Transcription and intragenic recombination in polar mutants of Escherichia coli.

Mitotic recombination within the beta-galactosidase gene was followed in F' merodiploids grown in the presence and absence of inducer. Inducer inhibited recombination when the two mutations in the merodiploid were nonpolar but had no effect when the mutations were strongly polar. These results are interpreted to mean that transcription inhibits intragenic mitotic recombination and that strongly polar mutations inhibit transcription.

UNC-115, a conserved protein with predicted LIM and actin-binding domains, mediates axon guidance in C. elegans.

Axon guidance receptors modulate the growth cone cytoskeleton through signaling pathways that are not well understood. Here, we describe the C. elegans unc-115 gene, which encodes a candidate cytoskeletal linker protein that acts in axon guidance. unc-115 mutants have defects in a subset of axons, particularly as the affected axons change environments during outgrowth. The unc-115 gene encodes a putative actin-binding protein that is similar to the human actin-binding protein abLIM/limatin; it has a villin headpiece domain and three LIM domains that could mediate protein interactions. unc-115 is expressed in neurons during their development and is required cell-autonomously in certain neurons for normal axon guidance. We propose that UNC-115 modulates the growth cone actin cytoskeleton in response to signals received by growth cone receptors.

The acetylcholinesterase genes of C. elegans: identification of a third gene (ace-3) and mosaic mapping of a synthetic lethal phenotype.

In C. elegans, the newly identified ace-3 is the third gene affecting acetylcholinesterase (AChE) activity. ace-3 II specifically affects class C AChE and is unlinked to ace-1 X or ace-2 I, which affect the other two AChE classes (A and B, respectively). Strains homozygous for an ace-3 mutation have no apparent behavioral or developmental defect; ace-1 ace-3 and ace-2 ace-3 double mutants are also nearly wild type. In contrast, ace-1 ace-2 ace-3 triple mutant animals are paralyzed and developmentally arrested; their embryonic development is relatively unimpaired, but they are unable to grow beyond the hatching stage. Based on the analysis of genetic mosaics, we conclude that in the absence of ace-2 and ace-3 function, the expression of ace-1(+) in muscle cells, but not in neurons, is essential for postembryonic viability.

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.

Analysis of smu-1, a gene that regulates the alternative splicing of unc-52 pre-mRNA in Caenorhabditis elegans.

Mutations in the smu-1 gene of Caenorhabditis elegans were previously shown to suppress mutations in the genes mec-8 and unc-52. mec-8 encodes a putative RNA binding protein that affects the accumulation of specific alternatively spliced mRNA isoforms produced by unc-52 and other genes. unc-52 encodes a set of basement membrane proteins, homologs of mammalian perlecan, that are important for body wall muscle assembly and attachment to basement membrane, hypodermis, and cuticle. We show that a presumptive null mutation in smu-1 suppresses nonsense mutations in exon 17 but not exon 18 of unc-52 and enhances the phenotype conferred by an unc-52 splice site mutation in intron 16. We have used reverse transcription-PCR and RNase protection to show that loss-of-function smu-1 mutations enhance accumulation in larvae of an alternatively spliced isoform that skips exon 17 but not exon 18 of unc-52. We have identified smu-1 molecularly; it encodes a nuclearly localized protein that contains five WD motifs and is ubiquitously expressed. The SMU-1 amino acid sequence is more than 60% identical to a predicted human protein of unknown function. We propose that smu-1 encodes a trans-acting factor that regulates the alternative splicing of the pre-mRNA of unc-52 and other genes.

Mosaic analysis of two genes that affect nervous system structure in Caenorhabditis elegans.

