A VECTOR FOR GENE TRAP, AND A METHOD FOR GENE TRAPPING BY USING THE VECTOR

Abstract: The present application provides a vector for trapping an unknown gene of Drosophila melanogaster, which is a recombinant plasmid comprising the following nucleotide sequences in this order: an artificial consensus splicing acceptor site; a synthetic "stop/start" sequence; a reporter gene; a drug resistance gene; a gene responsible for a detectable phenotype of the Drosophila melanogaster; and a synthetic splicing donor site. The present application also provides a method for trapping an unknown gene of Drosophila melanogaster by using the vector.

Description: Description A Vector for Gene Trap, and A Method for Gene Trapping by Using The Vector Technical Field The present invention relates to a new vector system to facilitate the cloning and functional analysis of new genes of a fly, Drosophila melanogaster, and a method for gene trapping with the vector system. Background Art There are numerous examples for application of gene trapping methods in wide range of living organisms including maize and mouse (Gossler et al, . Science, 244:463-465, 1989). With respect to tools for gene trapping, the application of different types of enhancer trap P-element vectors (Wilson et al . , Genes & Development, 3:1301-1313, 1989) for cloning and analyzing trapped genes, as well their use for mosaic analysis with the help of the Gal4/UAS transcription activator system has proven fruitful. However, sometimes the expression pattern of the Gal4 or other reporter gene of the vector construct is affected by enhancers belonging to more than one gene. Similarly, in some cases it is difficult to determine whether the enhancer trap insertion effects the function of one or more of the neighboring genes . These circumstances altogether with the fact that in some cases the mutant phenotype could be attributed to the changed expression of a gene with its nearest exon located more than 30 kB apart from the insertion site, can lead in unfortunate cases to an ordeal when it' s time to clone and analyze the affected gene. One object of this application is to provide a vector system that includes specifically designed artificial regulatory sequences as well as selection methods for easy screening of positive recombinant lines. More especially, this application intends to provide a vector system of this invention offering much easier and faster cloning opportunities of the affected gene, compared to the widely used enhancer trap P-element vectors. Another object of this application is to provide easier detection method possibilities of the successful trapping events and much higher chance to get more characteristic ("functional") expression patterns of the reporter gene because in the contrary with much of the cases with enhancer trap lines, when using the vector system of this invention, the reporter gene expression is influenced only by a single endogenous transcription unit and effects only the expression of the very same gene. Disclosure of Invention The first invention of this application is a vector for trapping an unknown gene of Drosophila melanogaster, which is a recombinant plasmid comprising the following nucleotide sequences in this order: an artificial consensus splicing acceptor site; a synthetic "stop/start" sequence; a reporter gene; a drug resistance gene; a gene responsible for a detectable phenotype of the Drosophila melanogaster; and a synthetic splicing donor site. One embodiment of the first invention is that the recombinant plasmid is derived from pCasper3. Other embodiments of the first invention are that the reporter gene is the Gal4 gene, Gal4 DNA binding domaιn-P53 fusion gene or the Gal4-firefly luciferase fusion gene. Further embodiment of this first invention is that the gene responsible for a detectable phenotype of the Drosophila melanogaster is mini-white gene. More further embodiment of the first invention is that the drug resistance gene is neomycin-phosphotranspherase gene and its promoter is a heatshock promoter. The second invention of this application is a method for trapping an unknown gene of Drosophila melanogaster by using a vector which is a recombinant plasmid comprising the following nucleotide sequences in this order: an artificial consensus splicing acceptor site; a synthetic "stop/start" sequence; a reporter gene; a drug resistance gene; a gene responsible for a detectable phenotype of the Drosophila melanogaster; and a synthetic splicing donor site, which method comprises the steps of: (a) introducing the vector into the genome of a white minus fly; (b) selecting primary transformants resistant to a drug; (c) crossing the primary transformants with a transposase source strain to force the vector to jump into other locations ; (d) selecting secondary transformants by picking up