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.

A novel bacterial pathogen, Microbacterium nematophilum, induces morphological change in the nematode C. elegans.

The Dar (deformed anal region) phenotype, characterized by a distinctive swollen tail, was first detected in a variant strain of Caenorhabditis elegans which appeared spontaneously in 1986 during routine genetic crosses [1,2]. Dar isolates were initially analysed as morphological mutants, but we report here that two independent isolates carry an unusual bacterial infection different from those previously described [3], which is the cause of the Dar phenotype. The infectious agent is a new species of coryneform bacterium, named Microbacterium nematophilum n. sp., which fortuitously contaminated cultures of C. elegans. The bacteria adhere to the rectal and post-anal cuticle of susceptible nematodes, and induce substantial local swelling of the underlying hypodermal tissue. The swelling leads to constipation and slowed growth in the infected worms, but the infection is otherwise non-lethal. Certain mutants of C. elegans with altered surface antigenicity are resistant to infection. The induced deformation appears to be part of a survival strategy for the bacteria, as C. elegans are potentially their predators.

ETR-1, a homologue of a protein linked to myotonic dystrophy, is essential for muscle development in Caenorhabditis elegans.

Post-transcriptional gene processing by RNA-binding proteins (RBPs) has crucial roles during development [1] [2]. Here, we report the identification of ETR-1 (ELAV-type RNA-binding protein), a muscle-specific RBP in the nematode Caenorhabditis elegans. ETR-1 is related to the family of RBPs defined by the protein ELAV, which is essential for neurogenesis in the fruit fly Drosophila; members of the family possess two consecutive RNA recognition motifs (RRMs) separated from a third, carboxy-terminal RRM by a tether region of variable length [3] [4] [5] [6]. Its closest homologue, CUG-binding protein (CUG-bp), is a human RBP that has been implicated in the disease myotonic dystrophy and binds CUG repeats in the 3' untranslated region (UTR) of the mRNA for myotonic dystrophy protein kinase (DMPK) [7] [8]. Inactivation of etr-1 by RNA-mediated interference resulted in embryonic lethality. Embryos failed to elongate and became paralysed, a phenotype characteristic of C. elegans Pat mutants, which are defective in muscle formation and function [9]. The data indicate that etr-1 is essential for muscle development in C. elegans, perhaps by playing a role in post-transcriptional processing of some muscle component, and thus suggesting a possible conservation of gene function with human CUG-bp.

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.

Differential expression of five tRNA(UAGTrp) amber suppressors in Caenorhabditis elegans.

Caenorhabditis elegans has 12 tRNA(UGGTrp) genes as defined by Southern analysis. In order to evaluate the function of the individual members of this multigene family, we sought to recover amber (UAG)-suppressing mutations from reversion experiments with animals carrying amber mutations in a nervous system-affecting gene (unc-13) or a sex-determining gene (tra-3). Revertants were analyzed by Southern blot, exploiting the fact that the CCA to CTA change at the anticodon creates a new XbaI site. Five different members of the tRNATrp gene family were identified as suppressors: sup-7 X, sup-5 III, sup-24 IV, sup-28 X, and sup-29 IV. All five suppressor genes were sequenced and found to encode identical tRNA(UAGTrp) molecules with a single base change (CCA to CTA) at the anticodon compared with their wild-type counterparts. The flanking sequences had only limited homology. The relative expression of these five genes was determined by measuring the efficiencies of suppressers against amber mutations in genes affecting the nervous system, hypodermis, muscle, and sex determination. The results of these cross-suppression tests showed that the five members of the tRNA(Trp) gene family were differentially regulated in a tissue- or development stage-specific manner.

Genetic and molecular analysis of eight tRNA(Trp) amber suppressors in Caenorhabditis elegans.