The mutation mec-4(e 1611), identified by M. Chalfie, leads to the degeneration and death of the six neurons, called the microtubule cells, that mediate the response of wild-type animals to light touch. The fates of two of these cells, PLML and PLMR, which are responsible for response to light touch in the tail of the animal, have been monitored in animals mosaic for the mec-4(e 1611) mutation. The results are consistent with the view that the mutation behaves cell autonomously in its killing effect; in particular, none of the neurons that make either chemical synapses or gap junctions to PLML or PLMR is responsible for the deaths of PLML or PLMR. The results of gene dosage and dominance tests suggest that the mec-4(+) gene product, which is required for wild-type microtubule cell function, is altered by the e 1611 mutation into a novel product that kills the microtubule cells. Mutation in the gene unc-3 leads to the derangement of the processes of the motor neurons of the ventral cord. Mosaic analysis strongly suggests that unc-3(+) expression is required only in the motor neurons themselves for normal neuronal development. In particular, the hypodermis surrounding the ventral cord is not the primary focus of unc-3 action (body muscle was excluded in earlier work). Finally, the mosaic analysis supports an earlier suggestion that a sensory defect caused by a daf-6 mutation is localized to a non-neuronal cell called the sheath cell.

Crossover suppressors and balanced recessive lethals in Caenorhabditis elegans.

Two dominant suppressors of crossing over have been identified following X-ray treatment of the small nematode C. elegans. They suppress crossing over in linkage group II (LGII) about 100-fold and 50-fold and are both tightly linked to LGII markers. One, called C1, segregates independently of all other linkage groups and is homozygous fertile. The other is a translocation involving LGII and X. The translocation also suppresses crossing over along the right half of X and is homozygous lethal. C1 has been used as a balancer of LGII recessive lethal and sterile mutations induced by EMS. The frequencies of occurrence of lethals and steriles were approximately equal. Fourteen mutations were assigned to complementation groups and mapped. They tended to map in the same region where LGII visibles are clustered.

Analysis of genetic mosaics of the nematode Caneorhabditis 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 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.

The mec-8 gene of Caenorhabditis elegans affects muscle and sensory neuron function and interacts with three other genes: unc-52, smu-1 and smu-2.

Mutations in the Caenorhabditis elegans gene mec-8 were previously shown to cause defects in mechanosensation and in the structure and dye filling of certain chemosensory neurons. Using noncomplementation screens, we have identified eight new mec-8 alleles and a deficiency that uncovers the locus. Strong mec-8 mutants exhibit an incompletely penetrant cold-sensitive embryonic and larval arrest, which we have correlated with defects in the attachment of body muscle to the hypodermis and cuticle. Mutations in mec-8 strongly enhance the mutant phenotype of unc-52(viable) mutations; double mutants exhibit an unconditional arrest and paralysis at the twofold stage of embryonic elongation, a phenotype characteristic of lethal alleles of unc-52, a gene previously shown to encode a homolog of the core protein of heparan sulfate proteoglycan, found in basement membrane, and to be involved in the anchorage of myofilament lattice to the muscle cell membrane. We have identified and characterized four extragenic recessive suppressors of a mec-8; unc-52(viable) synthetic lethality. The suppressors, which define the genes smu-1 and smu-2, can weakly suppress all mec-8 mutant phenes. They also suppress the muscular dystrophy conferred by an unc-52(viable) mutation.

Recombination between small X chromosome duplications and the X chromosome in Caenorhabditis elegans.

Twelve new X chromosome duplications were identified and characterized. Eight are translocated to autosomal sites near four different telomeres, and four are free. Ten include unc-1(+), which in wild type is near the left end of the X chromosome, and two of these, mnDp72(X;IV) and mnDp73(X;f), extend rightward past dpy-3. Both mnDp72 and mnDp73 recombined with the one X chromosome in males in the unc-1-dpy-3 interval at a frequency 15- to 30-fold higher than was observed for X-X recombination in hermaphrodites in the same interval. Recombinant duplications and recombinant X chromosomes were both recovered. Recombination with the X chromosome in the unc-1-dpy-3 interval was also detected for five other unc-1(+) duplications, even though their right breakpoints lie within the interval. In hermaphrodites, mnDp72 and mnDp73 promoted meiotic X nondisjunction and recombined with an X chromosome in the unc-1-dpy-3 interval at frequencies comparable to that found for X-X recombination; mnDp72(X;IV) also promoted trisomy for chromosome IV. A mutation in him-8 IV was identified that severely reduced recombination between the two X chromosomes in hermaphrodites and between mnDp73 and the X chromosome in males. Recombination between the X chromosome and duplications of either the right end of the X or a region near but not including the left end was rare. We suggest that the X chromosome has one or more elements near its left end that promote meiotic chromosome pairing.

