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WO1999060022A1 - Regulateurs de canal calcium - Google Patents

Regulateurs de canal calcium Download PDF

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Publication number
WO1999060022A1
WO1999060022A1 PCT/US1999/010821 US9910821W WO9960022A1 WO 1999060022 A1 WO1999060022 A1 WO 1999060022A1 US 9910821 W US9910821 W US 9910821W WO 9960022 A1 WO9960022 A1 WO 9960022A1
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Prior art keywords
nucleotide
protein
seq
amino acid
set forth
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Inventor
William L. Pak
Chenjian Li
Chaoxian Geng
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Purdue Research Foundation
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Purdue Research Foundation
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Priority to AU39969/99A priority Critical patent/AU3996999A/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants

Definitions

  • Ca 2+ is both a critical molecule for homeostasis and an intracellular signaling molecule in many physiological processes such as muscle contraction, glandular secretion, transcriptionai activation, and neurotransmitter release [Berridge, M.J. (1993) Na ture 361: 315-325; Berridge, M.J. (1995) Biochem . J. , 312:1- 11; Clapham, D.E. (1995) Cell 80:259-268; Clapham, D.E. (1996) Neuron 16:1069-1072].
  • Mobilization of Ca 2+ is also involved in the immune response, such as autoimmune diseases and generation of an immune response after organ transplantation.
  • neuronal degeneration diseases such as Alzheimer's is caused by excessive Ca 2+ mobilization.
  • the cytosolic [Ca 2+ ]i is maintained at about 10-100 nM, but during stimulation the cytosolic [Ca2+] can rise rapidly to micromolar ranges.
  • the widely used signal transduction pathway the receptor-based, G protein-coupled, PLC-IP3 cascade,
  • Ca 2+ as a key signaling molecule.
  • excitable cells such as muscle cells, Purkinje neurons and Drosophila photoreceptor cells, as well as in nonexcitable cells such as mast cells and lymphocytes
  • extracellular stimuli activate receptors on the cell membrane, which in turn activate receptor-coupled G proteins.
  • the activated G protein then activates phospholipase C to hydrolyze PIP2 to IP3 and DAG.
  • IP3 binds to IP3
  • the Ca 2+ release from intracellular stores triggers, through unknown molecules and mechanisms, Ca 2+ influx from the extracellular space into the cell via Ca 2+ selective channels on the plasma membrane (reviewed by Berridge,
  • InaD binds to the Drosophila Trp protein.
  • InaD is a soluble protein with PDZ domains which are known to be important for protein/protein interaction and anchoring ion channels [Kim, E. et al. (1995) Na ture 378:85-88; Kim, E. et al. (1996) Neuron 17:103- 113; Kormau, H.C. et al. (1995) Science 269:1737-1740].
  • InaD has been shown by co-immunoprecipitation and gel- overlay assays to bind physically to the trp protein (Shieh, B. and Zhu, M.
  • InaC has been identified as an eye-specific protein kinase C (Smith, D.P. et al. (1991) Science 254:1478-1484). InaC binds to InaD, suggesting that InaD could be one of the substrates of InaC-mediated phophorylation (Huber et al. (1996) above).
  • InaF A novel protein, InaF, that functions in regulation of calcium ion entry into a cell, has been discovered. Accordingly, in one aspect of the invention, purified InaF proteins are provided.
  • isolated nucleic acid molecules that encode InaF proteins are provided.
  • the nucleic acid molecules may be incorporated into a vector to form a recombinant nucleic acid molecule.
  • recombinant nucleic acid molecules may be introduced into a host cell .
  • methods of expressing InaF proteins include transforming a host cell with a nucleotide sequence encoding a protein that functions in regulating calcium ion entry into a cell as provided herein, and culturing the transformed host cells under conditions effective in achieving expression of InaF proteins.
  • the proteins may then be purified by conventional techniques.
  • FIG. 1 depicts the cross scheme of single P local hopping mutagenesis for P69 and trol . Asterisks indicate chromosomes into which the P element could have transposed.
  • FIG. 2 depicts electroretinogram (ERG) recordings from inaF mutants as discussed in Example 2.
  • the top trace is an ERG of strong allele, ina plllx
  • the bottom trace is an ERG of weak allele, inaF 112 .
  • Stimulus duration was 4 seconds.
  • FIG. 3 depicts intracellularly recorded photoreceptor potentials as discussed in Example 2.
  • the voltage responses to 8 second light stimuli were measured in wild-type flies, trp P301 , and inaF plllx .
  • FIG. 4 depicts intracellularly recorded receptor potentials showing photoreceptor response latency as discussed in Example 2. Flies were dark-adapted for 2 minutes .
  • FIGS. 5A-B are views of photoreceptors obtained by transmission electron microscopy as discussed in
  • FIG. 5A left panel, depicts photoreceptors of wild-type flies;
  • FIG. 5A right panel, depicts photoreceptors of 19 day old inaF;bw;st reared light/dark;
  • FIG. 5B depicts an enlarged view of the region indicated by the arrow in FIG. 5A.
