Biomedical subjects

P E Kuwabara

Publications and source records attributed to P E Kuwabara.

At least 19 recordsLinked to original sources

PubMed2001

It ain't over till it's ova: germline sex determination in C. elegans.

Sex determination in most organisms involves a simple binary fate choice between male or female development; the outcome of this decision has profound effects on organismal biology, biochemistry and behaviour. In the nematode C. elegans, there is also a binary choice, either male or hermaphrodite. In C. elegans, distinct genetic pathways control somatic and germline sexual cell fate. Both pathways share a common set of globally acting regulatory genes; however, germline-specific regulatory genes also participate in the decision to make male or female gametes. The determination of sexual fate in the germline of the facultative hermaphrodite poses a special problem, because first sperm then oocytes are produced. It has emerged that additional layers of post-transcriptional regulation have been imposed to modulate the activities of the global sex-determining genes, tra-2 and fem-3; the balance between these activities is crucial in controlling sexual cell fate in the hermaphrodite germline.

Animals↗

PubMed2001

The use of functional genomics in C. elegans for studying human development and disease.

The 100 Mb Caenorhabditis elegans genome sequence is the first animal genome to be sequenced in its entirety. Many reverse-genetics tools have been developed to mine the genome sequence and to facilitate the jump between the identification of a gene sequence and the understanding of its function. Here we discuss how C. elegans can contribute to understanding of the function of genes involved in human development and disease.

Animals↗

PubMed2001

RNA-mediated interference as a tool for identifying drug targets.

The nematode Caenorhabditis elegans is the first multicellular organism with a fully sequenced genome. As a model organism, C. elegans is playing a special role in functional genomic analyses because it is experimentally tractable on many levels. Moreover, the lessons learned from C. elegans are often applicable across phyla because many of the key biologic processes involved in development and disease have been well conserved. Many global approaches for analysing gene activity are being pursued in C. elegans. RNA-mediated interference (RNAi) is an efficient high-throughput method to disrupt gene function. The basic technique of RNAi involves introducing sequence-specific double-stranded RNA into C. elegans in order to generate a nonheritable, epigenetic knockout of gene function that phenocopies a null mutation in the targeted gene. This technique drastically reduces the time needed to jump from the identification of an interesting gene sequence to achieving an understanding of its function. Thus, RNAi facilitates the high-throughput functional analysis of gene targets identified during drug discovery. RNAi can also help to identify the biochemical mode of action of a drug or pesticide and to identify other genes encoding products that may respond or interact with specific compounds.

Animals↗

PubMed2000

Direct protein-protein interaction between the intracellular domain of TRA-2 and the transcription factor TRA-1A modulates feminizing activity in C. elegans.

In the nematode Caenorhabditis elegans, the zinc finger transcriptional regulator TRA-1A directs XX somatic cells to adopt female fates. The membrane protein TRA-2A indirectly activates TRA-1A by binding and inhibiting a masculinizing protein, FEM-3. Here we report that a part of the intracellular domain of TRA-2A, distinct from the FEM-3 binding region, directly binds TRA-1A. Overproduction of this TRA-1A-binding region has tra-1-dependent feminizing activity in somatic tissues, indicating that the interaction enhances TRA-1A activity. Consistent with this hypothesis, we show that tra-2(mx) mutations, which weakly masculinize somatic tissues, disrupt the TRA-2/TRA-1A interaction. Paradoxically, tra-2(mx) mutations feminize the XX germ line, as do tra-1 mutations mapping to the TRA-2 binding domain. We propose that these mutations render tra-2 insensitive to a negative regulator in the XX germ line, and we speculate that this regulator targets the TRA-2/TRA-1 complex. The intracellular domain of TRA-2A is likely to be produced as a soluble protein in vivo through proteolytic cleavage of TRA-2A or through translation of an XX germ line-specific mRNA. We further show that tagged derivatives of the intracellular domain of TRA-2 localize to the nucleus, supporting the hypothesis that this domain is capable of modulating TRA-1A activity in a manner reminiscent of Notch and Su(H).

Animals↗

PubMed2000

A C. elegans patched gene, ptc-1, functions in germ-line cytokinesis.

Patched (Ptc), initially identified in Drosophila, defines a class of multipass membrane proteins that control cell fate and cell proliferation. Biochemical studies in vertebrates indicate that the membrane proteins Ptc and Smoothened (Smo) form a receptor complex that binds Hedgehog (Hh) morphogens. Smo transduces the Hh signal to downstream effectors. The Caenorhabditis elegans genome encodes two Ptc homologs and one related pseudogene but does not encode obvious Hh or Smo homologs. We have analyzed ptc-1 by RNAi and mutational deletion and find that it is an essential gene, although the absence of ptc-1 has no detectable effect on body patterning or proliferation. Therefore, the C. elegans ptc-1 gene is functional despite the lack of Hh and Smo homologs. We find that the activity and expression of ptc-1 is essentially confined to the germ line and its progenitors. ptc-1 null mutants are sterile with multinucleate germ cells arising from a probable cytokinesis defect. We have also identified a surprisingly large family of PTC-related proteins containing sterol-sensing domains, including homologs of Drosophila dispatched, in C. elegans and other phyla. These results suggest that the PTC superfamily has multiple functions in animal development.

