Genetic Organization of the Region around in Caenorhabditis Elegans

In the nematode Caenorhabditis elegans mutants in the gene unc-15 ( I ) affect the muscle protein paramyosin (WATERSTON, FISHPOOL and BRENNER 1977). We have characterized 20 ethyl methanesulfonate-induced mutations in essential genes closely linked to unc-15. These lethals defined 16 new complememtation groups. In the 0.65 map-unit interval around unc-I5 defined by dpy-14 and unc-56, seven newly identified genes have been mapped relative to five existing genes. At present, the average distance between genes in this region is approximately 0.05 map units. Two genes, unc-I5 and unc-13, are only 0.025 map units apart. Partial fine-structure maps of alleles of these t w o genes have been constructed. This analysis of unc-I5 and genes adjacent to it is the f i s t in a series of genetic and biochemical studies directed towards understanding the control of unc-I5 expression. NE approach to understanding the regulation of gene activity during tissue differentiation is the detailed study of a region around a k n o w n gene. In D. melanogaster, the elegant studies of CROVNICK and his colleagues (see CHOVNICK, GELBART and MCCARRON 1977 for review) have demonstrated the value of this approach. We have taken a similar approach in Caenorhabditis ekgans by studying the region around unc-15 ( I ) ; unc-15 mutants are paralyzed and have altered paramyosin. Paramyosin is a structural protein that forms the core of the thick muscle filament in invertebrates. While other models are not ruled out, evidence is consistent with the hypothesis that unc-15 contains the coding sequence for a paramyosin subunit (WATERSTON? FISHPOOL and BRENNER 1977). unc-15 is situated on chromosome I , closely linked to other genes that produce uncoordinated phenotypes. For example, another gene producing paralyzed mutants, unc-87, maps close to unc-15 (MACLEOD and WATERSON, personal communication). This observation has led us to speculate that other genes closely linked to unc-15 may also code for muscle-related proteins. Our ultimate goal is to recover mutations in all loci around unc-15 and analyze the organization of the unc-15 region. We hope that such an approach will provide insight into the genetic regulation of muscle differentiation. 'Present address: Department of Biochemishy, The University of British Columbia, Vancouver, B.C. V6T lW5, Canada. Genetics 96: 639648 November, 1980. 640 A. M. ROSE A N D D. L. BAILLIE In C. elegans, unconditional lethals have been induced and characterized on LG ZZ (HERMAN 1978) and the tip of the X chromosome (MENEELY and HERMAN 1979). We describe here the recovery and characterization of ethyl methane-sulfonate(EMS) and formaldehyde-induced mutations around unc-15. This is the first attempt in C. elegans to identify all the lethal sites in a small region. To further characterize the organization of this region, we have constructed fine-structure maps for unc-15 and its closest neighbor, unc-13. MATERIALS A N D METHODS Our wild-type strain of Caenorhubditis eZegans var. Bristol was obtained originally from S. BRENNER (MRC, Cambridge, England). Other mutant strains used in this study came from the laboratory indicated by the italicized letter prefixing the mutation number as described by HORVITZ e't al. (1979). We followed the procedure for strain maintenance and genetic outcrossing described previously by ROSE and BAILLIE (1979). Induction of lethals and deficiencies: A heterozygous strain, dpy-14(e188) + unc-I3 (e51)/+ unc-l5(e73) +, was constructed. This strain was maintained by picking wild-type-appearing heterozygotes each generation and allowing them to self-cross. In order to induce lethal mutations, this strain was treated with 0.025 M EMS for 4 hr, modified from BRENNER (1974). Treated hermaphrodites were placed 1 per plate. F, individuals were placed on separate plates to self-cross. F, progeny were screened for the absence of dumpyuncoordinated worms, indicating the presence of a lethal closely linked to dpy-14 and unc-13. In this way, 1,600 chromosomes were tested. Lethals induced by this method were maintained by transferring the heterozygotes each generation. Two deficiencies of this region were recovered. sDj5 was either spontaneous or induced with 0.05 M EMS, but sDj6 was induced by treating wild-type male sperm with 0.07% formaldehyde as follows. Some 200 to 300 dumpy-uncoordinated (dpy-5 unc-i?