  • FIG. 6 depicts the cross-scheme for remobilization of the P insertion in inaF P105p , as discussed in Example 4.
  • FIG. 7 depicts a cytogenetic map of the inaF mutation as discussed in Example 5.
  • Df (1) HA85 (inaF “ ) , Df (l)m259-4 (inaF “ ) and Df (1) (inaF “ ) are deficiency stocks as discussed in Example 5.
  • FIG. 8 depicts a genomic Southern analysis as discussed in Example 6. Genomic DNA was purified and digested by EcoRI (lanes 1-4), BamHI (lanes 5-8), and Hindlll (lanes 9-12), and loaded on a 0.7% agarose gel in the following order: wild-type (lanes 1, 5 and 9) ; mutator 3B (lanes 2, 6 and 10) ; mutator 3B1-2
  • FIG. 9 depicts a genomic Southern analysis as discussed in Example 6. Genomic DNA was purified and digested by EcoRI (lanes 1 and 2), BamHI (lanes 3 and 4), and Hindll (lanes 5 and 6), and loaded on a 0.7% agarose gel in the following order: mutator 3B (lanes 1, 3 and 5), and inaF pl05p (lanes 2, 4 and 6).
  • FIG. 10 depicts a polytene chromosome after an in situ hybridization procedure performed as described in Example 7.
  • the signal (arrowhead) detected on the polytene chromosome was localized in the 10 C2-E3 region of the X chromosome, which was consistent with the results obtained by using pCaSpeR3 and fragment 4 of A23 as probes.
  • FIG. 11 depicts a Northern blot probed with cDNA #1 insert as discussed in Example 7.
  • the lanes were loaded, from left to right, with polyA+ RNA from wild- type head, wild-type body, inaF ( inaF 1059 ) head and eya head.
  • RP49 a ribosomal protein universally expressed in all tissues.
  • FIG. 12 depicts restriction maps of inaF cDNA and of the corresonding genomic region in the A23 clone and three inaF mutants.
  • the unfilled inverted triangle in the inaF 105p map identifies the P element insertion.
  • the empty spaces to the right and left of the P insertion site in the inaF P106x and inaF 111 maps, respectively, represent the deletions caused by imprecise excision of the P element.
  • the broken dotted line indicates the extent of the intron, and the open rectangle identifies the open reading frame.
  • a composite genomic map at the top shows EcoRI sites (R) and the sizes of EcoRI fragments.
  • FIG. 13 depicts a Western blot analysis of null ( inaF 106 *, trp P3i3 ) and near-null ( inaF 9105? , trp p301 ) inaF and trp mutants, and wild-type and revertant controls.
  • the seven lanes were loaded with total protein prepared from (lanes 1-7) : wild-type heads, wild-type bodies, revertant heads, trp 301 heads, trp P343 heads, inaf pl05p heads, and inaF 5106 heads.
  • DESCRIPTION OF THE PREFERRED EMBODIMENTS DESCRIPTION OF THE PREFERRED EMBODIMENTS
  • a novel calcium channel regulator protein, InaF has been identified in the fruit fly, Drosophila melanogaster. Accordingly, the present invention provides purified InaF protein.
  • the invention further provides isolated nucleic acid molecules that include nucleotide sequences encoding functional InaF proteins. Recombinant nucleic acid molecules are also provided that include the novel inaF nucleotide sequence. The nucleic acid molecules may be incorporated in a host cell.
  • methods of expressing functional InaF protein are also provided.
  • novel, purified InaF proteins are provided that function in regulating cellular influx of calcium ions.
  • the InaF polypeptides are substantially pure (i.e., InaF proteins are essentially free, e.g., at least about 95% free, from other proteins with which they naturally occur) .
  • the amino acid sequence of an InaF protein, originally found in Drosophila melanogaster is set forth in SEQ ID:1.
  • InaF protein is used to refer generally to a protein having the features described herein and a preferred example includes a polypeptide having the amino acid sequence of SEQ ID NO:l. Also included within this definition, and in the scope of the invention, are variants of the polypeptide which function in regulating calcium ion movement into a cell, as described herein.
  • an amino acid sequence isolated from another species may differ to a certain degree from the sequences set forth in SEQ ID NOS : 1 and 2, and yet have similar functionality with respect to catalytic and regulatory function.
  • Amino acid sequences comprising such variations are included within the scope of the present invention and are considered substantially or sufficiently similar to a reference amino acid sequence.
  • the identity between amino acid sequences that is necessary to maintain proper functionality is related to maintenance of the tertiary structure of the polypeptide such that specific interactive sequences will be properly located and will have the desired activity.
  • the present invention be limited by any theory by which it achieves its advantageous result, it is contemplated that a polypeptide including these interactive sequences in proper spatial context will have good activity, even where alterations exist in other portions thereof.
  • an InaF protein variant is expected to be functionally similar to that set forth in SEQ ID N0:1, for example, if it includes amino acids which are conserved among a variety of species or if it includes non-conserved amino acids which exist at a given location in another species that expresses a functional InaF protein.