the flies having strong eye color, (e) crossing the secondary transformants with UAS (Upstream Activator Sequence) -luciferase harboring strain and measuring the reporter gene expression of the resultant flies; and (f) identifying the trapped gene by cloning and sequencing the cD As fused to the reporter gene and the gene responsible for a detectable phenotype of the fly. The third invention of this application is a method for trapping an unknown gene of Drosophila melanogaster by using a vector A which is a recombinant plasmid comprising the following nucleotide sequences in this order: an artificial consensus splicing acceptor site; a synthetic "stop/start" sequence; Gal4 DNA binding domaιn-P53 fusion gene as a reporter gene; a drug resistance gene; a gene responsible for a detectable phenotype of the Drosophila melanogaste ; and a synthetic splicing donor site, and a vector B derived from pCasperhs, which has the heatshock promoter directed Gal4 activator domain-large T antigen fusion gene within polycloning site of the pCasperhs, which method comprises the steps of: (a) introducing each of the vectors A and B into the genomes of separate white minus flies; r> (b) selecting primary transformants for the vector A which are resistant to the drug, and selecting primary transformants for the vector B which have an eye color; (c) crossing the primary transformants for the vector A with a transposase source strain to force the vector to jump into other locations; (d) selecting secondary transformants for the vector A by picking up the flies having strong eye color; (e) crossing the secondary transformants with the primary transformants for the vector B to obtain flies harboring both the vectors A and B; (f) crossing the flies obtained in the step (e) with an UAS-luciferase harboring fly strain and measuring the reporter gene expression of the resultant flies after a heatshock treatment; and (g) identifying the trapped gene by cloning and sequencing the cDNAs fused to the reporter gene and the gene responsible for a detectable phenotype of the fly. Embodiments of the second and third inventions are corresponded to the embodiments of the first invention, and they will be more precisely described in the following description. Brief Description of Drawings Figure 1 shows the schematic map of the vector of this invention, pTrap-hsneo. Figure 2 shows the schematic map of the vector of this invention, pTrap-G4-p53. Figure 3 shows the schematic map of the vector of this invention, pCasperhs-G4-LT. Figure 4 shows the schematic map of the vector of this invention, pTrap-G4-luc. Figure 5 shows the shematic drawing of a fly genome to which the vector of this invention is inserted for cloning. Figure 6 shows the results of sequencing RT-PCR products of aop-Gal4 and m-white-aop fusion mRNAs. Figure 7 presents pictures of characteristic beta- galactosidase staining patterns in different parts of the fly brain resulted from crossing positive gene trap lines with flies harboring a UAS-lacZ construct. Best Mode for Carrying Out the Invention A vector construct of the first invention, for example, can be based on the commonly used, P-element transformation vector, pCasper3 (Pirotta, Vectors: A survey of molecular cloning vectors and their uses, eds . Rodriguez, R.L. & Denhardt, D.T., Butterworths , Boston. 437-456, 1998) and the convenient Gal4-UAS expression system (Brand and Perrimon, Development, 118:401-415, 1993) . A promoterless Gal4 gene preceded by an artificial consensus splicing acceptor site and a synthetic "stop/start" sequence to govern the read through translation coming from upstream exon(s) of the trapped gene into the proper reading frame of Gal4 was inserted into the polycloning site of pCasper3. The removal of the whole 3' UTR (untranslated region) sequence of the mini-white gene and replacement by an artificial splicing donor site resulted in a truncated gene without its own poly-adenylation site. Without a successful gene trapping event this truncated mini-white gene was not expected to confer any eye color, therefore in this invention a heatshock promoter directed neomycin-phosphotransferase (hs-neo) gene for helping selection of primary transformants by antibiotic feeding has been inserted. Figure 1 shows the schematic map of the gene trap construct (pTrap-hsneo) , and SEQ ID No.l is the complete nucleotide sequence of the vector pTrap-hsneo. Another gene trap construct, pTrap-G4-p53 (Figure 2) is created by replacing the Gal4 coding sequence of plasmid pTrap-hsneo with a Gal4 DNA binding dorrιain-P53 fusion gene (Clontech, Matchmaker Two Hybrid System, #K1605-1) . When this construct coexists in the genome of the same fly with another vector, pCasperhs-G4-LT (Figure 3) containing a heatshock promoter directed Gal4 activator domain-large T antigen (Clontech, Matchmaker Two