Worm Breeder's Gazette 13(2): 23 (February 1, 1994)

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.

Cloning C. briggsae Homologues by Synteny

Patricia Kuwabara, Sheetal Shah

Figure 1

MRC-LMB, Hills Road, Cambridge CB2 2QH, England.

Phylogenetic comparisons of gene and protein sequences often reveal elements that are conserved throughout evolution because they arc important for gene expression, function or regulation. To identify such elements in the C. elegans sex-determining gene tra-2 ,we are comparing the C. elegans tra-2 sequence to homologues cloned from closely related nematode species. As a first step, we report on the cloning of the tra-2 homologue from C. briggsae. tra-2 plays a crucial role in a sex determination and encodes a putative integral membrane protein with no obvious similarities in current databases.

To identify the C. briggsae tra-2 homologue, we took two approaches (using the Baillie lab genomic library). First, we hybridized duplicate plaque lifts with probes corresponding to either the 5' half of the 3' half of the 4.7 Kb tra-2 cDNA using low stringency conditions. No plaques were found that hybridized to both probes. Our second approach, which proved successful, relied on synteny (conservation of gene order) between C. elegans and C. briggsae by using the gene upstream of the C. elegans tra-2 ,"gene X", as a probe to identify a phage clone that might also carry tra-2 .Our hope was that gene X, which shows similarity to a family of pyrophosphorylases, might be more conserved between species than tra-2 .Indeed, we easily found a phage carrying the C.b. gene X that hybridizes to both 5' and 3' gene X probes, lambd aPK10 .

Although we have not yet completed sequencing lambd aPK10 ,we believe that we have also obtained the C. briggsae tra-2 homologue for the following reasons. First, the St. Louis sequencing consortium have fortuitously identified a C. briggsae clone that is predicted by BLAST to have sequence similarity to C. elegans tra-2 .We have found by DNA sequencing that the St. Louis sequence, kindly provided by Rick Wilson, is present in lambd aPK10 .We have also sequenced other regions of lambd aPK10 that show 60% similarity to C.e. tra-2 at the protein level. Second, BLAST analysis of sequences generated from lambd aPK10 subclones reveals that lambd aPK10 contains an additional two genes that are predicted to map close to tra-2 on the physical map. Most significantly, we have identified the C.b. homologue of C.e. sod-1 ,which is upstream of tra-2 on the C. elegans physical map (Larsen, PNAS 1993). Using sod-1 sequence information, kindly provided by Pam Larsen, we have also determined that C.e. sod-1 lies in the same orientation as tra-2 .At the protein level, the C.e. and C.b. sod-1 genes are >90% identical and are more closely related to one another than to other sod genes. Surprisingly, we have also identified a gene downstream of C.b. sod-1 that is predicted to have >70% sequence similarity to rat SC2 ,a glycoprotein enriched in rat brain and neuronal tissues with similarity to steroid 5-alpha reductase Il. Preliminary Southern hybridization results suggest there is a C.e. SC2 homologue positioned between "gene X" and sod-1 .The figure below summarizes our findings.

Literature Cited:

Spieth et al., Cell 1993

Figure 1