  • Another manner in which similarity may exist between two amino acid sequences is where a given amino acid of one group (such as a non-polar amino acid, an uncharged polar amino acid, a charged polar acidic amino acid or a charged polar basic amino acid) is substituted with another amino acid from the same amino acid group.
  • a given amino acid of one group such as a non-polar amino acid, an uncharged polar amino acid, a charged polar acidic amino acid or a charged polar basic amino acid
  • the uncharged polar amino acid serine may commonly be substituted with the uncharged polar amino acid threonine in a polypeptide without substantially altering the functionality of the polypeptide. If one is unsure whether a given substitution will affect the functionality of the enzyme, then this may be determined without undue experimentation using synthetic techniques and screening assays known in the art.
  • inventive amino acid sequences similar to the amino acid sequences set forth herein that have at least about 30% identity thereto and function in regulating cellular influx of calcium ions.
  • inventive amino acid sequences have at least about 50% identity to these sequences, further preferably at least about 70% identity, more preferably at least about 80% identity and most preferably at least about 90% identity.
  • Percent identity may be determined, for example, by comparing sequence information using the advanced BLAST computer program, version 2.0.8, available from the National Institutes of Health.
  • the BLAST program is based on the alignment method of Karlin and Altschul, Proc. Natl . Acad. Sci . USA 87:2264-68 (1990) and as discussed in Altschul, et al., J. Mol . Biol . 215:403-10 (1990); Karlin and Altschul, Proc. Na tl . Acad. Sci . USA 90:5873-7 (1993); and Altschul et al. (1997) Nucleic Acids Res . 25:3389-3402.
  • the BLAST program defines identity as the number of identical aligned symbols (i.e., nucleotides or amino acids) , divided by the total number of symbols in the shorter of the two sequences .
  • the program may be used to determine percent identity over the entire length of the proteins being compared.
  • the program also uses an SEG filter to mask-off segments of the query sequence as determined by the SEG program of Wootton and Federhen (1993) Computers and Chemistry 17:149-163.
  • isolated nucleic acid molecules originally isolated from Drosophila melanogaster, are provided that encode a functional InaF protein that functions in regulating calcium ion entry into cells.
  • the nucleotide sequences are set forth in SEQ ID NOS : 1 and 2. It is preferred that the nucleotide sequence includes nucleotides spanning nucleotides 301 to 1036 in SEQ ID NO:l and nucleotides spanning nucleotides 528 to 1250 in SEQ ID NO: 2. It is not intended that the present invention be limited to these exemplary nucleotide sequences, but include sequences having substantial similarity thereto and sequences which encode variant forms of functional InaF protein as discussed above and as further discussed below.
  • isolated nucleic acid is intended to refer to nucleic acid which is not in its native environment.
  • the nucleic acid is separated from other contaminants that naturally accompany it, such as proteins, lipids and other nucleic acid sequences.
  • the term includes nucleic acid which has been removed or purified from its naturally- occurring environment or clone library, and further includes recombinant or cloned nucleic acid isolates and chemically synthesized nucleic acid.
  • nucleotide sequence is intended to refer to a natural or synthetic linear and sequential array of nucleotides and/or nucleosides, including deoxyribonucleic acid and ribonucleic acid, and derivatives thereof.
  • encoding and coding refer to the process by which a nucleotide sequence, through the mechanisms of transcription and translation, provides the information to a cell from which a series of amino acids can be assembled into a specific amino acid sequence to produce a functional polypeptide, such as, for example, an active enzyme or other protein that has a specific function.
  • the process of encoding a specific amino acid sequence may involve DNA sequences having one or more base changes (i.e., insertions, deletions, substitutions) that do not cause a change in the encoded amino acid, or which involve base changes which may alter one or more amino acids, but do not eliminate the functional properties of the polypeptide encoded by the DNA sequence. It is therefore understood that the invention encompasses more than the specific exemplary nucleotide sequence of inaF. For example, nucleic acid sequences encoding variant amino acid sequences, as discussed above, are within the scope of the invention.
  • Modifications to a sequence such as deletions, insertions, or substitutions in the sequence, which produce "silent" changes that do not substantially affect the functional properties of the resulting polypeptide molecule are expressly contemplated by the present invention.
  • alterations in a nucleotide sequence which reflect the degeneracy of the genetic code, or which result in the production of a chemically equivalent amino acid at a given site are contemplated.
  • a codon for the amino acid alanine, a hydrophobic amino acid may be substituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine.
  • changes which result in substitution of one negatively charged residue for another such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine, can also be expected to produce a biologically equivalent product.
  • Nucleotide changes which result in alteration of the N-terminal and C-terminal portions of the encoded polypeptide molecule would also not generally be expected to alter the activity of the polypeptide. In some cases, it may in fact be desirable to make mutations in the sequence in order to study the effect of alteration on the biological activity of the polypeptide. Each of the proposed modifications is well within the routine skill in the art.