Amino Acid Sequence↗

PubMed2000

Proteolysis in Caenorhabditis elegans sex determination: cleavage of TRA-2A by TRA-3.

The Caenorhabditis elegans tra-3 gene promotes female development in XX hermaphrodites and encodes an atypical calpain regulatory protease lacking calcium-binding EF hands. We report that despite the absence of EF hands, TRA-3 has calcium-dependent proteolytic activity and its proteolytic domain is essential for in vivo function. We show that the membrane protein TRA-2A, which promotes XX female development by repressing the masculinizing protein FEM-3, is a TRA-3 substrate. Cleavage of TRA-2A by TRA-3 generates a peptide predicted to have feminizing activity. These results indicate that proteolysis regulated by calcium may control some aspects of sexual cell fate in C. elegans.

Animals↗

PubMed2000

RNAi--prospects for a general technique for determining gene function.

Gene discovery programs centred around expressed sequence tag (EST) and genome sequencing projects have predictably led to an exponential surge in the number of parasite gene sequences deposited in public databases. To take advantage of this wealth of sequence information, it is essential to develop rapid methods for elucidating the biological function or mode of action of individual genes. Here, Patricia Kuwabara and Alan Coulson discuss the virtues of a powerful epigenetic gene disruption technique, RNA-mediated interference (RNAi), which was originally developed for the nematode Caenorhabditis elegans. It is anticipated that this technique will not only provide insights into gene function, but also help investigators to mine the genome for candidate drug intervention or vaccine development targets, some of which may not be readily apparent on the basis of sequence information alone.

Animals↗

PubMed1999

Negative regulation of male development in Caenorhabditis elegans by a protein-protein interaction between TRA-2A and FEM-3.

The tra-2 gene of the nematode Caenorhabditis elegans encodes a predicted membrane protein, TRA-2A, that promotes XX hermaphrodite development. Genetic analysis suggests that tra-2 is a negative regulator of three genes that are required for male development: fem-1, fem-2, and fem-3. We report that the carboxy-terminal region of TRA-2A interacts specifically with FEM-3 in the yeast two-hybrid system and in vitro. Consistent with the idea that FEM-3 is a target of negative regulation, we find that excess FEM-3 can overcome the feminizing effect of tra-2 and cause widespread masculinization of XX somatic tissues. In turn, we show that the masculinizing effects of excess FEM-3 can be suppressed by overproduction of the carboxy-terminal domain of TRA-2A. A FEM-3 fragment that retains TRA-2A-binding activity can masculinize fem-3(+) animals, but not fem-3 mutants, suggesting that it is possible to release and to activate endogenous FEM-3 by titrating TRA-2A. We propose that TRA-2A prevents male development by interacting directly with FEM-3 and that a balance between the opposing activities of TRA-2A and FEM-3 determines sex-specific cell fates in somatic tissues. When the balance favors FEM-3, it acts through or with the other FEM proteins to promote male cell fates.

Animals↗

PubMed1999

Regulation of dauer larva development in Caenorhabditis elegans by daf-18, a homologue of the tumour suppressor PTEN.

The tumour suppressor gene PTEN (also called MMAC1 or TEP1) is somatically mutated in a variety of cancer types [1] [2] [3] [4]. In addition, germline mutation of PTEN is responsible for two dominantly inherited, related cancer syndromes called Cowden disease and Bannayan-Ruvalcaba-Riley syndrome [4]. PTEN encodes a dual-specificity phosphatase that inhibits cell spreading and migration partly by inhibiting integrin-mediated signalling [5] [6] [7]. Furthermore, PTEN regulates the levels of phosphatidylinositol 3,4,5-trisphosphate (PIP3) by specifically dephosphorylating position 3 on the inositol ring [8]. We report here that the dauer formation gene daf-18 is the Caenorhabditis elegans homologue of PTEN. DAF-18 is a component of the insulin-like signalling pathway controlling entry into diapause and adult longevity that is regulated by the DAF-2 receptor tyrosine kinase and the AGE-1 PI 3-kinase [9]. Others have shown that mutation of daf-18 suppresses the life extension and constitutive dauer formation associated with daf-2 or age-1 mutants. Similarly, we show that inactivation of daf-18 by RNA-mediated interference mimics this suppression, and that a wild-type daf-18 transgene rescues the dauer defect. These results indicate that PTEN/daf-18 antagonizes the DAF-2-AGE-1 pathway, perhaps by catalyzing dephosphorylation of the PIP3 generated by AGE-1. These data further support the notion that mutations of PTEN contribute to the development of human neoplasia through an aberrant activation of the PI 3-kinase signalling cascade.