Hybrid System, #K1605-1) fusion gene, the assembly of a functional Gal4 molecule, through p53-large T antigen interaction, can be regulated by external heatshock. On this way, the possibility of an intentional temporary control of Gal4 activity became available. In other words the Gal4 expression in a pattern as already determined spatially by the promoter of the trapped gene now can be induced at any desired stage of development by external heatshock . In order to make the detection of Gal4 expression easier, the Gal4 gene in another construct is replaced with a Gal4-firefly luciferase fusion gene to get pTrap-G4-luc (Figure 4) . This artificial gene is coding for a fusion polypeptide which has preserved both enzymatic activities. The easy measuring of luciferase activity by luminoassay (Brandes et al . , Neuron, 16:687-694, 1996) makes the detection of Gal4 activity comfortable in every single living fly. Then, one of the best mode of the second or third invention, a method for gene trapping using the vector system, is described in detail . (1) Screening: The gene trap vector constructs can be introduced into the genome of a white minus fly by icroinjection. The selection of primary transformants is possible by using G418, an analog of neomycin, resistance conferred by hs-neo gene. (When performing transformation experiments with these constructs it's turned out that the truncated mini-white gene generally provides a very slight yellow eye color which could be distinguished from w-minus phenotype in most of the cases, therefore G418 selection apparently is not necessary.) After a line with the gene trap construct is being established, the secondary transformants can be generated on the usual way by crossing the original line with a so-called jumpstarter containing the transposase expressing delta 2-3 genetic element. Usually a certain percentage, between 4 and 8, of the secondary transformants have much stronger eye color (deep orange or reddish) than the ancestor fly indicating that the construct was being inserted downstream of a promoter and now the mini-white gene is using the transcriptional "facilities" of that gene (e.g.: poly-adenylation site and transcriptional y terminator) instead of its removed ones . They are the most likely candidates for successful gene trap events . In case of these lines the vector probably has been inserted either into an intron of a gene or upstream from the first intron into the 5' UTR in proper orientation (that is the direction of transcription is same for the "trapped gene" and the mini- white (and Gal4) genes as well) . The mini-white gene has its own promoter therefore its expression pattern is supposed to be largely independent from that of the trapped gene. These positive lines are to be checked in the next step for Gal4 expression by crossing them with a "marker" line harboring a UAS-luciferase reporter gene construct. (When using pTrap-G4-luc vector, this step is obviously not necessary.) Usually very strong correlation was found between eye color and Gal4 expression: more than 90% of the lines having strong eye color proved to be expressing Gal4 by means of luciferase assay using luminometer (Brandes et al . , Neuron, 16:687-692, 1996) . (2) Cloning: When the gene trap construct is being inserted into an intron of an endogenous gene, the marker genes of the construct are supposed to be spliced on mRNA level to the exons of the trapped gene by using the artificial splicing acceptor and donor sites. More exactly while the Gal4 mRNA should be joint to the exon(s) located upstream of the insertion site, at the same time the mini-white mRNA is fused to the following exon(s) accomplishing the dual tagging of the trapped gene (Figure 5) . This feature can be used for quickly and easily identifying the trapped gene by means of 3' and 5' RACE (Rapid Amplification of cDNA Ends) experiments. Even cloning and sequencing only a part of the caught mRNA still provides reasonable chance to find homologous mRNAs in the BDGP (Berkeley Drosophila Genome Project) EST (Expressed Sequence Tag) library. With these approaches, the identification of an already cloned gene can take less then a week compared to the usually more than one year period in average when analyzing a mutant created by some enhancer trap construct. It's well-known from the literature and the present inventors also have experienced that P-element vectors tend to integrate into or near the 5' UTR of active genes. (The present inventors found that in these cases if the insertion occurred upstream from the first intron, and therefore the artificial splicing acceptor site could not be utilized, the Gal4 gene was expressed by read-through transcription from the nearby promoter. ) The advantage of this tendency can be taken by cloning and sequencing the flanking genomic sequences of the