  • the nucleotide sequence has substantial similarity to the sequence set forth in SEQ ID:1 or SEQ ID: 2, preferably the sequence spanning nucleotides 314 to 1036 in SEQ ID:1 and preferably the sequence spanning nucleotides 528 to 1250 in SEQ ID: 2, and variants described herein.
  • the term "substantial similarity" is used herein with respect to a nucleotide sequence to designate that the nucleotide sequence has a sequence sufficiently similar to a reference nucleotide sequence that it will hybridize therewith under moderately stringent conditions. This method of determining similarity is well known in the art to which the invention pertains.
  • moderately stringent conditions are defined in Sambrook et al., Molecular Cloning: A Labora tory Manual , 2nd ed. Vol. 1, pp. 101-104, Cold Spring Harbor Laboratory Press (1989) as including the use of a prewashing solution of 5X SSC (a sodium chloride/sodium citrate solution), 0.5% sodium dodecyl sulfate (SDS) , 1.0 mM ethylene diaminetetraacetic acid (EDTA) (pH 8.0) and hybridization and washing conditions of 55°C, 5x SSC.
  • 5X SSC sodium chloride/sodium citrate solution
  • SDS sodium dodecyl sulfate
  • EDTA 1.0 mM ethylene diaminetetraacetic acid
  • hybridization and washing conditions 55°C, 5x SSC.
  • inventive polynucleotide is that it must encode a polypeptide having similar functionality to the InaF protein described herein, i.e., functioning to regulate influx
  • nucleotide sequences having selected percent identities to specified regions of the nucleotide sequence set forth in SEQ ID:1 are provided.
  • nucleotide sequences are provided that have at least about 60% identity, more preferably at least about 80% identity, and most preferably at least about 90% identity, to a nucleotide sequence of substantial length within the nucleotide sequence from nucleotides 314 to 1036 set forth in SEQ ID:1.
  • such length may be 100, 200 or 400 nucleotides, or may be the entire sequence from nucleotides 314 to 1036 of SEQ ID:1.
  • nucleotide sequence from nucleotide 314 to 1036 set forth in SEQ ID:1 encodes a protein that functions in regulating calcium entry into cells.
  • the percent identity may be determined, for example, by comparing sequence information using the advanced BLAST computer program, version 2.0.8., as described above with reference to amino acid identity.
  • a suitable DNA sequence may be obtained by cloning techniques using cDNA libraries.
  • cDNA libraries are available commercially or may be constructed using standard methods known in the art.
  • Suitable nucleotide sequences may be isolated from DNA libraries obtained from a wide variety of species by means of nucleic acid hybridization or polymerase chain reaction (PCR) procedures, using as probes or primers nucleotide sequences selected in accordance with the invention, such as those set forth in SEQ ID:1, nucleotide sequences having substantial similarity thereto, or portions thereof.
  • nucleic acid sequences encoding a functional InaF protein may be constructed by recombinant DNA technology, for example, by cutting or splicing nucleic acids using restriction enzymes and DNA ligase.
  • nucleic acid sequences may be constructed using chemical synthesis, such as solid-phase phosphoramidate technology. PCR may be used to increase the quantity of nucleic acid produced.
  • the particular nucleic acid sequence is of a length which makes chemical synthesis of the entire length impractical, the sequence may be broken up into smaller segments which may be synthesized and ligated together to form the entire desired sequence by methods known in the art.
  • InaF polypeptides functioning in regulating calcium ion entry into a cell and having the amino acid sequences encoded by nucleotide sequences having substantial similarity to the nucleotide sequences described above are also provided.
  • recombinant nucleic acid molecules, or recombinant vectors are provided.
  • the nucleic acid molecules include a nucleotide sequence encoding a functional InaF protein.
  • the nucleotide sequence has substantial similarity, as defined above, to the nucleotide sequence set forth in SEQ ID:1 or SEQ ID: 2, preferably the sequence spanning nucleotides 314 to 1036 in SEQ ID:1 or the identical sequence in SEQ ID: 2 spanning nucleotides 528 to 1250.
  • the protein produced has the amino acid sequence set forth in SEQ ID:1, or variants thereof as described above.
  • Recombinant vectors may be constructed by incorporating the desired nucleotide sequence within a vector according to methods well known to the skilled artisan and as described for example, in Maniatis et al . , Molecular Cloning: A Labora tory Manual , Cold Springs Laboratory, Cold Springs Harbor, New York (1982) .
  • a wide variety of vectors are known that have use in the invention.
  • various plasmid and phage vectors are known that are ideally suited for use in the invention.
  • pGEM, pBluesript, EMBL and ⁇ Gtll may be used in the invention.
  • the desired recombinant vector may be constructed by ligating DNA linker sequences to the 5' and 3' ends of the desired nucleotide insert, cleaving the insert with a restriction enzyme that specifically recognizes sequences present in the linker sequences and the desired vector, cleaving the vector with the same restriction enzyme, mixing the cleaved vector with the cleaved insert and using DNA ligase to incorporate the insert into the vector as known in the art.