/dpy-5 unc-13) hermaphrodites were mated for 18 hr to wild-type males. The mated hermaphrodites were treated with formaldehyde in M-9 buffer for 4 hr. Treated worms were spotted on a plate for 30 min. Individual dpy-5 unc-l3/dpy-5 unc-13 worms were then placed on small (35 mm) petri plates. The F, progeny were counted and screened for the presence of exceptional dumpy or uncoordinated progeny. sDj5 and sDj6 are maintained heterozygously over unc-15 (e73). Mapping studies: unc-37(e262), unc-87(e1489) and mec-8je398j were mapped by generating a heterozygous strain that carried the gene to be mapped over either dpy-5 dpy-14 or dpy-I4 unc-13. These strains were maintained by selecting wild-type appearing hermaphrodites each generation. Recombinants that were homozygous for either dpy-5, dpy-14 o r unc-13 were placed on individual plates and progeny tested. Mutations in dpy-5 and dpy-I4 give rise to very different phenotypes that are easily distinguished (for example, dpy-14 mutants are temperature sensitive, whereas dpy-5 mutants are not). The results from these progeny tests were used to determine the position of the gene being mapped relative to dp7-5, dpy-14 and unc-13. unc-87 was' further positioned by picking dumpy and uncoordinated recombinants from the heterozygous strain, dpy-14 + unc-37/ + unc-87 +. Mutations in essential genes were induced in a dpy-14 um-I3 chromosome, and maintained heterozygously over a unc-15 chromosome. Thus, the position of the gene could be determined from the number of the dumpy, uncoordinated and dumpy-uncoordinated progeny present in the self-cross. In addition, the occurrence of uncoordinated recombinants that do not carry unc-I5 proves that the essential gene maps outside the unc-15 unc-13 interval. Essential genes that mapped to the right of unc-13 were further positioned by constructing the heterozygous strain, dpy-I4 + unc-13 let-x +/ + unc-15 + f unc-56. THE U n C 1 5 REGION 641 Complementation analysis: Male strains of each lethal mutation were constructed by crossing dpy-I4 + unc-i3/ + unc 15 + males (generated by X-chromosome nondisjunction) to each lethal. Two types of males are produced by this cross, one of which carries the lethal to be tested. Individual males were backcrossed to hermaphrodites from the same strain. Males and hermaphrodites from crosses that gave male progeny, but no dumpy-uncoordinated worms, were mated each generation to maintain a male strain. Te ensure that the dumpy and uncoordinated mutations are present, each strain was mated to dpy-I4 + unc-13/ + unc-I5 + males and the progeny examined for the presence of dumpy-uncoordinated males. Mutant strains were mated inter se to determine if complementation occurred. Mapping alleles within a gene: Intragenic mapping methods were modified from MOERMAN and BAILLIE (1979). Alleles of unc-I5 and unc-I3 were mapped in the following way: Triplymarked chromosomes were constructed that carried 1 allele of the gene to be mapped. A strain heterozygous for the 2 alleles was produced by mating heterozygous males (e.g., unc-I5 (e1214)/+) to the triple mutant (e.g., dpy-5(ebl) unc-l5(e73) unc-56(e403)/ dpy-5(ell) unc15(e73) uc-56(e403). Paralyzed heterozygotes (Figure 1) were placed I per plate and allowed to self-cross. The number of progeny on a sample of plates was counted each generation, and the total number of screened chromosomes was estimated from these counts. All exceptional progeny (wild type, dumpy and uncoordinated) were progeny tested; only events showing exchange of flanking markers were used to calculate map distances between alleles (Figure 1). dPY 5 unc 15e73 unc 56 I I I -_ _ _ I unc 15e1214