What does a worm want with 20,000 genes?

The number of genes predicted for the Caenorhabditis elegans genome is remarkably high: approximately 20,000, if both protein-coding and RNA-coding genes are counted. This article discusses possible explanations for such a high value.

C. elegans EGL-9 and mammalian homologs define a family of dioxygenases that regulate HIF by prolyl hydroxylation.

HIF is a transcriptional complex that plays a central role in mammalian oxygen homeostasis. Recent studies have defined posttranslational modification by prolyl hydroxylation as a key regulatory event that targets HIF-alpha subunits for proteasomal destruction via the von Hippel-Lindau ubiquitylation complex. Here, we define a conserved HIF-VHL-prolyl hydroxylase pathway in C. elegans, and use a genetic approach to identify EGL-9 as a dioxygenase that regulates HIF by prolyl hydroxylation. In mammalian cells, we show that the HIF-prolyl hydroxylases are represented by a series of isoforms bearing a conserved 2-histidine-1-carboxylate iron coordination motif at the catalytic site. Direct modulation of recombinant enzyme activity by graded hypoxia, iron chelation, and cobaltous ions mirrors the characteristics of HIF induction in vivo, fulfilling requirements for these enzymes being oxygen sensors that regulate HIF.

Molecular identification of smg-4, required for mRNA surveillance in C. elegans.

Premature termination codons trigger a process in eukaryotes known as nonsense mediated decay or mRNA surveillance, resulting in the rapid decay of the aberrant transcript. Studies in C. elegans have shown this system is mediated by seven smg genes and can prevent the accumulation of toxic, truncated peptides. Here we report the cloning of smg-4 by physical mapping and functional rescue assays. The minimal rescuing activity is found within a genomic operon, encoding a novel protein. The final exon of the gene is alternatively spliced for expression of two different isoforms. Although no known genes were found to exhibit significant homology to smg-4, a novel conserved domain has been identified by alignment with sequences defined by expressed sequence tags (ESTs) from a variety of organisms. Furthermore, we describe a homolog from C. briggsae, which will rescue C. elegans smg-4 mutants. The C. elegans gene has been fused to green fluorescent protein (GFP). This SMG-4:GFP fusion exhibits nuclear accumulation and diffuse cytoplasmic staining, and further localizes to what appear to be perinuclear and cytoplasmic punctate structures.

gon-4, a cell lineage regulator required for gonadogenesis in Caenorhabditis elegans.

The gon-4 gene is required for gonadogenesis in the nematode Caenorhabditis elegans. Normally, two precursor cells, Z1 and Z4, follow a reproducible pattern of cell divisions to generate the mature somatic gonadal structures (e.g., uterus in hermaphrodites, vas deferens in males). In contrast, in gon-4 mutants, the Z1/Z4 cell lineages are variably aborted in both hermaphrodites and males: Z1 and Z4 divide much later than normal and subsequent divisions are either absent or severely delayed. In gon-4 adults, normal somatic gonadal structures are never observed, and germ-line and vulval tissues, which depend on somatic gonadal cues for their development, are also aberrant. In contrast, nongonadal tissues and the timing of other developmental events (e.g., molts) appear to be normal in gon-4 mutants. The gon-4 alleles are predicted to be strong loss-of-function or null alleles by both genetic and molecular criteria. We have cloned gon-4 in an attempt to learn how it regulates gonadogenesis. The gon-4 gene encodes a novel, acidic protein. A GON-4::GFP fusion protein, which rescues a gon-4 mutant to fertility, is expressed in somatic gonadal cells during early gonadal development. Furthermore, this fusion protein is nuclear. We conclude that gon-4 is a regulator of the early lineage of Z1 and Z4 and suggest that it is a part of a genetic program common to the regulation of both hermaphrodite and male gonadogenesis.

A conserved checkpoint pathway mediates DNA damage--induced apoptosis and cell cycle arrest in C. elegans.

To maintain genomic stability following DNA damage, multicellular organisms activate checkpoints that induce cell cycle arrest or apoptosis. Here we show that genotoxic stress blocks cell proliferation and induces apoptosis of germ cells in the nematode C. elegans. Accumulation of recombination intermediates similarly leads to the demise of affected cells. Checkpoint-induced apoptosis is mediated by the core apoptotic machinery (CED-9/CED-4/CED-3) but is genetically distinct from somatic cell death and physiological germ cell death. Mutations in three genes--mrt-2, which encodes the C. elegans homolog of the S. pombe rad1 checkpoint gene, rad-5, and him-7-block both DNA damage-induced apoptosis and cell proliferation arrest. Our results implicate rad1 homologs in DNA damage-induced apoptosis in animals.

The caenorhabditis elegans fate-determining gene mab-9 encodes a T-box protein required to pattern the posterior hindgut.

Caenorhabditis elegans mab-9 mutants are defective in hindgut and male tail development because of cell fate transformations in two posterior blast cells, B and F. We have cloned mab-9 and show that it encodes a member of the T-box family of transcriptional regulators. MAB-9 localizes to the nucleus of B and F and their descendents during development, suggesting that it acts cell autonomously in the posterior hindgut to direct cell fate. T-box genes related to brachyury have also been implicated in hindgut patterning, and our results support models for an evolutionarily ancient role for these genes in hindgut formation.

MRT-2 checkpoint protein is required for germline immortality and telomere replication in C. elegans.

The germ line is an immortal cell lineage that is passed indefinitely from one generation to the next. To identify the genes that are required for germline immortality, we isolated Caenorhabditis elegans mutants with mortal germ lines--worms that can reproduce for several healthy generations but eventually become sterile. One of these mortal germline (mrt) mutants, mrt-2, exhibits progressive telomere shortening and accumulates end-to-end chromosome fusions in later generations, indicating that the MRT-2 protein is required for telomere replication. In addition, the germ line of mrt-2 is hypersensitive to X-rays and to transposon activity. Therefore, mrt-2 has defects in responding both to damaged DNA and to normal double-strand breaks present at telomeres. mrt-2 encodes a homologue of a checkpoint gene that is required to sense DNA damage in yeast. These results indicate that telomeres may be identified as a type of DNA damage and then repaired by the telomere-replication enzyme telomerase.

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.

The gon-1 gene is required for gonadal morphogenesis in Caenorhabditis elegans.

In wild-type Caenorhabditis elegans, the gonad is a complex epithelial tube that consists of long arms composed predominantly of germline tissue as well as somatic structures specialized for particular reproductive functions. In gon-1 mutants, the adult gonad is severely disorganized with essentially no arm extension and no recognizable somatic structure. The developmental defects in gon-1 mutants are limited to the gonad; other cells, tissues, and organs appear to develop normally. Previous work defined the regulatory "leader" cells as crucial for extension of the gonadal arms (J. E. Kimble and J. G. White, 1981, Dev. Biol. 81, 208-219). In gon-1 mutants, the leader cells are specified correctly, but they fail to migrate and gonadal arms are not generated. In addition, gon-1 is required for morphogenesis of the gonadal somatic structures. This second role appears to be independent of that required for leader migration. Parallel studies have shown that gon-1 encodes a secreted metalloprotease (R. Blelloch and J. Kimble, 1999, Nature 399, 586-590). We discuss how a metalloprotease may control two aspects of gonadal morphogenesis.

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.

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.

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.

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.

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.

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.