The Genetics of CAENORHABDITIS ELEGANS

Methods are described for the isolation, complementation and mapping of mutants of Caenorhabditis elegans, a small free-living nematode worm. About 300 EMS-induced mutants affecting behavior and morphology have been characterized and about one hundred genes have been defined. Mutations in 77 of these alter the movement of the animal. Estimates of the induced mutation frequency of both the visible mutants and X chromosome lethals suggests that, just as in Drosophila, the genetic units in C. elegans are large.     

Chemotaxis-Defective Mutants of the Nematode CAENORHABDITIS ELEGANS

The technique of countercurrent separation has been used to isolate 17 independent chemotaxis-defective mutants of the nematode Caenorhabditis elegans. The mutants, selected to be relatively insensitive to the normally attractive salt NaCl, show varying degrees of residual sensitivity; some are actually weakly repelled by NaCl. The mutants are due to single gene defects, are autosomal and recessive, and identify at least five complementation groups.     

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.     

Choline Acetyltransferase-Deficient Mutants of the Nematode CAENORHABDITIS ELEGANS

We have identified five independent allelic mutations, defining the gene cha-1, that result in decreased choline acetyltransferase (ChAT) activity in Caenorhabditis elegans. Four of the mutant alleles, when homozygous, lead to ChAT reductions of>98%, as well as recessive phenotypes of uncoordinated behavior, small size, slow growth and resistance to cholinesterase inhibitors. Animals homozygous for the fifth allele retain approximately 10% of the wild-type enzyme level; purified enzyme from this mutant has altered K_m values for both choline and acetyl-CoA and is more thermolabile than the wild-type enzyme. These qualitative alterations, together with gene dosage data, argue that cha-1 is the structural gene for ChAT. cha-1 has been mapped to the left arm of linkage group IV and is within 0.02 map unit of the gene unc-17, mutant alleles of which lead to all of the phenotypes of cha-1 mutants except for the ChAT deficiency. Extensive complementation studies of cha-1 and unc-17 alleles reveal a complex complementation pattern, suggesting that both loci may be part of a single complex gene.     

An Acetylcholinesterase-Deficient Mutant of the Nematode CAENORHABDITIS ELEGANS

Within a set of five separable molecular forms of acetylcholinesterase found in the nematode Caenorhabditis elegans, previously reported differences in kinetic properties identify two classes, A and B, likely to be under separate genetic control. Using differences between these classes in sensitivity to inactivation by sodium deoxycholate, a screening procedure was devised to search for mutants affected only in class A forms. Among 171 previously isolated behavioral and morphological mutant strains examined by this procedure, one (PR946) proved to be of the expected type, exhibiting a selective deficiency of class A acetylcholinesterase forms. Although originally isolated because of its uncoordinated behavior, this strain was subsequently shown to harbor mutations in two genes; one in the previously identified gene unc-3, accounting for its behavior, and one in a newly identified gene, ace-1, accounting for its selective acetylcholinesterase deficiency. Derivatives homozygous only for the ace-1 mutation also lacked class A acetylcholinesterase forms, but were behaviorally and developmentally indistinguishable from wild type. The gene ace-1 has been mapped near the right end of the X chromosome. Gene dosage experiments suggest that it may be a structural gene for a component of class A acetylcholinesterase forms.     

Analysis of Genetic Mosaics of the Nematode CAENORHABDITIS ELEGANS

A new method for producing genetic mosaics, which involves the spontaneous somatic loss of free chromosome fragments, is demonstrated. Four genes that affect the behavior of C. elegans were studied in mosaic animals. The analysis was greatly aided by the fact that the complete cell lineage of wild-type animals is known. Two of the mutant genes affect certain sensory responses and prevent uptake of fluorescein isothiocyanate (FITC) by certain sensory neurons. Mosaic analysis indicated that one of these mutant genes is cell autonomous with respect to its effect on FITC uptake and the other is cell nonautonomous. In the latter case, the genotype of a non-neuronal supporting cell that surrounds the processes of the neurons that normally take up FITC probably is critical. The other two mutant genes affect animal movement. Mosaic analysis indicated that the expression of one of these genes is specific to certain neurons (motor neurons of the ventral and dorsal nerve cords are prime candidates) and the expression of the other gene is specific to muscle cells.     