insertion site by inverse or vectorette PCR or by plasmid rescue using suitable restriction digestion to recover the neomycin resistance gene of the construct. Then again the BDGP library can be searched to find any significant matching. (3) Rescue: The only reliable way to confirm that any observed mutant phenotype is really the consequence of the P-element insertion is to rescue that particular phenotype. Expectedly the phenotype (some alteration from wild type fly) is caused by changed expression of gene(s) disturbed by insertion of the P-element. The rescue can be made by expressing the cDNA of the suspected gene most preferable with identical spatial and temporary pattern than that of the gene itself. As it was expected, the vector constructs of the first invention usually cause strong phenotypes. It's not surprising at all because the trapped genes are supposed to be split into two parts on mRNA level resulting in null mutants in majority of the cases. Accordingly mutants obtained by this method frequently show homozygous lethality or sterility. Hypomorphic mutants can be obtained by forcing imprecise excision of the gene trap P-element construct. As mentioned above, the Gal4 expression is obliged to reflect precisely to that of the trapped gene simply because the Gal4 gene has no its own promoter and they share a common, fused mRNA. This identical expression provides unique opportunity to rescue the mutant phenotype by crossing this fly with another one harboring the UAS directed, cloned cDNA of the trapped gene. On this way either the original , homozygous null mutant gene trap fly or any transheterozygous derivative of that with some hypomorphic allele over the null mutant allele can be rescued. (4) Determination of spatial and developmental expression pattern of the trapped gene: Histochemical determination of the spatially and temporarily controlled expression of any trapped gene is also easy following introduction of a UAS-lacZ construct into the genome of the same fly and performing either X-gal or antibody staining for beta-galactosidase. (5) Mosaic analysis: Possession of a large collection of fly lines with different, characteristic and, in the case of the pTrap-G4- p53/pCasperhs-G4-TL vector system, mducible Gal4 expression pattern makes feasible carrying out mosaic analysis of virtually any gene of interest by directing the expression of their UAS-constructs on a mutant background with different Gal4 expression patterns. This approach can answer the question of where and when that particular gene is required to be expressed to rescue the mutant phenotype. Similarly, any gene can be expressed in different ectopic patterns to generate new dominant mutant phenotypes. This approach might help to conclude the role of that particular gene and to identify the pathway, in which it's involved. Example The following example illustrates a specific embodiment of the various aspects of the invention. This example is not intended to limit the invention in any manner. Figure 6 shows the results of sequencing RT-PCR products of aop-Gal4 and m-white-aop fusion mRNAs. The template was total RNA prepared from a positive gene trap line which has the vector pTrap-hsneo being integrated into the first intron of the well-known aop (anterior open/pokkuri/yan) developmental gene. The sequences confirm that both splicing occurred precisely at that particular nucleotides of the artificial regulatory sequences where it was expected. On Figure 7, there are pictures of characteristic beta- galactosidase staining patterns in different parts of the fly brain resulted from crossing positive gene trap lines with flies harboring a UAS-lacZ construct. Industrial Applicability The vector system of this invention offers an exceptional opportunity for easy and fast cloning of the gene responsible for the observed phenotype. Furthermore, by using the UAS-dnven coding sequence of any gene of interest, that particular gene can be expressed in identical patterns than those of the trapped genes and these expressions can be regulated temporarily at any desired developmental stage. Sequence Listing <110> Japan Science and Technology Corporation <120> A Vector for Gene Trap, and A Method for Gene Trapping by Using The Vector <150> Japan, Application No. 10-141952 <151> 22 May 1998 <160> 1 <170> Patentln Ver. 2.0 <210> 1 <211> 11206 <212> DNA <213> Artificial sequence <220> <221> 3'P sequence <222> (1) .. (237) <220> <221> synthetic splicing acceptor site and stop/start sequence <222> (238).. (274) <220> <221> Gal4 gene (coding region and 3'UTR) <222> (275) .. (3164) <220> <221> hsp70 terminator <222> (3165) .. (3426) <220> <221> synthetic junction sequence <222> 3427-3457 <220> <221> heat shock promoter directed neomycine resistance gene on complementer strand <222> (3458) .. (4907) <220> <221> mini-white gene <222> (4908) .. (8275) <220> <221> synthetic splicing donor site <222> (8276) .. (8299) <220> <221> 5'P sequence <222> (8300) .. (8446) <220> <221> bacterial part of pCasper3 shuttle vector including complete pUC8 sequence <222> (8447) .. (11206) <220> <221> synthetic DNA <222> (238) .. (274) (3427) .. (3457) (4908) .. (4914) (8276) .. (8299) <400> 1 catgatgaaa taacataagg tggtcccgtc ggcaagagac atccacttaa cgtatgcttg 60 caataagtgc gagtgaaagg aatagtattc tgagtgtcgt attgagtctg agtgagacag 120 cgatatgatt gttgattaac ccttagcatg tccgtggggt ttgaattaac tcataatatt 180 aattagacga aattattttt aaagttttat ttttaataat ttgcgagtac gcaaagctct 240 ttctcttaca ggtcgaattg atgtgatgga tccaatgaag ctactgtctt ctatcgaaca 300 agcatgcgat atttgccgac ttaaaaagct caagtgctcc aaagaaaaac cgaagtgcgc 360 caagtgctg aagaacaact gggagtgtcg ctactctccc aaaaccaaaa ggtctccgct 420 gactagggca catctgacag aagtggaatc aaggctagaa agactggaac agctatttct 480 actgattttt cctcgagaag accttgacat gattttgaaa atggattctt tacaggatat 540 aaaagcattg ttaacaggat tatttgtaca agataatgtg aataaagatg ccgtcacaga 600 tagattggct tcagtggaga ctgatatgcc tctaacattg agacagcata gaataagtgc 660 gacatcatca tcggaagaga gagtaacaa aggtcaaaga cagttgactg tatcgattga 720 ctcggcagct catcatgata actccacaat tccgttggat tttatgccca gggatgctct 780 tcatggattt gattggtctg aagaggatga catgtcggat ggcttgccct tcctgaaaac 840 ggaccccaac aataatgggt tctttggcga cggttctctc ttatgtattc ttcgatctat 900 tggctttaaa ccggaaaatt acacgaactc taacgttaac aggctcccga ccatgattac 960 ggatagatac acgttggctt ctagatccac aacatcccgt ttacttcaaa gttatctcaa 1020 taattttcac ccctactgcc ctatcgtgca ctcaccgacg ctaatgatgt tgtataataa 1080 ccagattgaa atcgcgtcga aggatcaatg gcaaatcctt tttaactgca tattagccat 1140 tggagcctgg tgtatagagg gggaatctac tgatatagat gttttttact atcaaaatgc 1200 taaatctcat ttgacgagca aggtcttcga gtcaggttcc ataattttgg tgacagccct 1260 acatcttctg tcgcgatata cacagtggag gcagaaaaca aatactagct ataattttca 1320 , ,_ 16 cagcttttcc ataagaatgg ccatatcatt gggcttgaat agggacctcc cctcgtcctt 1380 cagtgatagc agcattctgg aacaaagacg ccgaatttgg tggtctgtct actcttggga 1440 gatccaattg tccctgcttt atggtcgatc catccagctt tctcagaata caatctcctt 1500 cccttcttct gtcgacgatg tgcagcgtac cacaacaggt cccaccatat atcatggcat 1560 cattgaaaca gcaaggctct tacaagtttt cacaaaaatc tatgaactag acaaaacagt 1620 aactgcagaa aaaagtccta tatgtgcaaa aaaatgcttg atgatttgta atgagattga 1680 ggaggtttcg agacaggcac caaagttttt acaaatggat atttccacca ccgctctaac 1740 caatttgttg aaggaacacc cttggctatc ctttacaaga ttcgaactga agtggaaaca 1800 gttgtctctt atcatttatg tattaagaga ttttttcact aattttaccc agaaaaagtc 1860 acaactagaa caggatcaaa atgatcatca aagttatgaa gttaaacgat gctccatcat 1920 gttaagcgat gcagcacaaa gaactgttat gtctgtaagt agctatatgg acaatcataa 1980 tgtcacccca tattttgcct ggaattgttc ttattacttg ttcaatgcag tcctagtacc 2040 cataaagact ctactctcaa actcaaaatc gaatgctgag aataacgaga ccgcacaatt 2100 attacaacaa attaacactg ttctgatgct attaaaaaaa ctggccactt ttaaaatcca 2160 gacttgtgaa aaatacattc aagtactgga agaggtatgt gcgccgtttc tgttatcaca 2220 gtgtgcaatc ccattaccgc atatcagtta taacaatagt aatggtagcg ccattaaaaa 2280 tattgtcggt tctgcaacta tcgcccaata ccctactctt ccggaggaaa atgtcaacaa 2340 tatcagtgtt aaatatgttt ctcctggctc agtagggcct tcacctgtgc cattgaaatc 2400 aggagcaagt ttcagtgatc tagtcaagct gttatctaac cgtccaccct ctcgtaactc 2460 tccagtgaca ataccaagaa gcacaccttc gcatcgctca gtcacgcctt ttctagggca 2520 acagcaacag ctgcaatcat tagtgccact gaccccgtct gctttgtttg gtggcgccaa 2580 ttttaatcaa agtgggaata ttgctgatag ctcattgtcc ttcactttca ctaacagtag 2640 caacggtccg aacctcataa caactcaaac aaattctcaa gcgctttcac aaccaattgc 2700 ctcctctaac gttcatgata acttcatgaa taatgaaatc acggctagta aaattgatga 2760 tggtaataat tcaaaaccac tgtcacctgg ttggacggac caaactgcgt ataacgcgtt 2820 tggaatcact acagggatgt ttaataccac tacaatggat gatgtatata actatctatt 2880 cgatgatgaa gataccccac caaacccaaa aaaagagtaa aatgaatcgt agatactgaa 2940 aaaccccgca agttcacttc aactgtgcat cgtgcaccat ctcaatttct ttcatttata 3000 catcgttttg ccttctttta tgtaactata ctcctctaag tttcaatctt ggccatgtaa 3060 cctctgatct atagaatttt ttaaatgact agaattaatg cccatctttt ttttggacct 3120 aaattcttca tgaaaatata ttacgagggc ttattcagaa gcttatcgat accgtcgact 3180 aaagccaaat agaaattatt cagttctggc ttaagttttt aaaagtgata ttatttattt 3240 ggttgtaacc aaccaaaaga atgtaaataa ctaatacata attatgttag ttttaagtta 3300 gcaacaaatt gattttagct atattagcta cttggttaat aaatagaata tatttattta 3360 aagataattc gtttttattg tcagggagtg agtttgctta aaaactcgtt tagatccact 3420 agaaggaccg cggctcctcg accggatcga aaggagggcg aagaactcca gcatgagatc 3480 cccgcgctgg aggatcatcc agccggcgtc ccggaaaacg attccgaagc ccaacctttc 3540 atagaaggcg gcggtggaat cgaaatctcg tgatggcagg ttgggcgtcg cttggtcggt 3600 catttcgaac cccagagtcc cgctcagaag aactcgtcaa gaaggcgata gaaggcgatg 3660 cgctgcgaat cgggagcggc gataccgtaa agcacgagga agcggtcagc ccattcgccg 3720 ccaagctctt cagcaatatc acgggtagcc aacgctatgt cctgatagcg gtccgccaca 3780 cccagccggc cacagtcgat gaatccagaa aagcggccat tttccaccat