Worm Breeder's Gazette 11(4): 30
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.
In Volume 11,#2 of the WBG we reported the identification of a cosmid, K01B11, that detects a polymorphism associated with the unc-87 phenotype. Here, we report more evidence consistent with the proposal that this cosmid contains at least part of the unc-87 gene. We also describe the cloning and sequencing of a putative unc-87 cDNA, and make observations on the primary structure of the predicted amino acid sequence. st1005 is a spontaneous allele of unc-87 isolated in a BO mut-6 background.(1) Southern blots containing DNAs prepared from st1005 and coisogenic revertants, were hybridized with K01B11. The pattern observed was consistent with a 2.5 kb insertion into the wildtype gene causing the mutation. The wildtype member of this polymorphism is a 5. 9 kb BglII fragment; K01B11 also contains a 5.9 kb BglII fragment. The 5.9 kb BglII fragment was subcloned from the cosmid. We used Northern analysis to determine if this genomic clone contained coding region, and whether any message detected was expressed in a pattern in st1005 consistent with what one might expect for the unc-87 message in a mutator-induced allele. Total RNA was prepared from mixed-stage populations of st1005, a coisogenic revertant, and N2. Thirty g of each were analyzed on Northern blots. Hybridization of the genomic clone detected a 1.4 kb transcript in N2 and the coisogenic revertant but nothing in the strain carrying st1005. A control hybridization with an unrelated probe indicated that intact st1005 RNA was present on the blot. These results suggest that the 1.4 kb transcript might be the unc-87 message. Inability to detect a transcript also suggests that st1005 is a hypomorphic, and possibly a null allele of unc-87.We next set out to obtain a cDNA, knowing that we could test any positive clones for the expected hybridization pattern on Northern blots. The genomic clone was used to screen Bob Barstead's cDNA library. We identified a cDNA clone large enough to account for a full length message. This clone was hybridized to a Northern blot, and the pattern observed was the same as that obtained with the genomic clone. This cDNA clone has been sequenced. A portion of the trans-spliced leader SL1(2) present at the 5' end indicates that the cDNA contains the entire coding region. The sequence predicts a 358 amino acid protein. A diagonal comparison of the predicted amino acid sequence with itself revealed seven regularly spaced motifs of approximately 41 residues. These motifs have identities ranging from 27 to 60 percent, with an average identity of 42 percent. A consensus derived from these motifs was used to search the NBRF protein database. Fifty percent identity exists between approximately the first 35 amino acids of the consensus and a similar length stretch of the protein SM22alpha, a 22 kD peptide isolated from chicken gizzard smooth muscle.(3) The function of the chicken protein is not known, but it appears to be smooth muscle specific. The entire predicted amino acid sequence was used to search a translation of the DNA database, and no other significant similarity was detected. DNA from other alleles of unc-87 will be examined by the PCR and/or chemical mismatch analysis. Detection of DNA rearrangements or sequence changes in the region covered by the cDNA will provide further support that we have in fact identified the unc-87 gene, and not a linked gene affected by the st1005 rearrangement. Future plans include production of fusion protein to generate antibodies, sequencing of the genomic clone, and transformation rescue.