Biomedical subjects
J Hodgkin
Publications and source records attributed to J Hodgkin.
Genetic and molecular analysis of fox-1, a numerator element involved in Caenorhabditis elegans primary sex determination.
fox-1 was previously identified as a candidate numerator element based on its overexpression phenotype. FOX-1 is an RRM-type RNA-binding protein, which can bind RNAs in vitro. Western analysis detects FOX-1 throughout development. fox-1::lacZ comes on ubiquitously early during embryogenesis. Postembryonically, fox-1::lacZ is expressed sex specifically in a subset of cells in the head and tail. We describe a Tc1-derived deletion allele [fox-1(Delta)] that removes the RRM domain. fox-1(Delta) confers no phenotype in XXs, but can rescue XO-specific lethality and feminization caused by duplications of the left end of the X. fox-1(Delta) synergizes with putative numerators, resulting in abnormal XX development. Genetic analysis indicated that fox-1(Delta) leads to a slight increase in xol-1 activity, while fox-1(gf) leads to partial loss of xol-1 activity, and xol-1 is epistatic to fox-1. RNase protection experiments revealed increased levels of the 2.2-kb xol-1 message in fox-1(Delta) animals, and reduced levels in fox-1(gf) animals. Additionally, fox-1(Delta) impairs male mating efficiency, which, we propose, represents another function of fox-1, independent of xol-1 and its role in sex determination.
Evidence for evolutionary conservation of sex-determining genes.
Most metazoans occur as two sexes. Surprisingly, molecular analyses have hitherto indicated that sex-determining mechanisms differ completely between phyla. Here we present evidence to the contrary. We have isolated the male sexual regulatory gene mab-3 from the nematode Caenorhabditis elegans and found that it is related to the Drosophila melanogaster sexual regulatory gene doublesex (dsx)2. Both genes encode proteins with a DNA-binding motif that we have named the 'DM domain'. Both genes control sex-specific neuroblast differentiation and yolk protein gene transcription; dsx controls other sexually dimorphic features as well. The form of DSX that is found in males can direct male-specific neuroblast differentiation in C. elegans. This structural and functional similarity between phyla suggests a common evolutionary origin of at least some aspects of sexual regulation. We have identified a human gene, DMT1, that encodes a protein with a DM domain and find that DMT1 is expressed only in testis. DMT1 maps to the distal short arm of chromosome 9, a location implicated in human XY sex reversal. Proteins with DM domains may therefore also regulate sexual development in mammals.
Changing styles in C. elegans genetics.
The past 30 years have taken the nematode Caenorhabditis elegans from obscurity, as a nondescript member of a large but unglamorous invertebrate phylum, to a position as one of the major model organisms. This year, it will acquire a particular celeberity as the owner of the first animal genome to be sequenced in its entirety. In this review we consider the ways in which genetical investigations of this species have begun to change and what some of the consequences of the completion of the sequence are likely to be.
Seven types of pleiotropy.
Pleiotropy, a situation in which a single gene influences multiple phenotypic traits, can arise in a variety of ways. This paper discusses possible underlying mechanisms and proposes a classification of the various phenomena involved.
Natural variation and copulatory plug formation in Caenorhabditis elegans.
Most of the available natural isolates of the nematode Caenorhabditis elegans have been examined and compared with the standard laboratory wild type (Bristol N2). Molecular markers, in particular transposon restriction fragment length polymorphisms, were used to assign these isolates to 22 different races, for which brood size and spontaneous male frequency were determined. Several distinctive traits were observed in some of these races. One example is mab-23, in a race from Vancouver, which leads to severe distortion of male genitalia and prevents male mating. Another is gro-1, segregating in a Californian race, which is associated with slow growth, heat resistance and longevity. Many races differ from N2 in carrying a dominant allele at the plg-1 locus, causing copulatory plug formation by males. Properties and possible advantages of the plugging trait have been investigated. The dominant plg-1 allele does not lead to increased male mating efficiency, but males from a Stanford race (CB4855), in which the plugging trait was first observed, are much more virile than N2 males. Crosses between N2 and CB4855 indicate that the higher virility is due to multiple factors. Size differences between N2 and CB4855 are associated with factors mapping to LGV and LGX.
The tra-3 sex determination gene of Caenorhabditis elegans encodes a member of the calpain regulatory protease family.