gatattcggc 3840 aagcaggcat cgccatgggt cacgacgaga tcctcgccgt cgggcatgcg cgccttgagc 3900 ctggcgaaca gttcggctgg cgcgagcccc tgatgctctt cgtccagatc atcctgatcg 3960 acaagaccgg cttccatccg agtacgtgct cgctcgatgc gatgtttcgc ttggtggtcg 4020 aatgggcagg tagccggatc aagcgtatgc agccgccgca ttgcatcagc catgatggat 4080 actttctcgg caggagcaag gtgagatgac aggagatcct gccccggcac ttcgcccaat 4140 agcagccagt cccttcccgc ttcagtgaca acgtcgagca cagctgcgca aggaacgccc 4200 gtcgtggcca gccacgatag ccgcgctgcc tcgtcctgca gttcattcag ggcaccggac 4260 aggtcggtct tgacaaaaag aaccgggcgc ccctgcgctg acagccggaa cacggcggca 4320 tcagagcagc cgattgtctg ttgtgcccag tcatagccga atagcctctc cacccaagcg 4380 gccggagaac ctgcgtgcaa tccatcttgt tcaatcatgc gaaacgatcc tcatcctgtc 4440 tcttgatcag atcccctatt cagagttctc ttcttgtatt caataattac ttcttggcag 4500 atttcagtag ttgcagttga tttacttggt tgctggttac ttttaattga ttcactttaa 4560 cttgcacttt actgcagatt gtttagcttg ttcagctgcg cttgtttatt tgcttagctt 4620 tcgcttagcg acgtgttcac tttgcttgtt tgaattgaat tgtcgctccg tagacgaagc 4680 gcctctattt atactccggc gctcttttcg cgaacattcg aggcgcgctc tctcgaacca 4740 acgagagcag tatgccgttt actgtgtgac agagtgagag agcattagtg cagagaggga 4800 gagacccaaa aagaaaagag agaataacga ataacggcca gagaaatttc tcgagttttc 4860 tttctgccaa acaaatgacc taccacaata accagtttgt tttgggatct agtccctaat 4920 tctagtatgt atgtaagtta ataaaaccct tttttggaga atgtagattt aaaaaaacat 4980 attttttttt tattttttac tgcactggac atcattgaac ttatctgatc agttttaaat 5040 ttacttcgat ccaagggtat ttgaagtacc aggttctttc gattacctct cactcaaaat 5100 gacattccac tcaaagtcag cgctgtttgc ctccttctct gtccacagaa atatcgccgt 5160 ctctttcgcc gctgcgtccg ctatctcttt cgccaccgtt tgtagcgtta cctagcgtca 5220 atgtccgcct tcagttgcac tttgtcagcg gtttcgtgac gaagctccaa gcggtttacg 5280 ccatcaatta aacacaaagt gctgtgccaa aactcctctc gcttcttatt tttgtttgtt 5340 ttttgagtga ttggggtggt gattggtttt gggtgggtaa gcaggggaaa gtgtgaaaaa 5400 tcccggcaat gggccaagag gatcaggagc tattaattcg cggaggcagc aaacacccat 5460 ctgccgagca tctgaacaat gtgagtagta catgtgcata catcttaagt tcacttgatc 5520 tataggaact gcgattgcaa catcaaattg tctgcggcgt gagaactgcg acccacaaaa 5580 atcccaaacc gcaatcgcac aaacaaatag tgacacgaaa cagattattc tggtagctgt 5640 gctcgctata taagacaatt tttaagatca tatcatgatc aagacatcta aaggcattca 5700 ttttcgacta cattcttttt tacaaaaaat ataacaacca gatattttaa gctgatccta 5760 gatgcacaaa aaataaataa aagtataaac ctacttcgta ggatacttcg ttttgttcgg 5820 ggttagatga gcataacgct tgtagttgat atttgagatc ccctatcatt gcagggtgac 5880 agcggacgct tcgcagagct gcattaacca gggcttcggg caggccaaaa actacggcac 5940 gctcctgcca cccagtccgc cggaggactc cggttcaggg agcggccaac tagccgagaa 6000 cctcacctat gcctggcaca atatggacat ctttggggcg gtcaatcagc cgggctccgg 6060 atggcggcag ctggtcaacc ggacacgcgg actattctgc aacgagcgac acataccggc 6120 gcccaggaaa catttgctca agaacggtga gtttctattc gcagtcggct gatctgtgtg 6180 aaatcttaat aaagggtcca attaccaatt tgaaactcag tttgcggcgt ggcctatccg 6240 ggcgaacttt tggccgtgat gggcagttcc ggtgccggaa agacgaccct gctgaatgcc 6300 cttgcctttc gatcgccgca gggcatccaa gtatcgccat ccgggatgcg actgctcaat 6360 ggccaacctg tggacgccaa ggagatgcag gccaggtgcg cctatgtcca gcaggatgac 6420 ctctttatcg gctccctaac ggccagggaa cacctgattt tccaggccat ggtgcggatg 6480 ccacgacatc tgacctatcg gcagcgagtg gcccgcgtgg atcaggtgat ccaggagctt 6540 tcgctcagca aatgtcagca cacgatcatc ggtgtgcccg gcagggtgaa aggtctgtcc 6600 ggcggagaaa ggaagcgtct ggcattcgcc tccgaggcac taaccgatcc gccgcttctg 6660 atctgcgatg agcccacctc cggactggac tcatttaccg cccacagcgt cgtccaggtg 6720 ctgaagaagc tgtcgcagaa gggcaagacc gtcatcctga ccattcatca gccgtcttcc 6780 gagctgtttg agctctttga caagatcctt ctgatggccg agggcagggt agctttcttg 6840 ggcactccca gcgaagccgt cgacttcttt tcctagtgag ttcgatgtgt ttattaaggg 6900 tatctagcat tacattacat ctcaactcct atccagcgtg ggtgcccagt gtcctaccaa 6960 ctacaatccg gcggactttt acgtacaggt gttggccgtt gtgcccggac gggagatcga 7020 gtcccgtgat cggatcgcca agatatgcga caattttgct attagcaaag tagcccggga 7080 tatggagcag ttgttggcca ccaaaaattt ggagaagcca ctggagcagc cggagaatgg 7140 gtacacctac aaggccacct ggttcatgca gttccgggcg gtcctgtggc gatcctggct 7200 gtcggtgctc aaggaaccac tcctcgtaaa agtgcgactt attcagacaa cggtgagtgg 7260 ttccagtgga aacaaatgat ataacgctta caattcttgg aaacaaattc gctagatttt 7320 agttagaatt gcctgattcc acacccttct tagttttttt caatgagatg tatagtttat 7380 agttttgcag aaaataaata aatttcattt aactcgcgaa catgttgaag atatgaatat 7440 taatgagatg cgagtaacat tttaatttgc agatggttgc catcttgatt ggcctcatct 7500 ttttgggcca acaactcacg caagtgggcg tgatgaatat caacggagcc atcttcctct 7560 tcctgaccaa catgaccttt caaaacgtct ttgccacgat aaatgtaagt cttgtttaga 7620 atacatttgc atattaataa tttactaact ttctaatgaa tcgattcgat ttaggtgttc 7680 acctcagagc tgccagtttt tatgagggag gcccgaagtc gactttatcg ctgtgacaca 7740 tactttctgg gcaaaacgat tgccgaatta ccgctttttc tcacagtgcc actggtcttc 7800 acggcgattg cctatccgat gatcggactg cgggccggag tgctgcactt cttcaactgc 7860 ctggcgctgg tcactctggt ggccaatgtg tcaacgtcct tcggatatct aatatcctgc 7920 gccagctcct cgacctcgat