Over 100 revertants of five different amber mutants were analyzed by Southern blot hybridization using synthetic oligomers as probes to detect a single base change at the anticodon, CCA to CTA (amber), of tRNA(Trp) genes of Caenohrabditis elegans. Of the 12 members of the tRNA(Trp) gene family, a total of eight were converted to amber suppressor alleles. All eight encode identical tRNAs; three of these are new tRNA(Trp) suppressors, sup-21, sup-33 and sup-34. Previous results had suggested that individual suppressor tRNA genes were expressed differentially in a cell-type- or developmental stage-specific manner. To extend these observations to the new genes and to test the specificity of expression against additional genes, cross suppression tests of these eight amber suppressors were carried out against amber mutations in several different genes including genes likely to be expressed in the same cell-type: three nervous system-affecting genes, two muscle structure-affecting genes and two genes presumed to be expressed in hypodermis. Seven out of eight suppressors could be distinguished one from another by the spectrum of their suppression efficiencies. These results also provide further evidence of cell-type-specific patterns of expression in the nervous system, muscle and hypodermis. The suppression pattern of the suppressor against the two muscle-affecting genes, unc-15 and unc-52, suggested that either the suppressors are expressed in a developmental stage-specific manner or that the unc-52 products are expressed in cell-types other than muscle, possibly hypodermis.

More sex-determination mutants of Caenorhabditis elegans.

Sex determination in Caenorhabditis elegans is controlled by the X chromosome : autosome ratio, i.e. 2A;XX animals are hermaphrodite, and 2A;XO animals are male. A procedure for isolating 2A;XO animals that are transformed in;to hermaphrodites has been developed. Nine mutations causing this transformation have been obtained: eight are recessive, and all of these fall into a new autosomal complementation group, her-1 V. The remaining mutation (her-2) is dominant and has a genetic map location similar to that of tra-1 III. Recessive mutations of tra-1 cause the reverse transformation, transforming 2A;XX animals into males. Therefore, the her-2 mutation may result in constitutive expression of tra-1. Mutations in her-1 are without effect on XX animals, but the her-2 mutation prevents sperm production in both XX and XO animals, in addition to its effect on the sexual phenotype of XO animals. The epistatic relationships between tra and her genes are used to deduce a model for the action of these genes in controlling sex determination.

Novel nematode amber suppressors.

Nine amber suppressor mutations were isolated in the nematode Caenorhabditis elegans by reverting amber alleles of a sex-determining gene, tra-3. One suppressor maps to a known locus, sup-5 III , but the other eight map to three new loci, sup-21 X (five alleles), sup-22 IV (two alleles) and sup-23 IV (one allele). Amber alleles of tra-3 and of a dumpy gene, dpy-20, were used to measure the efficiency of suppression; the sup-21 and the sup-22 alleles were both shown to be heterogeneous and generally weaker suppressors than sup-5 alleles, which are homogeneous. The spectrum of mutations suppressed by a strong sup-21 allele, e1957, was investigated and compared to the spectra for the amber suppressors sup-5 III and sup-7 X, using amber alleles in 13 assorted genes. Some of the differences between these spectra may be due to limited tissue specificity in sup-21 expression.-Suppression of dpy-20 was used to show that the sex-linked suppressors sup-7 and sup-21 are not dosage compensated in male (XO) relative to hermaphrodite (XX).-Several uses of amber suppressors are critically discussed: for identifying null mutations, for varying levels of gene activity and for detecting maternal mRNA.

Sex determination in the nematode C. elegans: analysis of tra-3 suppressors and characterization of fem genes.

Mutations of the gene tra-3 result in partial masculinization of XX animals of C. elegans, which are normally hermaphrodites (males are XO). A total of 43 tra-3 revertants (one intragenic, 42 extragenic) have been isolated and analyzed, in the hope of identifying new sex-determination loci. Most (38) of the extra-genic suppressors cause partial or complete feminization of XX and XO animals; the remaining four are weak suppressors. The feminizing suppressors are mostly alleles of known sex-determining genes: tra-1 (11 dominant alleles), tra-2 (one dominant allele), fem-1 (four alleles) and fem-2 (four alleles), but 18 are alleles of a new gene, fem-3. Additional alleles have been isolated for the fem-2 and fem-3 genes, as well as fem-3 deficiencies. Mutations in fem-3 resemble alleles of fem-1 (previously characterized): putative null alleles result in complete feminization of XX and XO animals, transforming them into fertile females. Severe alleles of fem-2 also cause complete feminization of XX animals at all temperatures, but feminization of fem-2 XO animals is temperature-sensitive: complete at 25 degrees, incomplete at 20 degrees. As with fem-1, severe mutations of fem-2 and fem-3 are wholly epistatic to masculinizing alleles of tra-2 and tra-3, and epistatic to tra-1 masculinizing alleles in the germline, but not in the soma. All three fem genes are essential for male development and appear to have a dual role in promoting spermatogenesis and repressing tra-1 activity. All three fem genes exhibit strong maternal effects; the maternal contribution of fem gene products may be inactivated in XX animals by a posttranscriptional mechanism. Maternal contributions of wild-type fem-3 product are necessary for normal XO male development and XX hermaphrodite (as opposed to female) development.