The Caenorhabditis elegans sex determination gene tra-3 is required for the correct sexual development of the soma and germ line in hermaphrodites, while being fully dispensable in males. Genetic analysis of tra-3 has suggested that its product may act as a potentiator of another sex determination gene, tra-2. Molecular analysis reported here reveals that the predicted tra-3 gene product is a member of the calpain family of calcium-regulated cytosolic proteases, though it lacks the calcium binding regulatory domain. Calpains are regulatory processing proteases, exhibiting marked substrate specificity, and mutations in the p94 isoform underlie the human hereditary condition limb-girdle muscular dystrophy type 2A. The molecular identity of TRA-3 is consistent with previous genetic analysis which suggested that tra-3 plays a very selective modulatory role and is required in very small amounts. Based on these observations and new genetic data, we suggest a refinement of the position of tra-3 within the sex determination cascade and discuss possible mechanisms of action for the TRA-3 protein.
Evolution of sex determination in caenorhabditis: unusually high divergence of tra-1 and its functional consequences.
The tra-1 gene is a terminal regulator of somatic sex in Caenorhabditis elegans: high tra-1 activity elicits female development, low tra-1 activity elicits male development. To investigate the function and evolution of tra-1, we examined the tra-1 gene from the closely related nematode C. briggsae. Ce-tra-1 and Cb-tra-1 are unusually divergent. Each gene generates two transcripts, but only one of these is present in both species. This common transcript encodes TRA-1A, which shows only 44% amino acid identity between the species, a figure much lower than that for previously compared genes. A Cb-tra-1 transgene rescues many tissues of tra-1(null) mutants of C. elegans but not the somatic gonad or germ line. This transgene also causes nongonadal feminization of XO animals, indicating incorrect sexual regulation. Alignment of Ce-TRA-1A and Cb-TRA-1A defines several conserved regions likely to be important for tra-1 function. The phenotypic differences between Ce-tra-1(null) mutants rescued by Cb-tra-1 transgenes and wild-type C. elegans indicate significant divergence of regulatory regions. These molecular and functional studies suggest that evolution of sex determination in nematodes is rapid and genetically complex.
The nematode Caenorhabditis elegans and its genome.
Over the past two decades, the small soil nematode Caenorhabditis elegans has become established as a major model system for the study of a great variety of problems in biology and medicine. One of its most significant advantages is its simplicity, both in anatomy and in genomic organization. The entire haploid genetic content amounts to 100 million base pairs of DNA, about 1/30 the size of the human value. As a result, C. elegans has also provided a pilot system for the construction of physical maps of larger animal and plant genomes, and subsequently for the complete sequencing of those genomes. By mid-1995, approximately one-fifth of the complete DNA sequence of this animal had been determined. Caenorhabditis elegans provides a test bed not only for the development and application of mapping and sequencing technologies, but also for the interpretation and use of complete sequence information. This article reviews the progress so far toward a realizable goal--the total description of the genome of a simple animal.
Genome maps. VI. Caenorhabditis elegans. Wall chart.
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Dominant feminizing mutations implicate protein-protein interactions as the main mode of regulation of the nematode sex-determining gene tra-1.
The tra-1 gene is the terminal global selector of somatic sex in Caenorhabditis elegans: High tra-1 activity elicits female somatic development while low tra-1 activity elicits male development. Previous genetic studies defined a cascade of negatively interacting genes that regulates tra-1 activity in response to the primary sex-determining signal. Here, we investigate the last step in this regulatory cascade, by studying rare gain-of-function (gf) mutations of tra-1 that direct female somatic development irrespective of the upstream sex-determining signal. These mutations appear to abolish negative regulation of tra-1 in male tissues. We identify the lesions associated with 29 of these mutations and find that all affect a short stretch of amino acid residues present in both protein products of the tra-1 gene. Twenty-six alleles are associated with single nonconservative amino acid substitutions. Two alleles affect tra-1 RNA splicing and generate messages that omit part or all of the exon encoding this short stretch. These results suggest that sexual regulation of tra-1 is achieved post-translationally, by an inhibitory protein-protein interaction. The amino acid stretch altered by the tra-1(gf) mutations may define a site of interaction for negative regulators of tra-1. The stretch includes a potential phosphorylation site for glycogen synthase kinase 3 and may be conserved in the human gene GLI3, a homolog of tra-1 identified previously.
Isolation of dominant XO-feminizing mutations in Caenorhabditis elegans: new regulatory tra alleles and an X chromosome duplication with implications for primary sex determination.
