Takashi Ohsako - Academia.edu (original) (raw)
Papers by Takashi Ohsako
Genetic Analysis of Male Sterility in the Hybrid Between Drosophila Albomicans Ans D. Nasuta. IV
The Japanese Journal of Genetics, Dec 1, 1995
A comprehensive Drosophila resource to identify key functional interactions between SARS-CoV-2 factors and host proteins
Cell Reports, Aug 1, 2023
Genes & Genetic Systems, 2021
Sperm are modified substantially in passing through both the male and the female reproductive tra... more Sperm are modified substantially in passing through both the male and the female reproductive tracts, only thereafter becoming functionally competent to fertilize eggs. Drosophila sperm become motile in the seminal vesicle; after ejaculation, they interact with seminal fluid proteins and undergo biochemical changes on their surface while they are stored in the female sperm storage organs. However, the molecular mechanisms underlying these maturation processes remain largely unknown. Here, we focused on Drosophila Neprilysin genes, which are the fly orthologs of the mouse Membrane metallo-endopeptidase-like 1 (Mmel1) gene. While Mmel1 knockout male mice have reduced fertility without abnormality in either testis morphology or sperm motility, there are inconsistent results regarding the association of any Neprilysin gene with male fertility in Drosophila. We examined the association of the Nep1-5 genes with male fertility by RNAi and found that Nep4 gene function is specifically required in germline cells. To investigate this in more detail, we induced mutations in the Nep4 gene by the CRISPR/Cas9 system and isolated two mutants, both of which were viable and female fertile, but male sterile. The mutant males had normal-looking testes and sperm; during copulation, sperm were transferred to females and stored in the seminal receptacle and paired spermathecae. However, following sperm transfer and storage, three defects were observed for Nep4 mutant sperm. First, sperm were quickly discarded by the females; second, the proportion of eggs fertilized was significantly lower for mutant sperm than for control sperm; and third, most eggs laid did not initiate development after sperm entry. Taking these observations together, we conclude that the Nep4 gene is essential for sperm function following sperm transfer to females.
A cross-species approach for the identification of Drosophila male sterility genes
G3 Genes|Genomes|Genetics
Male reproduction encompasses many essential cellular processes and interactions. As a focal poin... more Male reproduction encompasses many essential cellular processes and interactions. As a focal point for these events, sperm offer opportunities for advancing our understanding of sexual reproduction at multiple levels during development. Using male sterility genes identified in human, mouse, and fruit fly databases as a starting point, 103 Drosophila melanogaster genes were screened for their association with male sterility by tissue-specific RNAi knockdown and CRISPR/Cas9-mediated mutagenesis. This list included 56 genes associated with male infertility in the human databases, but not found in the Drosophila database, resulting in the discovery of 63 new genes associated with male fertility in Drosophila. The phenotypes identified were categorized into six distinct classes affecting sperm development. Interestingly, the second largest class (Class VI) caused sterility despite apparently normal testis and sperm morphology suggesting that these proteins may have functions in the matur...
Humanized Flies and Resources for Cross-Species Study
Advances in Experimental Medicine and Biology
G3: Genes|Genomes|Genetics
In Drosophila, mature sperm are transferred from males to females during copulation, stored in th... more In Drosophila, mature sperm are transferred from males to females during copulation, stored in the sperm storage organs of females, and then utilized for fertilization. Here, we report a gene named sheepish (shps) of Drosophila melanogaster that is essential for sperm storage in females. shps mutant males, although producing morphologically normal and motile sperm that are effectively transferred to females, produce very few offspring. Direct counts of sperm indicated that the primary defect was correlated to failure of shps sperm to migrate into the female sperm storage organs. Increased sperm motion parameters were seen in the control after transfer to females, whereas sperm from shps males have characteristics of the motion parameters different from the control. The few sperm that occasionally entered the female sperm storage organs showed no obvious defects in fertilization and early embryo development. The female postmating responses after copulation with shps males appeared no...