  • the vectors may include other nucleotide sequences, such as those encoding selectable markers, including those for antibiotic resistance or color selection.
  • the vectors also preferably include a promoter nucleotide sequence.
  • the desired nucleic acid insert is preferably operably linked to the promoter.
  • a nucleic acid is "operably linked" to a another nucleic acid sequence, such as a promoter sequence, when it is placed in a specific functional relationship with the other nucleic acid sequence.
  • the functional relationship between a promoter and a desired nucleic acid insert typically involves the nucleic acid and the promoter sequences being contiguous such that transcription of the nucleic acid sequence will be facilitated.
  • Two nucleic acid sequences are further said to be operably linked if the nature of the linkage between the two sequences does not (1) result in the introduction of a frame-shift-mutation; (2) interfere with the ability of the promoter region sequence to direct the transcription of the desired nucleotide sequence, or (3) interfere with the ability of the desired nucleotide sequence to be transcribed by the promoter sequence region.
  • the promoter element is generally upstream (i.e., at the 5' end) of the nucleic acid insert coding sequence.
  • promoters are known in the art, including cell-specific promoters, inducible promoters, and constitutive promoters.
  • the promoters may further be selected such that they require activation by activating elements known in the art, so that production of the protein encoded by the nucleic acid sequence insert may be regulated as desired.
  • the vectors may further include other regulatory elements, such as enhancer sequences, which cooperate with the promoter to achieve transcription of the nucleic acid insert coding sequence.
  • enhancer sequences nucleotide sequence elements which can stimulate promoter activity in a cell, such as a bacterial or eukaryotic host cell.
  • the vectors may include another nucleotide sequence insert that encodes a protein that may aid in purification of the desired protein encoded by the desired nucleotide sequence.
  • the additional nucleotide sequence is positioned in the vector such that a fusion, or chimeric, protein is obtained.
  • an InaF protein may be produced having at its C-terminal end linker amino acids, as known in the art, joined to the other protein.
  • the additional nucleotide sequence may include, for example, the nucleotide sequence encoding glutathione-S-transferase (GST) . After purification procedures known to the skilled artisan, the additional amino acid sequence is cleaved with an appropriate enzyme.
  • the additional amino acid sequence is that of GST
  • thrombin is used to separate the InaF protein from GST.
  • the InaF protein may then be isolated from the other proteins, or fragments thereof, by methods known in the art.
  • the inventive recombinant vectors may be used to transform a host cell. Such methods include, for example, those described in Maniatis et al . , Molecular Cloning: A Labora tory Manual , Cold Springs Laboratory, Cold Springs Harbor, New York (1982) . Once the desired nucleic acid has been introduced into the host cell, the host cell may produce the inventive InaF protein, or variants thereof, as described above.
  • a host cell that includes the inventive recombinant vectors described above.
  • a wide variety of host cells may be used in the invention, including prokaryotic and eukaryotic host cells.
  • Bacterial host cells such as Escherichia coli , HB 101 and XL-1 blue may be advantageously used in the present invention.
  • Typical eukaryotic host cells include SF9, S2, NIH 3T3 and NIH 293.
  • the method includes providing a nucleotide sequence described above, or variants thereof, that encodes a functional InaF protein that regulates calcium ion entry into cells, and introducing the nucleotide sequence into a host cell, as described above.
  • the desired nucleotide sequence may be advantageously incorporated into a vector to form a recombinant vector.
  • the recombinant vector may then be introduced into a host cell according to known procedures in the art. Such host cells are then cultured under conditions, well known to the skilled artisan, effective to achieve expression of the InaF polypeptide.
  • the InaF polypeptide may then be purified using conventional techniques.
  • the first inaF mutant was generated through P- element mediated mutagenesis, as described below, on a whi te ' background.
  • the actual eye color of the mutant was light orange because the P element insertion causing the inaF mutation contains a mini -whi te gene.
  • the original inaF mutant was placed in a bw; st background, so that inaF; bw; st flies would have no eye-color pigment.
  • the mutator, 3B was chosen for local hopping mutagenesis, because it has an insertion in 3B1-2, which is very close to 3A3-5 where the P69 gene is localized.
  • This fly has a mutation in an eye-pigment gene whi te, and thus originally has a white eye color background.
  • the actual eye color of 3B is orange, because the fly also carries a P element, pCaspeR3, which has the mini-white gene as a marker.
  • the shades of eye color, from dark red to light lemon, are dependent on where the insertions are. The eye color is darker when the insertion is in the vicinity of a strong enhancer, and the eye color is lighter when the insertion is close to a weaker enhancer. This location-sensitive eye color change is a very good indication of whether the P element has been mobilized to a new place.
  • the mutator, y w/P [lacW] was chosen for random targeting mutagenesis. This fly carries a whi te mutation, and thus has a white eye color background. Its actual orange eye color is from the P[lacW], an engineered P element with the plasmid rescue feature as well as the enhancer trap capability as described in Bier et al., Genes and Development, 3:1273-1287 (1989).