The ncl-1 gene and genetic mosaics of Caenorhabditis elegans.

A ncl-1 mutation results in enlarged nucleoli, which can be detected in nearly all cells of living animals by Nomarski microscopy. Spontaneous mitotic loss of a ncl-1(+)-containing free duplication in an otherwise homozygous ncl-1 mutant animal results in mosaicism for ncl-1 expression, and the patterns of mosaicism lead us to conclude that ncl-1 acts cell autonomously. The probability of mitotic loss of the duplication sDp3 is approximately constant over many cell divisions. About 60% of the losses of sDp3 at the first embryonic cell division involve nondisjunction. Frequencies of mitotic loss of different ncl-1(+)-bearing free duplications varied over a 200-fold range. The frequencies of mitotic loss were enhanced by a chromosomal him-10 mutation. We have used ncl-1 as a cell autonomous marker in the mosaic analysis of dpy-1 and lin-37. The focus of action of dpy-1 is in hypodermis. A mutation in lin-37 combined with a mutation in another gene results in a synthetic multivulva phenotype. We show that lin-37 acts cell nonautonomously and propose that it plays a role, along with the previously studied gene lin-15, in the generation of an intercellular signal by hyp7 that represses vulval development.

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.

Lethals, steriles and deficiencies in a region of the X chromosome of Caenorhabditis elegans.

Twenty-one X-linked recessive lethal and sterile mutations balanced by an unlinked X-chromosome duplication have been identified following EMS treatment of the small nematode, Caenorhabditis elegans. The mutations have been assigned by complementation analysis to 14 genes, four of which have more than one mutant allele. Four mutants, all alleles, are temperature-sensitive embryonic lethals. Twelve mutants, in ten genes, are early larval lethals. Two mutants are late larval lethals, and the expression of one of these is influenced by the number of X chromosomes in the genotype. Two mutants are maternal-effect lethals; for both, oocytes made by mutant hermaphrodites are rescuable by wild-type sperm. One of the maternal-effect lethals and two larval lethals are allelic. One mutant makes defective sperm. The lethals and steriles have been mapped by recombination and by complementation testing against 19 deficiencies identified after X-ray treatment. The deficiencies divide the region, about 15% of the X-chromosome linkage map, into at least nine segments. The deficiencies have also been used to check the phenotypes of hemizygous lethal and sterile hermaphrodites.

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 degrees; 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.

Suppression and function of X-linked lethal and sterile mutations in Caenorhabditis elegans.

We have expanded our collection of recessive lethal and sterile mutants in the region of the X chromosome balanced by mnDp1(X;V), about 15% of the X linkage map, to a total of 54 mutants. The mutations have been mapped with respect to 20 overlapping deficiencies and five X duplications, and they have been assigned to 24 genes by complementation testing. Nine mutants are hermaphrodite-sterile: one of these is a sperm-defect mutant, two have abnormal gonadogenesis and six, in five genes, are maternally influenced mutants, producing inviable zygote progeny. One of the gonadogenesis mutants and two of the maternally influenced mutants are male fertile. All but one of the maternally influenced mutants give cross progeny when mated with wild-type males. Forty-three mutants were tested for suppression by homozygous sup-5 (e1464), which is believed to be specific for null alleles. Ten mutants that were judged by independent criteria not to be null mutants are not suppressed. Nine of the other 33 mutants, in nine genes, are suppressed, five in both heterozygous and homozygous suppressor stocks and four only in homozygous suppressor stocks.

Segregation of Induced Frameshift Mutations and the Sequence of Gene Replication in ESCHERICHIA COLI K12.