Genetics, 1999
We have constructed a P-element-based gene search vector for efficient detection of genes in Dros... more We have constructed a P-element-based gene search vector for efficient detection of genes in Drosophila melanogaster. The vector contains two copies of the upstream activating sequence (UAS) enhancer adjacent to a core promoter, one copy near the terminal inverted repeats at each end of the vector, and oriented to direct transcription outward. Genes were detected on the basis of phenotypic changes caused by GAL4-dependent forced expression of vector-flanking DNA, and the transcripts were identified with reverse transcriptase PCR (RT-PCR) using the vector-specific primer and followed by direct sequencing. The system had a greater sensitivity than those already in use for gain-of-function screening: 64% of the vector insertion lines (394/613) showed phenotypes with forced expression of vector-flanking DNA, such as lethality or defects in adult structure. Molecular analysis of 170 randomly selected insertions with forced expression phenotypes revealed that 21% matched the sequences of ...
The Gene Search System: Its Application to Functional Genomics in Drosophila Melanogaster
Journal of Neurogenetics, 2001
In Drosophila melanogaster, gain-of-function mutagenesis utilizing the GAL4-UAS system has been e... more In Drosophila melanogaster, gain-of-function mutagenesis utilizing the GAL4-UAS system has been established, allowing identification of genes that may not be easily detectable by loss-of-function screening approaches. The conditional features of misexpression systems are especially useful for studying late-stage biological processes, such as those involving adult behavior or lifespan. The gene search system, incorporating a bidirectional misexpression vector, was used to screen for genes critical for longevity determination. We have identified several genes whose misexpression in adulthood extends the fly's lifespan. Phenotypic characterization of fly lines carrying a mis-expression vector, in conjunction with obtaining information about the genomic insertion sites, creates valuable resources for the systematic functional genomics in Drosophila.
Genetics, 2004
Proper segregation of homologous chromosomes in meiosis I is ensured by pairing of homologs and m... more Proper segregation of homologous chromosomes in meiosis I is ensured by pairing of homologs and maintenance of sister chromatid cohesion. In male Drosophila melanogaster, meiosis is achiasmatic and homologs pair at limited chromosome regions called pairing sites. We screened for male meiotic mutants to identify genes required for normal pairing and disjunction of homologs. Nondisjunction of the sex and the fourth chromosomes in male meiosis was scored as a mutant phenotype. We screened 2306 mutagenized and 226 natural population-derived second and third chromosomes and obtained seven mutants representing different loci on the second chromosome and one on the third. Five mutants showed relatively mild effects (Ͻ10% nondisjunction). mei(2)yh149 and mei(2)yoh7134 affected both the sex and the fourth chromosomes, mei(2)yh217 produced possible sex chromosome-specific nondisjunction, and mei(2)yh15 and mei(2)yh137 produced fourth chromosome-specific nondisjunction. mei(2)yh137 was allelic to the teflon gene required for autosomal pairing. Three mutants exhibited severe defects, producing Ͼ10% nondisjunction of the sex and/or the fourth chromosomes. mei(2)ys91 (a new allele of the orientation disruptor gene) and mei(3)M20 induced precocious separation of sister chromatids as early as prometaphase I. mei(2)yh92 predominantly induced nondisjunction at meiosis I that appeared to be the consequence of failure of the separation of paired homologous chromosomes.