Animals↗

PubMed1999

Developmental genetics of Caenorhabditis elegans sex determination.

The nematode Caenorhabditis elegans has two naturally occurring sexes: a self-fertile XX hermaphrodite that first produces sperm, then oocytes, and an XO male. The primary determinant of sex is the X:A ratio, the number of X chromosomes to sets of autosomes. The X:A ratio regulates not only sex determination, but also dosage compensation. In the intervening years since the identification of the X:A ratio, most of the key regulatory genes that respond to the X:A ratio have been genetically identified and ordered into regulatory hierarchies. Advances have also been made in identifying the X chromosome numerator elements of the X:A ratio. This review highlights the genetic, molecular, and biochemical approaches that have led to an understanding of how these genes interact to control sex determination and dosage compensation. The review also discusses the differences between the control of sexual cell fate in the soma and germ line of C. elegans and addresses the role of germ-line-specific regulation in controlling the sperm-oocyte decision in the hermaphrodite germ line. Finally, strategies that take advantage of the availability of the entire C. elegans genome sequence, which is expected to be completed in 1998, are discussed for identifying hitherto unidentified genes that may play a role in the control of sexual cell fate.

Animals↗

PubMed1998

Germ-line regulation of the Caenorhabditis elegans sex-determining gene tra-2.

The Caenorhabditis elegans sex-determining gene tra-2 promotes female development of the XX hermaphrodite soma and germ line. We previously showed that a 4.7-kb tra-2 mRNA, which encodes the membrane protein TRA-2A, provides the primary feminizing activity of the tra-2 locus. This paper focuses on the germ-line activity and regulation of tra-2. First, we characterize a 1.8-kb tra-2 mRNA, which is hermaphrodite-specific and germ-line-dependent. This mRNA encodes TRA-2B, a protein identical to a predicted intracellular domain of TRA-2A. We show that the 1.8-kb mRNA is oocyte-specific, suggesting that it is involved in germ-line or embryonic sex determination. Second, we identify a tra-2 maternal effect on brood size that may be associated with the 1.8-kb mRNA. Third, we investigate seven dominant tra-2(mx) (for mixed character) mutations that sexually transform hermaphrodites to females by eliminating hermaphrodite spermatogenesis. Each of the tra-2(mx) mutants possesses a nonconserved missense change in a 22-amino-acid region common to both TRA-2A and TRA-2B, called the MX region. We propose that the MX region mediates a posttranslational regulation of tra-2 essential for the onset of hermaphrodite spermatogenesis. Finally, we discuss aspects of tra-2 function and regulation that are specific to the unusual control of cell fate in the hermaphrodite germ line.

Amino Acid Sequence↗

PubMed1998

Gametogenesis: keeping the male element under control.

Caenorhabditis elegans hermaphrodites switch from making sperm to oocytes. This switch involves repression of fem-3 mRNA, mediated by a protein that binds RNA through a conserved motif; a similar motif mediates RNA binding by the Drosophila pattern-regulatory protein Pumilio.

Animals↗

PubMed1997

Worming your way through the genome.

The 100 Mb sequence of the nematode Caenorhabditis elegans genome will be completed in 1998. More than 10,000 predicted genes have been identified to date, so it should come as no surprise to find a C. elegans homologue of your favourite gene in current databases. For some investigators, the discovery of a C. elegans homologue represents a unique opportunity to adopt a genetic approach and to take advantage of the extensive repertoire of C. elegans gene characterization and manipulation tools. RNA injection provides a quick and efficient method for obtaining clues about wild-type gene function. Reverse genetic approaches also make it feasible to screen de novo for mutations in specific gene sequences. This review highlights the resources available for analysing a C. elegans homologue, starting from the gene sequence and proceeding to the biological function.

Animals↗

PubMed1996

Interspecies comparison reveals evolution of control regions in the nematode sex-determining gene tra-2.