Molecular cloning and duplication of the nematode sex-determining gene tra-1.

The autosomal sex-determining gene tra-1 plays a major role in controlling sexual phenotype in the nematode Caenorhabditis elegans. This gene is the terminal global regulator in a well-characterized cascade of sex-determining genes. It governs all aspects of somatic sexual differentiation, and it also has important functions in governing germ-line differentiation. Previous genetic analyses have led to the characterization of many loss-of-function (masculinizing) and gain-of-function (dominant feminizing) alleles, and to models for the functions and regulation of tra-1. The gene was cloned by identifying linked transposon insertions, about 200 kb away from tra-1. From this starting point a series of YAC, cosmid and phage clones were assembled into a genomic walk covering over 400 kb. Much of this region was found to be unrepresented in the cosmid database that covers most of the C. elegans genome. This deficit is largely or wholly due to the presence of sequences that cannot be cloned in rec+bacterial hosts. The ratio of physical map distances to recombinational map distances in the tra-1 region of the genome appears to be unusually low, indicating considerable local map expansion. The location of tra-1 within the cloned region was determined using a variety of tra-1 mutations that are associated with physical rearrangements of the gene. One of these is a 14-kb deletion, which behaves as a null allele. Another rearrangement, eDp24, is a tandem duplication of 22kb. Genetic analysis demonstrates that eDp24 carries two incomplete copies of tra-1, and that these copies appear to interact, suggesting some form of negative autoregulation at this locus. Three variant forms of the tra-1 locus have been identified in different natural isolates of C. elegans.

Male Phenotypes and Mating Efficiency in CAENORHABDITIS ELEGANS.

Mating behavior in adult male nematodes can be assayed by mating efficiency, i.e., the number of cross progeny sired by males under standard conditions. Mutant males from 220 strains, representing most of the known complementation groups of C. elegans, have been examined for mating efficiency and for anatomical abnormalities of the specialized male copulatory organs. These data extend the phenotypic description of these mutants and indicate what anatomical and behavioral components are necessary for the ability to mate successfully. Also, mutants with specific defects in the male were sought by establishing superficially wild-type hermaphrodite stocks after mutagenesis and testing the males segregated by these stocks for mating efficiency. Forty-nine of 1119 stocks yielded abnormal males. Seventeen were characterized in detail and found to be abnormal in sensory behavior (carrying mutations in the genes che-2 or che-3) or male genital anatomy (carrying mutations in one of the genes mab-1 to mab-10). Four of the mab (male abnormal) genes affect specific postembryonic cell lineages.

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.

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.

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.

Mutations causing transformation of sexual phenotype in the nematode Caenorhabditis elegans.

Ten mutations are described that transform genotypic hermaphrodites of the nematode Caenorhabditis elegans into phenotypic males. These fall into three autosomal complementation groups, termed tra-1, tra-2, and tra-3. Two alleles of tra-1 produce almost complete transformation, to a fertile male phenotype; such transformed animals are useful for analyzing sex-linked genes. All alleles of tra-1 and tra-2 are recessive; the one known allele of tra-3 is both recessive and maternal in effect. Where tested, both XX and XXX hermaphrodites are transformed into males, but XO males (true males) are unaffected by these mutations. It is suggested that these genes are actually involved in hermaphrodite development and have no role in male development.

Nondisjunction Mutants of the Nematode CAENORHABDITIS ELEGANS.