A rapid decrease in number of the complete ninja element and concomitant increase of the defective element in a strain of Drosophila simulans
Genetica, 2005
The ninja element, originally isolated from an unstable white mutant strain white-milky (w(mky)) ... more The ninja element, originally isolated from an unstable white mutant strain white-milky (w(mky)) of Drosophila simulans, is a member of the retrotransposon family with long terminal repeats (LTRs). We show that ninja is present in high copy numbers in the w(mky)-derivative sublines white-chocolate (w(cho)) and white-persimmonl (w(psm1)), in a low copy number in another derivative subline white-milky 3 (w(mky3)), and in only a few copies in a wild type strain. We have cloned the ninja elements from these sublines and examined their structures. Most of the elements cloned (38 out of 41 independent clones) from w(cho) were full length. In contrast, only 9 of 23 independent clones from w(mky3) were full length. We hypothesize that ninja elements were integrated and lost frequently in the w(mky) strain and its derivative genomes, and that a rapid decrease in numbers of the ninja element was caused not by an increased rate of loss but by a reduction of integration of full length ninja elements in w(mky3). Each defective element had a unique deletion and/or an insertion except for the three from w(mky3), which had exactly the same 81-bp deletion in each of the 5' and 3' LTRs. The 5' and 3' ends of the deletion appeared to represent sequences similar to those of Drosophila consensussplicing sites. Ectopic splicing may have produced these defective ninja elements.
Genes & Genetic Systems, 2011
Females of many animal species store sperm after copulation for use in fertilization, but the mec... more Females of many animal species store sperm after copulation for use in fertilization, but the mechanisms controlling sperm storage and utilization are largely unknown. Here we describe a novel male sterile mutation of Drosophila melanogaster, wasted (wst), which shows defects in various processes of sperm utilization. The sperm of wst mutant males are stored like those of wild-type males in the female sperm storage organs, the spermathecae and seminal receptacles, after copulation and are released at each ovulation. However, an average of thirteen times more wst sperm than wild type sperm are released at each ovulation, resulting in rapid loss of sperm stored in seminal receptacles within a few days after copulation. wst sperm can enter eggs efficiently at 5 hr after copulation, but the efficiency of sperm entry decreases significantly by 24 hr after copulation, suggesting that wst sperm lose their ability to enter eggs during storage. Furthermore, wst sperm fail to undergo nuclear decondensation, which prevents the process of fertilization even when sperm enter eggs. Our results indicate that the wst gene is essential for independent processes in the utilization of stored sperm; namely, regulation of sperm release from female storage organs, maintenance of sperm efficiency for entry into eggs, and formation of the male pronucleus in the egg at fertilization.
Genes & Genetic Systems, 2003
The male sterile mutation, misfire (mfr), of Drosophila melanogaster is a novel paternal effect, ... more The male sterile mutation, misfire (mfr), of Drosophila melanogaster is a novel paternal effect, fertilization defective mutant that effects sperm head decondensation. mfr sperm were motile, appeared normal morphologically and were transferred to the female during copulation. However, less than 0.1% of eggs laid by females mated to mfr males hatched. Although mfr sperm entered eggs at a high frequency (93%), 99% of the inseminated eggs did not initiate the first nuclear division. Unlike wild type fertilizing sperm, the position and shape of mfr sperm tails within the egg were not constant, but varied in a seemingly random manner. The heads of inseminating mutant sperm were always located near the surface of eggs just underlying the egg plasma membrane, and maintained their needle-like shape indicating the failure of nuclear decondensation. Further observations revealed that plasma membrane of inseminating sperm appeared intact, including the head region. These phenotypes were equivalent to those of sneaky (snky), another fertilization defective male sterile mutation. Our observations strongly suggest that mfr mutant males are sterile because their inseminating sperm fail to form a male pronucleus due to the inability of the sperm to properly respond to egg factors responsible for the breakdown of the plasma membrane. Although mfr and snky mutations were phenotypically identical, they mapped to cytologically distinct genetic loci and no genetic interactions were observed, suggesting that at least two distinct paternal gene products are involved in the early stages of pronuclear formation.