Osmotic Avoidance Defective Mutants of the Nematode CAENORHABDITIS ELEGANS

A wild-type strain of the nematode Caenorhabditis elegans has been shown to avoid high concentrations of a number of sugars and salts. Individual and population assays for this response were developed and mutants were selected for their inability to avoid high concentrations of fructose or NaCl. Seven nonavoiding mutants representing six complementation groups were isolated and characterized. Genetic studies indicate that the mutants each carry a single recessive mutation responsible for the defective osmotic avoidance behavior. The map locations of the six complementation groups identified by these mutations have been determined. Mutants isolated for their inability to avoid fructose are also unable to avoid NaCl and vice versa. The mutants move normally, exhibit normal touch sensitivity, and, like wild type, follow isotherms in a radial thermal gradient. All of the mutants are at least partially defective in the attraction to sodium chloride exhibited by wild type. None of the mutants is temperature sensitive, and all exhibit defective osmotic avoidance behavior as young L1 larvae. Preliminary anatomical studies indicate selective sensory neuron changes in at least one mutant.     

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.     

A Second Class of Acetylcholinesterase-Deficient Mutants of the Nematode CAENORHABDITIS ELEGANS

In Johnson et al. (1981), the Caenorhabditis elegans mutant strain PR1000, homozygous for the ace-1 mutation p1000, is shown to be deficient in the class A subset of acetylcholinesterases, which comprises approximately one-half of the total C. elegans acetylcholinesterase activity. Beginning with this strain, we have isolated 487 new behavioral and morphological mutant strains. Two of these, independently derived, lack approximately 98% of the wild-type acetylcholinesterase activity and share the same specific uncoordinated phenotype; both move forward in a slow and uncoordinated manner, and when mechanically stimulated to induce reversal, both hypercontract and become temporarily paralyzed. In addition to the ace-1 mutation, both strains also harbor recessive mutations in the same newly identified gene, ace-2, which maps to chromosome I and is therefore not linked to ace-1. Gene dosage experiments suggest that ace-2 is a structural gene for the remaining class B acetylcholinesterases, which are not affected by ace-1.—The uncoordinated phenotype of the newly isolated, doubly mutant strains depends on both the ace-1 and ace-2 mutations; homozygosity for either mutation alone produces normally coordinated animals. This result implies functional overlap of the acetylcholinesterases controlled by ace-1 and ace-2, perhaps at common synapses. Consistent with this, light microscopic histochemical staining of permeabilized whole mounts indicates some areas of possible spatial overlap of these acetylcholinesterases (nerve ring, longitudinal nerve cords). In addition, there is at least one area where only ace-2-controlled acetylcholinesterase activity appears (pharyngeo-intestinal valve).     

Characterization of Temperature-Sensitive, Fertilization-Defective Mutants of the Nematode CAENORHABDITIS ELEGANS

The isolation and characterization of three Caenorhabditis elegans temperature-sensitive mutants that are defective at fertilization are described. All three are alleles of the gene fer-1. At the restrictive temperature of 25 degrees, mutant hermaphrodites make sperm and oocytes in normal numbers. No oocytes are fertilized, although they pass through the spermatheca and uterus normally. The oocytes can be fertilized by sperm transferred by wild-type males, indicating that the mutant defect is in the sperm. The temperature-sensitive period for the mutants coincides with spermatogenesis. Sperm made by mutants at 25 degrees cannot be distinguished from wild-type sperm by light microscopy. The sperm do contact oocytes in mutant hermaphrodites, but do not fertilize. Mutant sperm appear to be nonmotile. Mutant males are also steril when grown at 25 degrees. They trnasfer normal numbers of sperm to hermaphrodites at mating, but these sperm fail to migrate to the spermatheca and are infertile. The phenotype of these mutants is consistent with a primary defect in sperm motility, but the cause of this defect is not known.     