  • the jumpstarter, P3629 carries a functional transposase gene which lacks the end inverted repeats (delta2-3) .
  • the delta2-3 is inserted on the 3rd chromosome, which also carries a visible dominant marker Sb. This visible marker is useful in indicating the presence or absence of the delta2-3.
  • the trol mutation is lethal.
  • females are balanced over FM7, and males carry, by translocation, a 2D-3C segment of the X chromosome on the Y chromosome, which rescues the trol lethality.
  • C (1) RM y w/w/Y fly stock
  • females have a special type of genome that contains two linked X chromosomes. These two linked chromosomes will segregate together. If a male fly is mated to C (1) RM, y w/Y females, all the male offspring will carry the same X chromosome as the PI male.
  • the P element mediated local hopping mutagenesis was undertaken with the aim of isolating lethal troJ alleles or viable ERG mutants (FIG. 1) . There were three generations of crosses before the mutagenesis result could be tested by ERG.
  • Cross I The mutator, 3B, which carries a pCaspeR3 in whi te background was used. Its eye-color is light orange.
  • the jumpstarter stock carries delta2-3 on the third chromosome, which is marked with a dominant marker Sb.
  • 20 mutator males and 20 jumpstarter females were combined.
  • Parent flies in each bottle were transferred after 4-5 days to a new bottle once, and then discarded. All flies were raised at 25°C.
  • Cross II Among the progeny of cross I, virgin females with Sb marker were selected to mate with males from a FMO containing stock. The females carried both the mutator on the X chromosome, and the jumpstarter on the 3rd chromosome. In the germ line cells of these flies, the P element in the mutator could be mobilized to new chromosomal locations because of the transposase activity conferred by delta2-3. In each round of the mutagenesis, 20 bottles of cross II were set up, each containing 20 virgin females and 20 males. Parent flies were transferred once to new food after 4-5 days, and then discarded. All flies were raised at 25 C.
  • Cross III From the progeny of cross II, flies were selected for remobilized pCaspeR3, by selecting for flies with changed eye color shades (presence of whi te ) . Flies were also selected against Sb-marked delta2-3, so that pCaspeR3 insertions would be stabilized.
  • Both male and female progeny of cross II were used: 1) Males were single-mated to C (1) RM, y w/Y females carrying an attached X chromosome to establish stable lines. After 7 days, male parents were scored by an electroretinogram (ERG) as described in Example 2. If the ERG showed a mutant phenotype, the line was saved for further study; and if the ERG were wild type, the line was discarded.
  • ERG electroretinogram
  • Cross II yielded about 2% of offspring that showed changes of eye color, indicating that the P element was mobilized to new chromosomal locations. Approximately 2X10 F 2 flies were scored.
  • Male F 2 flies with eye color changes were single male mated to C(l) RM, y w/Y. Among the offspring of this single-male-mating, all males carried the same X chromosome as the single male parent. 1-2 males of each line were scored by ERG. In 255 such single male mating lines, one was identified as a new mutant and designated as inaF.
  • the electroretinogram is an extracellular measurement of the light-induced responses in the eyes.
  • the ERGs were recorded as described in Pak, W.L. et al . Na ture 222:351-354 (1969).
  • a xenon arc lamp (Oriel) served as the light source with an infrared filter
  • the intracellular recording technique was performed as described in detail by Johnson, E.C. et al. (1986) J. Gen . Physiol . 88 (5) : 651-673. Flies anaesthetized with C02 were mounted on a glass coverslip with myristic acid. A small portion ( ⁇ 10%) of the cornea was cut off with a vibrating razor blade. A thin layer of inert vacuum grease was applied to the cut end to prevent desiccation of the retina. Both the reference and the recording electrodes were inserted into the eye through the cut end of the cornea. The reference electrode was a low resistance glass microelectrode filled with physiological saline and was placed into the retinal layer of the eye.
  • the intracellular recording electrode (FHC Borosil 1.2 mm) was pulled on a vertical Narashige puller, filled with 2 M KCl, and selected for resistance ranging between 30 to 100 mega ohm.
  • the recording electrode was inserted into the retinal layer with a Leitz micromanipulator .
  • Penetration of a photoreceptor was done by a minute forward movement of the electrode and a simultaneous delivery of a brief overdriving negative capacitance current to induce oscillation at the tip of the electrode.
  • Successful penetration of a photoreceptor cell was indicated by a drop in voltage of more than 30 mV as seen on the oscilloscope and a receptor potential of more than 20 mV in response to a bright light stimulus.
  • the preparation was dark adapted for more than 2 minutes before any further experiments.
  • the measured voltage was fed to a WPI preamplifier from which the signals were directed to both an oscilloscope and a digitizer (Digidata 1200, Axon Instrument) .
  • the digitized signals were filtered at 100 Hz and were recorded by Axoscope in a Pentium computer. Analysis
  • the rate of decay was allele-dependent . Strong alleles such as inaF ⁇ caused the receptor potential to decay to base line within 4-5 seconds under bright
  • FIG. 3 shows that the receptor potential of wild-type flies is maintained at a steady state.