The Japanese Journal of Genetics, 1994
Gene, 2003
Alternative splicing is an important mechanism contributing to the increased proteome diversity i... more Alternative splicing is an important mechanism contributing to the increased proteome diversity in higher eukaryotes. We have explored the alternative splicing events in the Drosophila longitudinals lacking (lola) gene by means of 5 0 RACE, 3 0 RACE, genome sequence searches, and EST sequencing. We demonstrated that the lola locus is comprised of 32 exons spanning over 60 kb, and encodes a total of 80 alternatively spliced variants consisting of 5 0 and 3 0 variable sequences and constitutive common exons. All the variants shared a common sequence (exons 5-8) encoding the N-terminal region containing the BTB domain, but both the 5 0 and 3 0 ends were variable. There were four promoters responsible for the variation in the 5 0 end (exons 1-4). Alternative splicing was involved in the variation in the 3 0 end corresponding to the C-terminal variable region, which was encoded by one or two exons that were selected from 20 groups of exons in a mutually exclusive manner (exons 9-32). Seventeen of the 20 isoforms contained C 2 H 2-like zinc finger motifs in the C-terminal variable region. Analyses of the 3 0 variant-specific cDNA pools revealed that all combinations of 5 0 and 3 0 variable sequences were expressed in both the embryonic and third instar larval stages. Since the BTB domain mediates dimerization, lola encodes a family of transcription regulators with a large variety of DNA-or protein-binding specificities, and could be involved in various developmental processes, including the embryonic neural pathfindings. We also showed that the structures of Lola isoforms were highly conserved in Drosophila pseudoobscura.
The Gene Search System: Its Application to Functional Genomics in Drosophila Melanogaster
Journal of Neurogenetics, Feb 1, 2001
In Drosophila melanogaster, gain-of-function mutagenesis utilizing the GAL4-UAS system has been e... more In Drosophila melanogaster, gain-of-function mutagenesis utilizing the GAL4-UAS system has been established, allowing identification of genes that may not be easily detectable by loss-of-function screening approaches. The conditional features of misexpression systems are especially useful for studying late-stage biological processes, such as those involving adult behavior or lifespan. The gene search system, incorporating a bidirectional misexpression vector, was used to screen for genes critical for longevity determination. We have identified several genes whose misexpression in adulthood extends the fly's lifespan. Phenotypic characterization of fly lines carrying a mis-expression vector, in conjunction with obtaining information about the genomic insertion sites, creates valuable resources for the systematic functional genomics in Drosophila.
Genetic Analysis of Male Sterility in the Hybrid Between Drosophila Albomicans Ans D. Nasuta. IV
The Japanese Journal of Genetics, Dec 1, 1995
A comprehensive Drosophila resource to identify key functional interactions between SARS-CoV-2 factors and host proteins
Cell Reports, Aug 1, 2023
Genes & Genetic Systems, 2021
Sperm are modified substantially in passing through both the male and the female reproductive tra... more Sperm are modified substantially in passing through both the male and the female reproductive tracts, only thereafter becoming functionally competent to fertilize eggs. Drosophila sperm become motile in the seminal vesicle; after ejaculation, they interact with seminal fluid proteins and undergo biochemical changes on their surface while they are stored in the female sperm storage organs. However, the molecular mechanisms underlying these maturation processes remain largely unknown. Here, we focused on Drosophila Neprilysin genes, which are the fly orthologs of the mouse Membrane metallo-endopeptidase-like 1 (Mmel1) gene. While Mmel1 knockout male mice have reduced fertility without abnormality in either testis morphology or sperm motility, there are inconsistent results regarding the association of any Neprilysin gene with male fertility in Drosophila. We examined the association of the Nep1-5 genes with male fertility by RNAi and found that Nep4 gene function is specifically required in germline cells. To investigate this in more detail, we induced mutations in the Nep4 gene by the CRISPR/Cas9 system and isolated two mutants, both of which were viable and female fertile, but male sterile. The mutant males had normal-looking testes and sperm; during copulation, sperm were transferred to females and stored in the seminal receptacle and paired spermathecae. However, following sperm transfer and storage, three defects were observed for Nep4 mutant sperm. First, sperm were quickly discarded by the females; second, the proportion of eggs fertilized was significantly lower for mutant sperm than for control sperm; and third, most eggs laid did not initiate development after sperm entry. Taking these observations together, we conclude that the Nep4 gene is essential for sperm function following sperm transfer to females.
