Worm Breeder's Gazette 11(4): 38
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.
We have been studying genes that are involved in the development of the HSN neurons. One of these genes, ham-1 IV, appears to be involved not only in HSN development but also in the development of the phasmid neuron PHB, the sister cell of the HSN. The HSN/PHB phenotypes led us to propose a model in which the distribution of developmental potential between the HSN and PHB is abnormal in ham-1 animals (Desai et al., 1988). In order to understand how the ham-1 gene product functions in HSN and PHB development, we have begun a molecular analysis of this gene. Using Bristol (N2)/Bergerac (N62) RFLPs as genetic markers, we mapped the ham-1 gene to the contig containing unc-31. A more detailed analysis mapped ham-1 between RFLPs recognized by the cosmids C08G1 and C13H6; of the 74 recombinants analyzed, ham-1 was not separated from the RFLP recognized by C03F9 (C08G1 and C13H6 overlap C03F9). To see if we could rescue the ham-1 defects with genomic DNA from this region, we coinjected into ham-1 animals the rol-6 plasmid pRF4 together with cosmids that mapped near ham-1. From the progeny of the injected animals, we established roller lines and scored them for two ham-1 phenotypes, egg laying and the ability of the phasmids to fill with fluorescent dyes. Two cosmids, C03F9 and C13H6, rescued ham-1 animals for both egg-laying and phasmid loading defects, indicating that both C03F9 and C13H6 contain the ham-1 gene. In additional experiments we have rescued the ham-1 defects with a 23 kbp fragment of C03F9 and partially rescued the ham-1 defects with a 12 kbp fragment. On Northern blots of poly(A)+ RNA using probes spanning C03F9, we detected a 1.8 kb RNA that we believe is the ham-1 mRNA for two reasons. First, it is the only transcript that is detected on Northern blots when the 12 kbp DNA that partially rescues ham-1 is used as a probe. Second, it is the only transcript recognized by the probes spanning C03F9 that is significantly reduced (>95%) in RNA from the mutant ham-1(n1438). Genomic DNA from ham-1(n1438) contains a small deletion in this region. In additional Northern blot experiments, we have shown that the ham-1 mRNA is enriched in poly(A)+ RNA prepared from embryos. We screened 100,000 plaques from Stuart Kim's cDNA library with the 23 kbp fragment that rescues ham-1 and isolated four cDNAs. One appears to be a ham-1 cDNA because it comes from the 12 kbp region that partially rescues ham-1 and it recognizes the 1.8 kb ham-1 mRNA on Northern blots. Since the cDNA is 1.8 kbp, it contains most of the ham-1 RNA. Sequence analysis of this cDNA revealed a single long open reading frame that could encode a protein of 414 amino acids. The putative initiator methionine is preceded by three in-frame stop codons indicating that we have defined the entire ham-1 coding region. This protein has no significant sequence similarity to any protein in the gene bank (TRANSGEN). Add ham-1 to the scores of novel developmentally important genes defined by C. elegans' genetics.