  • the latency is defined as the delay between the onset of the light stimulus and the beginning of photoreceptor depolarization. This delay is light intensity dependent and has been interpreted as the time required by the phototransduction pathway to proceed from photoconversion of rhodopsin to the opening of light- activated channels on the plasma membrane. In both trp and inaF ⁇ , the latency was prolonged compared to that of the wild type, and the delay was greater in ina IlF 111 * .
  • inaF compound eyes Retinal degeneration was observed in inaF compound eyes (FIGS. 5A-B) .
  • inaF ⁇ was put in a bw; st background to eliminate eye color.
  • Confocal microscopy and EM were used to examine photoreceptor structures .
  • the photoreceptors in inaF ⁇ ,• bw; st showed no detectable abnormality at 1 day posteclosion, suggesting that they had developed normally.
  • flies raised in a 12 hour-light/12 hour-dark cycle to 19 days posteclosion showed retinal degeneration.
  • Rabdomeres were absent in some ommatidia; most of the rabdomeres were much reduced in size; microvilli (membrane which contains rhodopsin) were disrupted by vacuolized structures; and the base of the microvilli was no longer smooth and regular.
  • inaF ⁇ • bw; st flies were raised in complete darkness from the embryo stage to 19 days posteclosion. EM study of these fly eyes indicated that the photoreceptor structure was largely intact (data not shown) . Hence the retinal degeneration in inaF is light dependent.
  • Type D offspring in each single-female line were selected for ERG. If the ERG showed the inaF phenotype, the excision event was an imprecise one. Types A and D were saved to establish stable lines of new inaF alleles. If the ERG showed a wild-type response, the excision event was a precise one. Types A and D were saved to establish stable lines of these revertants. These lines were used in chromosomal in si tu hybridization with the P element as a probe to confirm that the P insertion in inat ⁇ no longer existed. Analysis
  • This example shows that, by analyzing genomic Southern and Northern blots, clone A23 was shown to contain the inaF structural gene.
  • Genomic DNA of wild-type flies, the 3B mutators and the inaF ⁇ " flies was isolated by homogenizing fifteen to twenty flies and using the Puregene kit from Gentra Co. following recommended protocols.
  • 3 ⁇ g of genomic DNA of each type was digested with restriction enzymes of choice and loaded on a 0.7% agarose gel for electrophoresis .
  • the agarose gel was denatured in 1.5 M NaCl, 0.5 M NaOH solution for 30 minutes, neutralized in 1 M Tris-Cl, 3 M NaCl, pH 7.5 solution for 40 minutes, blotted overnight onto Hybond-N Nylon membrane (Amersham Co.), and UV cross-linked.
  • Prehybridization treatment was carried out in 0.5 M NaH2P04, 0.7% SDS, 1% BSA, 0.01 M EDTA solution at
  • the poly (A) RNA was extracted with a PolyATtract- 1000 kit from Promega Co. following their recommended protocol. 3 ⁇ g of poly (A) RNA was loaded in each lane of the agarose gel unless otherwise specified. 1 ⁇ g of genomic DNA or cDNA fragment was used as template for
  • Genomic Southern analyses were used to determine whether A23 contains DNA fragments flanking the P element insertion that causes the inaF mutation. Since the P insertion is in inaF, A23 could not contain the inaF gene if it were far removed from the P insertion.
  • Genomic DNA from wild-type flies, mutator 3B, and inaF ⁇ P was purified and digested with multiple restriction enzymes, electrophoresed and blotted. A genomic Southern blot was probed with pCaSpeR3 (FIG. 8) .
  • Restriction fragment length polymorphism was observed and can be interpreted as follows: 1) The RFLP between wild type and 3B is due to an additional P element in 3B; and 2) The RFLP between 3B and inalT " is due to the fact that DNA fragments of different sizes flank the P element insertion sites in
  • fragment 4 (3.6 kb) detected RFLPs between wild-type flies, 3B, and inaF ⁇ & that were similar to those detected by the pCaSpeR3 probe as seen in FIG. 8, except that the EcoRI lanes showed same size signals.
  • Northern blots were used to examine whether fragment 4 of A23 could detect alterations of transcripts between wild-type flies and inaF ⁇ .
  • Poly (A) RNA was purified from wild-type fly heads, wild-type fly bodies, eya heads, and inaF ⁇ " heads.
  • polyA RNA was purified from 1-3 days old inaF ⁇ flies. Fragment 4 of A23 was used as a probe for Northern analysis, and detected a 3.0 kb eye- specific transcript which was drastically reduced in
  • Fragment 4 of A23 a genomic DNA fragment, was used as a template for 32P-dCTP labeling with random primers.
  • the labeled probe was purified with a
  • cDNA#l had the biggest insert and thus was used for further experiments.
  • the insert of cDNA#l was labeled with biotin-dUTP and used as a probe for chromosomal in si tu hybridization and detected a hybridization signal in the 10 C2-E3 region as described in Example 8.