A cross-species approach for the identification of Drosophila male sterility genes
G3 Genes|Genomes|Genetics
Male reproduction encompasses many essential cellular processes and interactions. As a focal poin... more Male reproduction encompasses many essential cellular processes and interactions. As a focal point for these events, sperm offer opportunities for advancing our understanding of sexual reproduction at multiple levels during development. Using male sterility genes identified in human, mouse, and fruit fly databases as a starting point, 103 Drosophila melanogaster genes were screened for their association with male sterility by tissue-specific RNAi knockdown and CRISPR/Cas9-mediated mutagenesis. This list included 56 genes associated with male infertility in the human databases, but not found in the Drosophila database, resulting in the discovery of 63 new genes associated with male fertility in Drosophila. The phenotypes identified were categorized into six distinct classes affecting sperm development. Interestingly, the second largest class (Class VI) caused sterility despite apparently normal testis and sperm morphology suggesting that these proteins may have functions in the matur...
Humanized Flies and Resources for Cross-Species Study
Advances in Experimental Medicine and Biology
G3: Genes|Genomes|Genetics
In Drosophila, mature sperm are transferred from males to females during copulation, stored in th... more In Drosophila, mature sperm are transferred from males to females during copulation, stored in the sperm storage organs of females, and then utilized for fertilization. Here, we report a gene named sheepish (shps) of Drosophila melanogaster that is essential for sperm storage in females. shps mutant males, although producing morphologically normal and motile sperm that are effectively transferred to females, produce very few offspring. Direct counts of sperm indicated that the primary defect was correlated to failure of shps sperm to migrate into the female sperm storage organs. Increased sperm motion parameters were seen in the control after transfer to females, whereas sperm from shps males have characteristics of the motion parameters different from the control. The few sperm that occasionally entered the female sperm storage organs showed no obvious defects in fertilization and early embryo development. The female postmating responses after copulation with shps males appeared no...
Genetics, 1999
We have constructed a P-element-based gene search vector for efficient detection of genes in Dros... more We have constructed a P-element-based gene search vector for efficient detection of genes in Drosophila melanogaster. The vector contains two copies of the upstream activating sequence (UAS) enhancer adjacent to a core promoter, one copy near the terminal inverted repeats at each end of the vector, and oriented to direct transcription outward. Genes were detected on the basis of phenotypic changes caused by GAL4-dependent forced expression of vector-flanking DNA, and the transcripts were identified with reverse transcriptase PCR (RT-PCR) using the vector-specific primer and followed by direct sequencing. The system had a greater sensitivity than those already in use for gain-of-function screening: 64% of the vector insertion lines (394/613) showed phenotypes with forced expression of vector-flanking DNA, such as lethality or defects in adult structure. Molecular analysis of 170 randomly selected insertions with forced expression phenotypes revealed that 21% matched the sequences of ...
The Gene Search System: Its Application to Functional Genomics in Drosophila Melanogaster
Journal of Neurogenetics, 2001
In Drosophila melanogaster, gain-of-function mutagenesis utilizing the GAL4-UAS system has been e... more In Drosophila melanogaster, gain-of-function mutagenesis utilizing the GAL4-UAS system has been established, allowing identification of genes that may not be easily detectable by loss-of-function screening approaches. The conditional features of misexpression systems are especially useful for studying late-stage biological processes, such as those involving adult behavior or lifespan. The gene search system, incorporating a bidirectional misexpression vector, was used to screen for genes critical for longevity determination. We have identified several genes whose misexpression in adulthood extends the fly's lifespan. Phenotypic characterization of fly lines carrying a mis-expression vector, in conjunction with obtaining information about the genomic insertion sites, creates valuable resources for the systematic functional genomics in Drosophila.