We have constructed a strain of E. coli K12 carrying six mutations induced by the acridine half-mustard ICR-191. The mutations are widely spaced on the E. coli linkage map and are all easily reverted by ICR-191. Mapping of ten independent revertants for each of five markers indicated that the reversions induced by ICR-191 occurred near the original mutations. Exponentially and nonsynchronously growing cultures of this strain were exposed to ICR-191 for 0.85 generation, quickly washed free of mutagen, and resuspended in the original medium minus mutagen. Total viable cell number maintained its exponential increase both during and immediately after exposure to mutagen, whereas the number of revertants of any particular type remained constant for a characteristic period after removal of mutagen before finally assuming an exponential increase. Theoretically, the length of such a segregation lag should depend on the position of the particular reverted gene in the sequence of gene replication: the earlier a gene is replicated in the chromosome replication cycle, the longer its segregation lag should be. Our results are consistent with this prediction and fit a unidirectional, clockwise replication scheme with an origin between 55 and 74 min on the E. coli linkage map. The results also fit a very asymmetric bidirectional replication scheme.

Analysis of the Caenorhabditis elegans axonal guidance and outgrowth gene unc-33.

Mutations in the unc-33 gene of the nematode Caenorhabditis elegans lead to severely uncoordinated movement, abnormalities in the guidance and outgrowth of the axons of many neurons, and a superabundance of microtubules in neuronal processes. We have cloned unc-33 by tagging the gene with the transposable element Tc4. Three unc-33 messages, which are transcribed from a genomic region of at least 10 kb, were identified and characterized. The three messages have common 3' ends and identical reading frames. The largest (3.8-kb) message consists of the 22-nucleotide trans-spliced leader SL1 and 10 exons (I-X); the intermediate-size (3.3-kb) message begins with SL1 spliced to the 5' end of exon V and includes exons V-X; and the smallest (2.8-kb) message begins within exon VII and also includes exons VIII-X. A gamma-ray-induced deletion mutation situated within exon VIII reduces the sizes of all three messages by 0.5 kb. The three putative polypeptides encoded by the three messages overlap in C-terminal sequence but differ by the positions at which their N termini begin; none has significant similarity to any other known protein. A Tc4 insertion in exon VII leads to alterations in splicing that result in three approximately wild-type-size messages: the Tc4 sequence and 28 additional nucleotides are spliced out of the two larger messages; the Tc4 sequence is trans-spliced off the smallest message such that SL1 is added 13 nucleotides upstream of the normal 5' end of the smallest message.

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.

Molecular and genetic analysis of unc-7, a Caenorhabditis elegans gene required for coordinated locomotion.

Mutations in the Caenorhabditis elegans gene unc-7 confer an uncoordinated phenotype. Wild-type animals trace smooth, sinuous waves as they move; unc-7 mutants make irregular bends or kinks along their bodies, particularly when they move forward. The unc-7 locus has also been implicated in the nematode's response to volatile anesthetics. We have cloned unc-7 by transposon tagging: an unc-7 mutation was correlated with the insertion of the transposon Tc1, and reversion of the mutant phenotype was correlated with loss of the Tc1 element. We have physically mapped the region flanking the sites of Tc1 insertion and identified DNA rearrangements corresponding to eight additional unc-7 alleles. Northern analysis indicates that a 2.7-kb unc-7 message is present in all developmental stages but is most abundant in L1-L3 larvae. The 5' end of the message contains a trans-spliced leader SL1. An 18-kb intron is located upstream of the predicted translational start site of the gene, and DNA breakpoints of four gamma-ray-induced alleles were located within this intron. We determined the sequence of a cDNA corresponding to the unc-7 message. The message may encode a 60-kd protein whose amino acid sequence is unrelated to any other available protein sequence; a transmembrane location for the unc-7 protein is predicted. We predict from our analysis of unc-7 genetic mosaics that the unc-7 gene product is not required in muscle cells for wild-type coordination but is probably required in motor neurons (although a hypodermal role has not been excluded). We speculate that unc-7 may be involved in the function of neuronal ion channels.

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.