The Caenorhabditis elegans sex-determining gene tra-2 promotes female development and expresses 4.7-, 1.9- and 1.8-kb mRNAs. The 4.7-kb mRNA encodes the major feminizing activity of the locus, a predicted membrane receptor that mediates cell-to-cell communication, named TRA-2A. The tra-2 gene was characterized from a close relative, C. briggsae. The Cb-tra-2 gene expresses only a 4.7-kb mRNA and alternatively spliced variants, which encode TRA-2A homologues. The Cb-TRA-2A and Ce-TRA-2A sequences are highly diverged, sharing only 43% identity, although their hydropathy profiles remain remarkably similar. Three potential regulatory sites of Ce-tra-2 activity were previously identified by analyzing tra-2(eg), tra-2(gf), and tra-2(mx) mutations. Two of these sites, the EG site and MX region, are conserved in Cb-tra-2. By contrast, the two direct repeat elements in the Ce-tra-2 3' untranslated region, which are disrupted in tra-2(gf) mutants, are absent. Injection of Cb-tra-2 antisense RNA into C.briggsae mimics the Ce-tra-2 loss-of-function phenotype. Thus, antisense RNA permits studies of gene activity in nematodes that lack extensive genetics.

Alternative Splicing↗

PubMed1996

A novel regulatory mutation in the C. elegans sex determination gene tra-2 defines a candidate ligand/receptor interaction site.

Sex determination in the nematode C. elegans is dependent on cell-to-cell communication, which appears to be mediated by the predicted membrane protein TRA-2A and the secreted protein HER-1. In XO males, HER-1 is hypothesised to function as a repressive ligand that inactivates the TRA-2A receptor. In XX animals, HER-1 is absent and TRA-2A promotes hermaphrodite development by negatively regulating the FEM proteins. This paper describes the molecular and genetic characterisation of a novel class of feminising mutations called tra-2(eg), for enhanced gain-of-function. In XX animals, mutant tra-2(eg) activity promotes entirely normal hermaphrodite development. However, the tra-2(eg) mutations generate an XO-specific gain-of-function phenotype, because they transform XO mutants from male into hermaphrodite. Therefore, the tra-2(eg) mutations identify a major regulatory site, which may be the TRA-2A/HER-1 interaction site. All ten tra-2(eg) mutations encode identical missense changes in a predicted extracellular domain of TRA-2A, named the EG site. It is proposed that the tra-2(eg) mutation encodes a TRA-2A protein that functions constitutively in XO animals, because it is defective in HER-1 binding. Phenotypic characterisation of sexually transformed XO tra-2(eg) hermaphrodites reveals that their fertility is strongly affected by dosage compensation mutations, suggesting that dosage compensation plays a role in normal gametogenesis.

Animals↗

PubMed1995

A predicted membrane protein, TRA-2A, directs hermaphrodite development in Caenorhabditis elegans.

The nematode C. elegans naturally develops as either an XO male or XX hermaphrodite. The sex-determining gene, tra-2, promotes hermaphrodite development in XX animals. This gene encodes a predicted membrane protein, named TRA-2A, which has been proposed to provide the primary feminising activity of the tra-2 locus. Here, we show that transgenic TRA-2A driven from a heat shock promoter can fully feminise the somatic tissues of XX tra-2 loss-of-function mutants, which would otherwise develop as male. TRA-2A is thus likely to provide a component of the tra-2 locus that is both necessary and sufficient to promote female somatic development. Transgenic TRA-2A driven by the heat shock promoter can also transform XO animals from male to self-fertile hermaphrodite. This result establishes the role of tra-2 as a developmental switch that controls somatic sexual cell fate. We show that a carboxy-terminal region of TRA-2A, predicted to be intra-cellular, can partially feminise XX tra-2 loss-of-function mutants and XO tra-2(+) males. We suggest that this intra-cellular domain of TRA-2A promotes hermaphrodite development by negatively regulating the FEM proteins.

Animals↗

PubMed1994

Cloning by synteny: identifying C. briggsae homologues of C. elegans genes.

Phylogenetic comparisons of gene and protein sequences between related species are often used to identify evolutionarily conserved elements that are important for gene expression, function, or regulation. However, homologoues may sometimes be difficult to identify by conventional low stringency hybridisation techniques, if they have undergone substantial sequence divergence. A new approach, cloning by synteny, is described that was used to identify the C. briggsae homologue of the C. elegans sex-determining gene tra-2. We show that four genes tra-2, ppp-1, art-1, and sod-1 are organised in a syntenic cluster and suggest that extensive conservation of gene linkage may exist between C. briggsae and C. elegans. We have also constructed a C. briggsae cDNA library to facilitate characterisation of these genes. Given the rapid progress in the physical mapping and sequencing of the C. elegans genome, cloning by synteny may provide the fastest method for identifying C. briggsae gene homologues, especially for genes encoding novel proteins.

Amino Acid Sequence↗

PubMed1992

Molecular genetics of sex determination in C. elegans.

Sexual fate in the nematode Caenorhabditis elegans is controlled by a group of genetically well-characterized genes. Several of these sex-determining genes have now been analysed at the molecular level. Transcriptional regulation is likely to control both commitment to a single sexual fate and maintenance of that decision; in addition, intercellular signalling appears to coordinate the sexual fates of cells throughout the animal to adopt a single sexual fate.

Animals↗