Genetics, 2004
Proper segregation of homologous chromosomes in meiosis I is ensured by pairing of homologs and m... more Proper segregation of homologous chromosomes in meiosis I is ensured by pairing of homologs and maintenance of sister chromatid cohesion. In male Drosophila melanogaster, meiosis is achiasmatic and homologs pair at limited chromosome regions called pairing sites. We screened for male meiotic mutants to identify genes required for normal pairing and disjunction of homologs. Nondisjunction of the sex and the fourth chromosomes in male meiosis was scored as a mutant phenotype. We screened 2306 mutagenized and 226 natural population-derived second and third chromosomes and obtained seven mutants representing different loci on the second chromosome and one on the third. Five mutants showed relatively mild effects (Ͻ10% nondisjunction). mei(2)yh149 and mei(2)yoh7134 affected both the sex and the fourth chromosomes, mei(2)yh217 produced possible sex chromosome-specific nondisjunction, and mei(2)yh15 and mei(2)yh137 produced fourth chromosome-specific nondisjunction. mei(2)yh137 was allelic to the teflon gene required for autosomal pairing. Three mutants exhibited severe defects, producing Ͼ10% nondisjunction of the sex and/or the fourth chromosomes. mei(2)ys91 (a new allele of the orientation disruptor gene) and mei(3)M20 induced precocious separation of sister chromatids as early as prometaphase I. mei(2)yh92 predominantly induced nondisjunction at meiosis I that appeared to be the consequence of failure of the separation of paired homologous chromosomes.
A rapid decrease in number of the complete ninja element and concomitant increase of the defective element in a strain of Drosophila simulans
Genetica, 2005
The ninja element, originally isolated from an unstable white mutant strain white-milky (w(mky)) ... more The ninja element, originally isolated from an unstable white mutant strain white-milky (w(mky)) of Drosophila simulans, is a member of the retrotransposon family with long terminal repeats (LTRs). We show that ninja is present in high copy numbers in the w(mky)-derivative sublines white-chocolate (w(cho)) and white-persimmonl (w(psm1)), in a low copy number in another derivative subline white-milky 3 (w(mky3)), and in only a few copies in a wild type strain. We have cloned the ninja elements from these sublines and examined their structures. Most of the elements cloned (38 out of 41 independent clones) from w(cho) were full length. In contrast, only 9 of 23 independent clones from w(mky3) were full length. We hypothesize that ninja elements were integrated and lost frequently in the w(mky) strain and its derivative genomes, and that a rapid decrease in numbers of the ninja element was caused not by an increased rate of loss but by a reduction of integration of full length ninja elements in w(mky3). Each defective element had a unique deletion and/or an insertion except for the three from w(mky3), which had exactly the same 81-bp deletion in each of the 5' and 3' LTRs. The 5' and 3' ends of the deletion appeared to represent sequences similar to those of Drosophila consensussplicing sites. Ectopic splicing may have produced these defective ninja elements.
Genes & Genetic Systems, 2011
Females of many animal species store sperm after copulation for use in fertilization, but the mec... more Females of many animal species store sperm after copulation for use in fertilization, but the mechanisms controlling sperm storage and utilization are largely unknown. Here we describe a novel male sterile mutation of Drosophila melanogaster, wasted (wst), which shows defects in various processes of sperm utilization. The sperm of wst mutant males are stored like those of wild-type males in the female sperm storage organs, the spermathecae and seminal receptacles, after copulation and are released at each ovulation. However, an average of thirteen times more wst sperm than wild type sperm are released at each ovulation, resulting in rapid loss of sperm stored in seminal receptacles within a few days after copulation. wst sperm can enter eggs efficiently at 5 hr after copulation, but the efficiency of sperm entry decreases significantly by 24 hr after copulation, suggesting that wst sperm lose their ability to enter eggs during storage. Furthermore, wst sperm fail to undergo nuclear decondensation, which prevents the process of fertilization even when sperm enter eggs. Our results indicate that the wst gene is essential for independent processes in the utilization of stored sperm; namely, regulation of sperm release from female storage organs, maintenance of sperm efficiency for entry into eggs, and formation of the male pronucleus in the egg at fertilization.