  • the insert was also used to probe a genomic Southern blot and detected the same RFLP as those revealed by pCaSpeR3 and A23 probes.
  • the insert was labeled with 32P-dCTP and used to probe a Northern blot.
  • cDNA#l insert was labeled with 32 P-dCTP.
  • a 3.0 kb transcript was detected in the poly (A) + RNA from wild-type fly heads but not that from wild-type fly bodies and eya heads, indicating that the 3.0 kb transcript is eye specific (FIG. 11) .
  • the same transcript was absent from the poly (A) + RNA from inaF pl05p , indicating that the cDNA most likely contains the inaF gene.
  • the same blot was boiled to eliminate the radioactive probe and used again for a control experiment in which RP49, a ribosomal protein universally expressed in all tissues, was used as a probe.
  • Sequencing cDNA clone #1 cDNA clone #1 was partially digested with EcoRI and subcloned into the pBluescript-SK vector. T3 and T7 primers were used for initial sequencing reactions, and internal sequencing primers were designed and synthesized according to the sequence data obtained from each gel reading. The sequencing reactions were carried out at the DNA Sequencing Center at Iowa State University, Ames, Iowa. Both strands were sequenced, and every nucleotide has been confirmed from at least three independent reactions. The sequence of cDNA clone 1 is set forth in SEQ ID:1. A similar nucleotide sequence, differing only in certain 5' regions, and including a linker sequence at the 3' end, is set forth in SEQ ID: 2.
  • the cDNA has a poly (A) tail immediately before the 3 ' -end EcoRI cloning site, and a consensus polyadenylation signal (AATAAA) preceding the polyA .
  • the translation start site was determined on the basis that an in- frame stop codon is present about 15 amino acids upstream to the methionine assigned as the +1 site.
  • the putative protein has 241 amino acids with an estimated molecular weight of 26 kd. It appears to be a soluble protein since the Kyte-Doolittle plot did not reveal any hydrophobic segments which can serve as transmembrane domains .
  • a BLAST search of the NCBI A BLAST search of the NCBI ,
  • FIG. 12 shows a restriction map of inaF cDNA and of the corresponding genomic region in the A23 clone and three inaF mutants.
  • GST- InaF glutathione-S-transferase-InaF polypeptide
  • fusion construct was made by ligating the inaF coding region in frame with the glutathione transferase gene in the pGEX-KG vector [ (Guan and Dixon, Anal . Biochem . , 192:262-267 (1991)].
  • E. coli BL-21 over expression of the fusion protein was achieved by induction with IPTG.
  • the fusion protein was partially purified by using immobilized glutathione [Guan and Dixon (1991) , cited above] . Further purification can be achieved by ion exchange chromatography.
  • the fusion protein can be digested with thrombin (Sigma) and the InaF protein can be eluted from an immobilized glutathione agarose column as known in the art.
  • inaF cDNA- 1/XL-l Blue A deposit of inaF cDNA, designated as inaF cDNA- 1/XL-l Blue was deposited with the American Type Culture Collection, 10801 University Boulevard., Manassas, VA 20110-2209. The deposit is Epicurian Coli XL-1 Blue (Stratagene) harboring inaF cDNA (SEQ ID:1, nucleotides 314 to 1036) from Drosophila melanogaster (Berlin) in a pBluescript II KS (Stratagene) vector. The accession number is ATCC 207232.

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Abstract

L'invention concerne des protéines InaF purifiées, qui fonctionnent par isolation d'une entrée ionique de calcium dans des cellules. L'invention concerne également des séquences nucléotidiques codant pour des protéines fonctionnelles InaF. L'invention concerne, en outre, la séquence nucléotidique codant pour InaF, des cellules hôtes qui comprennent les vecteurs de recombinaison décrits dans le descriptif, et des procédés d'expression des protéines InaF par culture de cellules hôtes.
PCT/US1999/010821 1998-05-18 1999-05-18 Regulateurs de canal calcium Ceased WO1999060022A1 (fr)

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AU39969/99A AU3996999A (en) 1998-05-18 1999-05-18 Calcium channel regulators

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Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CHAOXIAN ET AL: "A novel protein required for store-operated calcium entry", SOCIETY FOR NEUROSCIENCE ABSTRACTS, vol. 24, 1998, pages 2030, SEE ABSTRACT 812.1 *
DATABASE EMBL-EST58-GENBANK-EST111 8 February 1999 (1999-02-08), HARVEY ET AL: "BDGP/HHMI Drosophila EST Project" *
MONTELL C.: "New light on TRP and TRPL", MOLECULAR PHARMACOLOGY, vol. 52, 1997, pages 755 - 763 *
MONTELL C.: "TRP trapped in fly signalling web", CURRENT OPINION IN NEUROBIOLOGY, vol. 8, no. 3, June 1998 (1998-06-01), pages 389 - 397, XP002921547, DOI: doi:10.1016/S0959-4388(98)80066-4 *

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