The frequency of males (5AA; XO) among the self progeny of wild-type Caenorhabditis elegans hermaphrodites (5AA; XX) is about one in 500. Fifteen him (for "high incidence of males") mutations have been identified that increase this frequency by a factor of ten to 150, as a result of increased X-chromosome nondisjunction. The mutations define ten complementation groups, which have been mapped: nine are autosomal, and one sex linked. Most of the mutants are superficially wild type in anatomy and behavior; however, him-4 mutants display gonadal abnormalities, and unc-86 mutants, which have a Him phenotype, exhibit a variety of anatomical and behavioral abnormalities. All the mutants segregate fertile 3X hermaphrodite progeny as well as XO male progeny. Some produce large numbers of inviable zygotes. Mutants in all ten genes produce diplo-X and nullo-X exceptional ova, and in the four strains tested, diplo-X and nullo-X exceptional sperm are produced by 2X "transformed" males. It appears likely that most of the mutants have defects in both gamete lines of the hermaphrodite. XO males of him strains other than him-4 and unc-86 are similar to wild-type males in anatomy and behavior, and all produce equal or almost equal numbers of haplo-X and nullo-X sperm, and no diplo-X sperm. Male fertility is reduced to varying extents in all him mutants. In four of the strains, nondisjunction during oogenesis has been shown to occur at a reductional division, and in three of these strains, abnormalities in recombination have been demonstrated. One mutant also exhibits autosomal nondisjunction, but many of the others probably do not. Therefore, the X chromosome of C. elegans may differ from the autosomes in the mechanisms controlling its meiotic behavior.--3X hermaphrodites are shorter and less fertile than 2X hermaphrodites, and they produce many inviable zygotes among their self progeny: these are probably 4X zygotes. Haplo-X and diplo-X ova are produced in 2:1 ratio by 3X hermaphrodites. him mutations are expressed in these animals, increasing the frequency of self-progeny males and 2X hermaphrodites.

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.

A new kind of informational suppression in the nematode Caenorhabditis elegans.

Independent reversions of mutations affecting three different Caenorhabditis elegans genes have each yielded representatives of the same set of extragenic suppressors. Mutations at any one of six loci act as allele-specific recessive suppressors of certain allels of unc-54 (a myosin heavy chain gene), lin-29 (a heterochronic gene), and tra-2 (a sex determination gene). The same mutations also suppress certain alleles of another sex determination gene, tra-1, and of a morphogenetic gene, dpy-5. In addition to their suppression phenotype, the suppressor mutations cause abnormal morphogenesis of the male bursa and the hermaphrodite vulva. We name these genes smg-1 through smg-6 (suppressor with morphogenetic effect on genitalia), in order to distinguish them from mab (male abnormal) genes that can mutate to produce abnormal genitalia but which do not act as suppressors (smg-1 and smg-2 are new names for two previously described genes, mab-1 and mab-11). The patterns of suppression, and the interactions between the different smg genes, are described and discussed. In general, suppression is recessive and incomplete, and at least some of the suppressed mutations are hypomorphic in nature. A suppressible allele of unc-54 contains a deletion in the 3' noncoding region of the gene; the protein coding region of the gene is apparently unaffected. This suggests that the smg suppressors affect a process other than translation, for example mRNA processing, transport, or stability.

Stu-7/air-2 is a C. elegans aurora homologue essential for chromosome segregation during embryonic and post-embryonic development.

We have isolated a new sterile uncoordinated C. elegans mutant, stu-7, which is defective in post-embryonic cell divisions in a regionally-specific fashion. The anterior of the worm is relatively unaffected whereas the mid-body and/or posterior are markedly thin, often resulting in worms having a central 'waist'. We have cloned stu-7 and found that it encodes a member of the recently expanding aurora sub-family of serine/threonine kinases. Elimination of maternal as well as zygotic stu-7 expression reveals that stu-7 is essential for mitosis from the first embryonic cell cycle onwards and is required for chromosome segregation though not for centrosome separation or for setting up a bipolar spindle. Multicopy expression of stu-7 also causes mitotic defects, suggesting that the level of this protein must be tightly controlled in order to maintain genetic stability during development.

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.