The DNA of CAENORHABDITIS ELEGANS

Chemical analysis and a study of renaturation kinetics show that the nematode, Caenorhabditis elegans, has a haploid DNA content of 8 x 10(7) base pairs (20 times the genome of E. coli). Eighty-three percent of the DNA sequences are unique. The mean base composition is 36% GC; a small component, containing the rRNA cistrons, has a base composition of 51% GC. The haploid genome contains about 300 genes for 4S RNA, 110 for 5S RNA, and 55 for (18 + 28)S RNA.     

Isolation and Genetic Characterization of Cell-Lineage Mutants of the Nematode CAENORHABDITIS ELEGANS

Twenty-four mutants that alter the normally invariant post-embryonic cell lineages of the nematode Caenorhabditis elegans have been isolated and genetically characterized. In some of these mutants, cell divisions fail that occur in wild-type animals; in other mutants, cells divide that do not normally do so. The mutants differ in the specificities of their defects, so that it is possible to identify mutations that affect some cell lineages but not others. These mutants define 14 complementation groups, which have been mapped. The abnormal phenotype of most of the cell-lineage mutants results from a single recessive mutation; however, the excessive cell divisions characteristic of one strain, CB1322, require the presence of two unlinked recessive mutations. All 24 cell-lineage mutants display incomplete penetrance and/or variable expressivity. Three of the mutants are suppressed by pleiotropic suppressors believed to be specific for null alleles, suggesting that their phenotypes result from the complete absence of gene activity.     

Duplications in CAENORHABDITIS ELEGANS

Thirteen chromosomal duplications, all unlinked to their linkage of origin, have been identified following X-irradiation. Ten are X-chromosome duplications, of which six are half-translocations on three autosomomal linkage groups and four are free fragments. Five of the half-translocations are homozygous fertile and two are recognizable cytologically as chromosome satellites, both of which show some mitotic instability. The free-X duplications show varying tendencies for loss. Three appear not to overlap in extent previously identified free-X duplications. The fourth carries genes from linkage group V, as well as X. Three duplications of a portion of linkage group II were identified and found to be free and quite stable in hyperploids. Some of the free duplications tend to disjoin from the X chromosome in males. New X-chromosome map data are presented.     

Chromosome Rearrangements in CAENORHABDITIS ELEGANS

A method for selecting unlinked duplications of a part of the X chromosome of C. elegans is described. Five such duplications have been identified. One of them, Dp (X;V)1, is translocated to linkage group V, where it suppresses crossing over along the left half of linkage group V. Dp(X;V)1 homozygotes grow slowly and are sterile. The other four duplications are associated with chromosome fragments, as observed cytologically by fluorescence microscopy, and tend to be lost. Their frequency of loss is higher in strains homozygous for a mutation that promotes nondisjunction of X chromosomes. The recombination frequencies between two of these duplications and the X have been measured: the frequencies are at least 50 times less than for X-X recombination in the same region. The duplications may prove useful as balancers of recessive lethal mutations.     

Indirect Suppression in CAENORHABDITIS ELEGANS

Two cases of indirect suppression have been characterized. One case involves suppressors compensating for defects in muscle structure. Nine independent suppressor mutations were judged to lie in a single suppressor gene, sup-3. Suppression is dominant, but dose dependent, and results in improved locomotion, as well as in an increase in the ability of mutant animals to lay eggs. Mutations in six genes known to affect muscle structure were tested for suppression by representative sup-3 mutations. Alleles of three of the six genes are suppressed, two of which are known to code for thick filament proteins. One suppressor allele was identified as a deletion by genetic criteria. A second case of indirect suppression is not associated with muscle defects, but involves two mutant genes producing uncoordinated phenotypes very similar to one another. As in the first case, suppression is dominant but dose dependent and is not allele specific.     

