 CGAGTG 66 (2) INFORMATION FOR SEQ ID NO: 7: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 66 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (iii) HYPOTHETICAL: NO (iv) ANTICIPATION: NO (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 7: GATCCACTCG AGCGGCTATA AATACGTAX TACGCACCCT GCGCTACCAT CCCTAGAGCT 60 GCAGTG 66 (2) INFORMATION FOR SEQ ID NO: 8: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 92 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (iii) HYPOTHETICAL: NO (iv) ANTICIPATION: NO (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 8: GATCCTATGA CGTATGGTAT GACGTGTGTT CAAGATGATG ACTTCMñCC TACCTATGAC 60 GTATGGTATG ACGTGTGTCG ACTGATGACT TA 92 (2) INFORMATION FOR SEQ ID NO: 9: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 92 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 9: GATCTAAGTC ATCAGTCGAC ACACGTCATA CCATACGTCA TAGGTAGGTT TGAAGTCATC 60 ATCTTCAACA CACGTCA? AC CATACGTCAT AG 92 (2) INFORMATION FOR SEQ ID NO: 10: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 72 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (iii) HYPOTHETICAL: NO (iv) ANTICIPATION: NO (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 10: GGATCCACTC GAGCGGCTAT AAATASSTAS ATASSSASSS TSSSSTASSA TCCCGAGCAC 60 TGCAGTGTCG AC 72 It is noted that in relation to this date, the best method known by the applicant to carry out the present invention, is the conventional one for the manufacture of the objects to which it refers. Having described the invention as above, the following are claimed as property:

Claims (24)

1. A promoter sequence of synthetic DNA plant,. the sequence is characterized in that it comprises: a TATA motif; a transcription start site; and a region between the TATA motif and the start site that is at least 64% GC rich.

2. The promoter according to claim 1, characterized in that the promoter sequence is SEQ ID NO: 10.

3. The promoter according to claim 1, characterized in that the promoter sequence is SEQ ID NO: 1.

4. An expression cassette, characterized in that it comprises: a synthetic promoter comprising a TATA motif; a transcription start site and a region between them that is at least 64% GC rich; a structural gene operably linked to the promoter; and a polyadenylation signal of the final transcription site.

5. The expression cassette according to claim 4, characterized in that the promoter sequence is SEQ ID NO: 1.

6. The expression cassette according to claim 4, characterized in that the sequence of the promoter is SEQ ID NO: 10.

7. The expression cassette according to claim 4, characterized in that it also comprises an upstream element operatively linked to the promoter so that the transcription is increased or amplified.

8. The expression cassette according to claim 1, characterized in that the upstream element is SEQ ID NO: 2.

9. A nucleic acid vector, characterized in that it comprises the promoter according to claim 1, 2 or 3. operatively linked to a structural gene.

10. The vector according to claim 9, characterized in that the vector is a cloning vector.

11. The vector according to claim 9, characterized in that the vector is an expression vector.

12. The vector according to claim 9, characterized in that it also comprises a marker gene for the selection of transformed cells.

13. The vector according to claim 12, characterized in that the marker gene is a gene for resistance to antibiotics.

14. The vector according to claim 9, characterized in that it further comprises a polyadenylation signal.

15. The vector according to claim 9, characterized in that the vector further comprises an upstream element operably linked to the promoter.

16. The vector according to claim 15, characterized in that the upstream element is SEQ ID NO: 2.

17. A prokaryotic or eukaryotic host cell characterized as a scaffold was transformed with the nucleic acid vector according to claim 9.

18. A transgenic plant, characterized in that it comprises: a plant cell or ancestor thereof, which has been transformed with the vector according to claim 9.

19. An upstream synthetic element, characterized in that it has a sequence of the SEQ ID NO: 2.

20. An expression cassette, characterized in that it comprises: a promoter sequence; a structural gene operably linked to the promoter sequence; a polyadenylation signal; and an upstream synthetic element homologous to SEQ ID NO: 2 operably linked to the promoter so that expression is amplified or enhanced.

21. The expression cassette according to claim 20, characterized in that the synthetic promoter sequence is SEQ ID NO: 2.

22. A nucleic acid vector, characterized in that it comprises the expression cassette according to claim 20.

23. The vector according to claim 22, characterized in that the upstream synthetic element is SEQ ID NO: 2.

24. A prokaryotic or eukaryotic host cell, characterized in that it was transformed with the vector according to claim 22.