Worm Breeder's Gazette 11(2): 36
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
CapZ is one of a family of actin binding proteins that may regulate actin dynamics in eukaryotic cells. This heterodimeric protein is located at the Z-line of vertebrate skeletal muscle in situ (1). Since the ends of actin filaments are also located at the Z-line, CapZ could function in the assembly or tethering of thin filaments at this location. The Z-lines of vertebrate skeletal muscle also contain the proteins alpha-actinin and vinculin. These two proteins have been localized to the dense bodies of nematode body wall muscles (2,3). Collectively, the above observations suggest that a CapZ homolog might also be present at the dense bodies or attachment plaques of nematode muscle. On this premise, we have initiated a search for CapZ homologs in C. elegans.Sequence data from cDNA clones encoding the CapZ subunits of chicken, (4,5), D. discoideum (6) and S. cerevisiae (7,8) were used to design degenerate oligonucleotide primers for the polymerase chain reaction. Direct amplification of nematode CDNA libraries (kindly provided by R. Barstead) generated candidate fragments for both subunits. A comparison between the deduced amino acid sequence from the nematode PCR products and the homologous regions in other organisms yielded the following table of [See Figure 1] High stringency hybridization of labelled PCR fragments to Southern blots of total nematode genomic DNA suggests that both subunit genes are single copy. Using these same probes, we have determined a physical map position by hybridization to YAC clones. PCR analysis of positive and negative YACs near the putative map position were in full agreement with the filter hybridization data. The beta subunit gene localizes to LGII between cloned gpd-1 and vmp-1 (roughly in the unc-4 to bli-1 interval). The alpha subunit gene localizes to a region between rtw-6 and rpo-1 on LGIV. To align the physical and genetic maps in the region of interest on LGII, we will use a novel application of PCR (suggested by R. Barstead) to screen deficiencies in the area. First, balanced deficiencies are allowed to segregate arrested embryos. These embryos are then used directly in a PCR reaction with primers specific for the gene of interest. The absence of a product would indicate that the deficiency removes the sequence tested. Finally, we screened a nematode cDNA library using the PCR products as hybridization probes. We recovered 22 alpha and 6 beta subunit positive clones. We are currently in the process of characterizing these clones.