ggcgctgtct gtgggtccgc cggttatcat accattcctg 7980 ctctttggcg gcttcttctt gaactcgggc tcggtgccag tatacctcaa atggttgtcg 8040 tacctctcat ggttccgtta cgccaacgag ggtctgctga ttaaccaatg ggcggacgtg 8100 gagccgggcg aaattagctg cacatcgtcg aacaccacgt gccccagttc gggcaaggtc 8160 atcctggaga cgcttaactt ctccgccgcc gatctgccgc tggactacgt gggtctggcc 8220 attctcatcg tgagcttccg ggtgctcgca tatctggctc taagacttcg ggcccgacgc 8280 aaggagtaga aggtaagtag cggccgcacg taagggttaa tgttttcaaa aaaaaattcg 8340 tccgcacaca acctttcctc tcaacaagca aacgtgcact gaatttaagt gtatacttcg 8400 gtaagcttcg gctatcgacg ggaccacctt atgttatttc atcatgggcc agacccacgt 8460 agtccagcgg cagatcggcg gcggagaagt taagcgtctc caggatgacc ttgcccgaac 8520 tggggcacgt ggtgttcgac gatgtgcagc taatttcgcc cggctccacg tccgcccatt 8580 ggttaatcag cagaccctcg ttggcgtaac ggaaccatga gaggtacgac aaccatttga 8640 ggtatactgg caccgagccc gagttcaaga agaaggcgtt tttccatagg ctccgccccc 8700 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat 8760 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc 8820 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcaatgct 8880 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg 8940 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc 9000 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga 9060 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa 9120 ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta 9180 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc 9240 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg 9300 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga 9360 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg 9420 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct 9480 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg 9540 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc 9600 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa 9660 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc 9720 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt 9780 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc 9840 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt 9900 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc 9960 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt 10020 gtatgcggcg accgagttgc tcttgcccgg cgtcaacacg ggataatacc gcgccacata 10080 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga 10140 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag 10200 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa 10260 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt 10320 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga 10380 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag 10440 aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc 10500 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 10560 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 10620 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 10680 accatatgcg gtgtgaaata ccgcaccgaa tcgcgcggaa ctaacgacag tcgctccaag 10740 gtcgtcgaac aaaaggtgaa tgtgttgcgg agagcgggtg ggagacagcg aaagagcaac 10800 tacgaaacgt ggtgtggtgg aggtgaatta tgaagagggc gcgcgatttg aaaagtatgt 10860 atataaaaaa tatatcccgg tgttttatgt agcgataaac gagtttttga tgtaaggtat 10920 gcaggtgtgt aagtcttttg gttagaagac aaatccaaag tctacttgtg gggatgttcg 10980 aaggggaaat acttgtattc tataggtcat atcttgtttt tattggcaca aatataatta 11040 cattagcttt ttgagggggc aataaacagt aaacacgatg gtaataatgg taaaaaaaaa 11100 aacaagcagt tatttcggat atatgtcggc tactccttgc gtcgggcccg aagtcttaga 11160 gccagatatg cgagcacccg gaagctcacg atgagaatgg ccagac 11206

Claims: CLAIMS 1. A vector for trapping an unknown gene of Drosophila melanogaster, which is a recombinant plasmid comprising the following nucleotide sequences in this order: an artificial consensus splicing acceptor site; a synthetic "stop/start" sequence; a reporter gene; a drug resistance gene; a gene responsible for a detectable phenotype of the Drosophila melanogaster; and a synthetic splicing donor site. 2. The vector of claim 1 , wherein the recombinant plasmid is derived from pCasper3. 3. The vector of claim 1 or 2, wherein the reporter gene is the Gal4 gene. 4. The vector of claim 3, which has the nucleotide sequence of SEQ ID No. 1. 5. The vector of claim 1 or 2, wherein the reporter gene is Gal4 DNA binding domain-P53 fusion gene. 6. The vector of claim 1 or 2, wherein the reporter gene is the Gal4-firefly luciferase fusion gene. 7. The vector of any one of claims 1-6, wherein the gene responsible for a detectable phenotype of the Drosophila melanogaster is mini-white gene. 8. The vector of any one of claims 1-7, wherein the drug resistance gene is neomycin-phosphotranspherase gene and its promoter is a heatshock promoter. 9 A vector derived from pCasperhs , which has the heatshock promoter directed Gal4 activator domain-large T antigen fusion gene within polycloning site of the pCasperhs. 