Genes & Genetic Systems, 2003
The male sterile mutation, misfire (mfr), of Drosophila melanogaster is a novel paternal effect, ... more The male sterile mutation, misfire (mfr), of Drosophila melanogaster is a novel paternal effect, fertilization defective mutant that effects sperm head decondensation. mfr sperm were motile, appeared normal morphologically and were transferred to the female during copulation. However, less than 0.1% of eggs laid by females mated to mfr males hatched. Although mfr sperm entered eggs at a high frequency (93%), 99% of the inseminated eggs did not initiate the first nuclear division. Unlike wild type fertilizing sperm, the position and shape of mfr sperm tails within the egg were not constant, but varied in a seemingly random manner. The heads of inseminating mutant sperm were always located near the surface of eggs just underlying the egg plasma membrane, and maintained their needle-like shape indicating the failure of nuclear decondensation. Further observations revealed that plasma membrane of inseminating sperm appeared intact, including the head region. These phenotypes were equivalent to those of sneaky (snky), another fertilization defective male sterile mutation. Our observations strongly suggest that mfr mutant males are sterile because their inseminating sperm fail to form a male pronucleus due to the inability of the sperm to properly respond to egg factors responsible for the breakdown of the plasma membrane. Although mfr and snky mutations were phenotypically identical, they mapped to cytologically distinct genetic loci and no genetic interactions were observed, suggesting that at least two distinct paternal gene products are involved in the early stages of pronuclear formation.
The Japanese Journal of Genetics, 1994
Gene, 2003
Alternative splicing is an important mechanism contributing to the increased proteome diversity i... more Alternative splicing is an important mechanism contributing to the increased proteome diversity in higher eukaryotes. We have explored the alternative splicing events in the Drosophila longitudinals lacking (lola) gene by means of 5 0 RACE, 3 0 RACE, genome sequence searches, and EST sequencing. We demonstrated that the lola locus is comprised of 32 exons spanning over 60 kb, and encodes a total of 80 alternatively spliced variants consisting of 5 0 and 3 0 variable sequences and constitutive common exons. All the variants shared a common sequence (exons 5-8) encoding the N-terminal region containing the BTB domain, but both the 5 0 and 3 0 ends were variable. There were four promoters responsible for the variation in the 5 0 end (exons 1-4). Alternative splicing was involved in the variation in the 3 0 end corresponding to the C-terminal variable region, which was encoded by one or two exons that were selected from 20 groups of exons in a mutually exclusive manner (exons 9-32). Seventeen of the 20 isoforms contained C 2 H 2-like zinc finger motifs in the C-terminal variable region. Analyses of the 3 0 variant-specific cDNA pools revealed that all combinations of 5 0 and 3 0 variable sequences were expressed in both the embryonic and third instar larval stages. Since the BTB domain mediates dimerization, lola encodes a family of transcription regulators with a large variety of DNA-or protein-binding specificities, and could be involved in various developmental processes, including the embryonic neural pathfindings. We also showed that the structures of Lola isoforms were highly conserved in Drosophila pseudoobscura.
The Gene Search System: Its Application to Functional Genomics in Drosophila Melanogaster
Journal of Neurogenetics, Feb 1, 2001
In Drosophila melanogaster, gain-of-function mutagenesis utilizing the GAL4-UAS system has been e... more In Drosophila melanogaster, gain-of-function mutagenesis utilizing the GAL4-UAS system has been established, allowing identification of genes that may not be easily detectable by loss-of-function screening approaches. The conditional features of misexpression systems are especially useful for studying late-stage biological processes, such as those involving adult behavior or lifespan. The gene search system, incorporating a bidirectional misexpression vector, was used to screen for genes critical for longevity determination. We have identified several genes whose misexpression in adulthood extends the fly's lifespan. Phenotypic characterization of fly lines carrying a mis-expression vector, in conjunction with obtaining information about the genomic insertion sites, creates valuable resources for the systematic functional genomics in Drosophila.