A strain of Caenorhabditis elegans was constructed that permits selection of dominant or sex-linked mutations that transform XO animals (normally male) into fertile females, using a feminizing mutation, tra-2(e2046gf), which by itself does not sexually transform XO males. Twenty-three mutations were isolated after chemical mutagenesis and found to fall into both expected classes (four dominant tra-1 mutations and eight recessive xol-1 mutations) and novel classes. The novel mutations include 10 second-site mutations of tra-2, which are called eg mutations, for enhanced gain-of-function. The tra-2(gf, eg) alleles lead to complete dominant transformation of XO animals from fertile male into fertile female. Also isolated was a duplication of the left end of the X chromosome, eDp26, which has dominant XO lethal and feminizing properties, unlike all previously isolated duplications of the X chromosome. The properties of eDp26 indicate that it carries copies of one or more numerator elements, which act as part of the primary sex-determination signal, the X:A ratio. The eDp26 duplication is attached to the left tip of the X chromosome in inverted orientation and consequently can be used to generate unstable attached-X chromosomes.
Genetic nomenclature guide. Caenorhabditis elegans.
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Regulatory rearrangements and smg-sensitive alleles of the C. elegans sex-determining gene tra-1.
The tra-1 gene is the terminal regulator in the sex determination pathway in C. elegans, directing all aspects of somatic sexual differentiation. Recessive loss-of-function (lf) mutations in tra-1 masculinize XX animals (normally somatically female), while dominant gain-of-function mutations feminize XO animals (normally male). Most tra-1 (lf) mutations can be fitted into a simple allelic series of somatic masculinization, but a small number of lf alleles do not fit into this series. Here we show that three of these mutations are associated with DNA rearrangements 5' to the coding region. One allele is an inversion that may be subject to a position effect. We also report the isolation of a new class of tra-1 alleles that are responsive to mutations in the smg system of RNA surveillance. We show that two of these express RNAs of aberrant size. We suggest that the smg-sensitive mutations may identify a carboxy-terminal domain required for negative regulation of tra-1 activity.
Epigenetics and the maintenance of gene activity states in Caenorhabditis elegans.
The nematode Caenorhabditis elegans has been the subject of many detailed investigations in developmental biology. Molecular analyses have failed to detect covalent alterations to DNA, such as methylation or rearrangement, during development of C. elegans. Genetic experiments indicate that imprinting of gamete genomes does not occur to any significant extent. The maintenance of gene activity states in this organism may depend predominantly on regulatory gene circuitry. Some possible examples of maintenance circuits are discussed.
Identification of a candidate primary sex determination locus, fox-1, on the X chromosome of Caenorhabditis elegans.
Sex in Caenorhabditis elegans (XX hermaphrodite, XO male) is determined by the X:A ratio, which is the ratio of X chromosome number to autosomal set number. Recent genetic results with X chromosome duplications have suggested that there may be only a small number of major numerator sites on the X chromosome that contribute to this ratio. Mapping of duplication endpoints delimited a region of less than 300 kb, likely to contain one such element. Cosmid clones from this region were tested for numerator activity by constructing transgenic lines carrying extra copies of each tested cosmid. Most cosmid arrays have no effect on the viability of either XX or XO animals. One cosmid array was found to be viable in XX animals, but lethal and feminizing in XO animals, consistent with it containing a major numerator element. Further experiments defined a region of 12-30 kb with apparent numerator activity, which is designated fox-1, 'Feminizing locus On X'. A cDNA clone hybridizing across part of this region encodes a predicted RNA-binding protein.
Zinc fingers in sex determination: only one of the two C. elegans Tra-1 proteins binds DNA in vitro.
The tra-1 gene of Caenorhabditis elegans is a major developmental regulator that promotes female development. Two mRNAs are expressed from the tra-1 locus as a result of alternative mRNA processing. One mRNA encodes a protein with five zinc fingers and the other a protein with only the first two zinc fingers. We have derived a preferred in vitro DNA binding site for the five finger protein by selection from random oligonucleotides. The two finger protein does not bind to DNA in vitro. Moreover, removal of the first two fingers from the five finger protein does not eliminate binding and has little effect on its preferred binding site. We find that a protein sequence amino-terminal to the finger domain also appears to play a role in DNA binding.
Fluxes, doses and poisons: molecular perspectives on dominance.
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