Mutations with Dominant Effects on the Behavior and Morphology of the Nematode CAENORHABDITIS ELEGANS

We have analyzed 31 mutations that have dominant effects on the behavior or morphology of the nematode Caenorhabditis elegans. These mutations appear to define 15 genes. We have studied ten of these genes in some detail and have been led to two notable conclusions. First, loss of gene function for four of these ten genes results in a wild-type phenotype; if these genes represent a random sample from the genome, then we would estimate that null mutations in about half of the genes in C. elegans would result in a nonmutant phenotype. Second, the dominant effects of mutations in nine of these ten genes are caused by novel gene functions, and in all nine cases the novel function is antagonized by the wild-type function.     

Radiation-Sensitive Mutants of CAENORHABDITIS ELEGANS

Nine rad (for abnormal radiation sensitivity) mutants hypersensitive to ultraviolet light were isolated in the small nematode Caenorhabditis elegans. The mutations are recessive to their wild-type alleles, map to four of the six linkage groups in C. elegans and define nine new games named rad-1 through rad-9. Two of the mutants—rad-1 and rad-2—are very hypersensitive to X rays, and three—rad-2, rad-3 and rad-4—are hypersensitive to methyl methanesulfonate under particular conditions of exposure. The hypersensitivity of these mutants to more than one DNA-damaging agent suggests that they may be abnormal in DNA repair. One mutant—rad-5, a temperature-sensitive sterile mutant—shows an elevated frequency of spontaneous mutation at more than one locus; rad-4, which shows a cold-sensitive embryogenesis, reduces meiotic X-chromosome nondisjunction tenfold and partially suppresses some but not all mutations that increase meiotic X-chromosome nondisjunction; the viability of rad-6 hermaphrodites is half that of rad-6 males at 25°; and newly mature (but not older) rad-8 hermaphrodites produce many inviable embryo progeny. Meiotic recombination frequencies were measured for seven rad mutants and found to be close to normal.     

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 into 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.     

Identification and Characterization of 22 Genes That Affect the Vulval Cell Lineages of the Nematode CAENORHABDITIS ELEGANS

Ninety-five mutants of the nematode Caenorhabditis elegans altered in the cell lineages of the vulva have been isolated on the basis of their displaying one of two phenotypes, Vulvaless or Multivulva. In Vulvaless mutants, which define 12 genes, no vulva is present. In Multivulva mutants, which define ten genes, one or more supernumerary vulva-like protrusions are located along the ventral side of the animal. A single recessive mutation is responsible for the phenotypes of most, but not all, of these strains. Fifteen of these 22 genes are represented by multiple alleles. We have shown by a variety of genetic criteria that mutations that result in a Vulvaless or Multivulva phenotype in six of the 22 genes most likely eliminate gene function. In addition, Vulvaless or Multivulva mutations in seven of the other genes most likely result in a partial reduction of gene function; the absence of the activity of any of these genes probably results in lethality or sterility. Our results suggest that we may have identified most, or all, genes of these two classes.     

Polyploids and Sex Determination in CAENORHABDITIS ELEGANS

Tetraploid stocks of Caenorhabditis elegans var. Bristol carrying autosomal and X-linked markers have been produced. Tetraploid hermaphrodites fall into two categories: those that give about 1% male self-progeny and those that give 25 to 40% male self-progeny. The former are basically 4A;4X--four sets of autosomes and four sex chromosomes--and the latter are 4A;3X. Males are 4A;2X. (Diploid hermaphrodites are 2A;2X; males are 2A;1X.) Triploids were produced by crossing tetraploid hermaphrodites and diploid males. Triploids of composition 3A;3X are hermaphrodites; 3A;2X animals are fertile males. Different X-chromosome duplications were added to a 3A;2X chromosome constitution to increase the X-to-autosome ratio. Based on the resulting sexual phenotypes, we conclude that there exists on the C. elegans X chromosome at least three (and perhaps many more) dose-sensitive sites that act cumulatively in determining sex.