10. A method for trapping an unknown gene of Drosophila melanogaster by using a vector which is a recombinant plasmid comprising the following nucleotide sequences in this order: an artificial consensus splicing acceptor site; a synthetic "stop/start" sequence; a reporter gene; a drug resistance gene; a gene responsible for a detectable phenotype of the Drosophila melanogaster; and a synthetic splicing donor site, which method comprises the steps of: (a) introducing the vector into the genome of a white minus fly; (b) selecting primary transformants resistant to a drug; (c) crossing the primary transformants with a transposase source strain to force the vector to jump into other locations ; (d) selecting secondary transformants by picking up the flies having strong eye color, (e) crossing the secondary transformants with UAS (Upstream Activator Sequence) -luciferase harboring strain and measuring the reporter gene expression of the resultant flies; and (f) identifying the trapped gene by cloning and sequencing the cDNAs fused to the reporter gene and the gene responsible for a detectable phenotype of the fly. 11. The method according to claim 10, wherein the recombinant plasmid is derived from pCasper3. 12. The method according to claim 10 or 11, wherein the reporter gene in the vector is the Gal4 gene, and in the step (e) the Gal4 expression is measured. 13. The method according to claim 10 or 11, wherein the reporter gene of the vector is the Gal4-firefly luciferase fusion gene, and in the step (e) expression of said fusion gene is measured without crossing the secondary transformants with UAS-luciferase harboring strain. 14. The method according to any one of claims 10 to 14, wherein the gene responsible for a detectable phenotype of the Drosophila melanogaster is mini-white gene, and in the step (f) the cDNAs fused to the reporter gene and the mini- white gene are cloned and sequenced. 15. The method according to any one of claims 10 to 15, wherein the drug resistance gene is neomycm- phosphotranspherase gene and its promoter is a heatshock promoter, and in the step (b) the transformants resistant to G418 is selected. 16. A method for trapping an unknown gene of Drosophila melanogaster by using a vector A which is a recombinant plasmid comprising the following nucleotide sequences in this order : an artificial consensus splicing acceptor site; a synthetic "stop/start" sequence; Gal4 DNA binding domaιn-P53 fusion gene as a reporter gene; a drug resistance gene; a gene responsible for a detectable phenotype of the Drosophila melanogaster; and a synthetic splicing donor site, and a vector B derived from pCasperhs, which has the heatshock promoter directed Gal4 activator domain-large T antigen fusion gene within polycloning site of the pCasperhs, which method comprises the steps of: (a) introducing each of the vectors A and B into the genomes of separate white minus flies; (b) selecting primary transformants for the vector A which are resistant to a drug, and selecting primary transformants for the vector B which have an eye color; (c) crossing the primary transformants for the vector A with a transposase source strain to force the vector to jump into other locations; (d) selecting secondary transformants for the vector A by picking up the flies having strong eye color; (e) crossing the secondary transformants with the primary transformants for the vector B to obtain flies harboring both the vectors A and B; (f) crossing the flies obtained in the step (e) with an UAS-luciferase harboring fly strain and measuring the reporter gene expression of the resultant flies after a heatshock treatment; and (g) identifying the trapped gene by cloning and sequencing the cDNAs fused to the reporter gene and the gene responsible for a detectable phenotype of the fly. 17. The method according to claim 16, wherein the vector A is derived from pCasper3. 18. The method according to claim 16 or 17, wherein the gene responsible for a detectable phenotype of the Drosophila melanogaster is mini-white gene, and in the step (g) the cDNAs fused to the reporter gene and the mini-white gene are cloned and sequenced. 19. The method according to any one of claims 16 to 18, wherein the drug resistance gene is neomycin- phosphotranspherase gene and its promoter is a heatshock promoter, and in the step (b) the transformant resistant to G418 is selected.

Inventors: ASZTALOS ZOLTAN, AWANO WAKAE, LUKACSOVICH TAMAS, YAMAMOTO DAISUKE, ASZTALOS, ZOLTAN, AWANO, WAKAE, LUKACSOVICH, TAMAS, YAMAMOTO, DAISUKE
Applicants: ASZTALOS ZOLTAN, AWANO WAKAE, JAPAN SCIENCE & TECH CORP, LUKACSOVICH TAMAS, YAMAMOTO DAISUKE, ASZTALOS, ZOLTAN, AWANO, WAKAE, JAPAN SCIENCE AND TECHNOLOGY CORPORATION, LUKACSOVICH, TAMAS, YAMAMOTO, DAISUKE
Application year: 1999
Main classification: C12N15
IPC codes: C07K14, C12N15, C12Q1, C12R1