Worm Breeder's Gazette 13(3): 21 (June 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.

Use of an expression library to clone genes by complementation.

RE. Palmer, P.W. Sternberg

HHMI and Division of Biology, Caltech, Pasadena, CA 91125

We identified a C. elegans cDNA that was able to bypass the requirement of the intact protein kinase signaling cascade that is required for the activation of the Saccharomyces cerevisiae mating pheromone-responsive gene FUS1 .We identified this cDNA during our efforts to clone the C. elegans homolog of the S. cerevisiae protein kinase STE11 .The STE11 gene is not essential for cell viability and a null mutation results in a defect in mating. The STE11 deleted strain contained an integrated copy of the upstream activating sequence (UAS) of the FUS1 gene fused to a reporter construct (HIS3 or beta-galactosidase). FUS1 transcription is absolutely dependent on the STE11 gene and therefore in the absence of STE11 the reporter construct is not expressed. The approach we undertook was to transform a ste11 deleted strain with a high-copy number plasmid library of C. elegans cDNAs expressed from the yeast adh1 promoter and identify cDNAs that were capable of inducing FUS1 transcription as measured by HIS3 and beta-galactosidase expression.

We screened approximately 58,000 transformants. Of those, 3 were able to grow on both SD-His plates and were blue on x-gal indicator plates. Restriction enzyme mapping of the insert and Southern hybridization showed that all three transformants contained the same insert. The inserts varied in size from approximately 1.35 - 1.40 kb. To confirm that the 1.40 kb insert was conferring the His+ phenotype we made a frameshift mutation in the cDNA. This plasmid, when reintroduced into a ste11 deleted strain, was unable to confer a His+ phenotype, suggesting that this phenotype is dependent on the intact open reading frame of the cDNA.

We asked whether the ability of the cDNA to confer the His+ phenotype was due to complementation or bypass of the ste11 mutation. We tested the ability of the cDNA to confer a His+ phenotype to other mutations in the pheromone response pathway. We selected deletion mutations of genes that have been shown to act downstrearn of the STE11 step in the pathway, i.e. STE7 and FUS3 / KSS1 protein kinases. These strains contained an integrated copy of the FUS1 UAS gene fused to a reporter construct the HIS3 gene. We expressed the cDNA in these strains and tested for HIS3 expression. For all strains the cDNA was able to confer a His+ phenotype. The ability of the cDNA to confer the His+ phenotype suggested that the cDNA was acting to bypass the signal transduction pathway and elicit its effect at the level of FUS1 gene expression. We have chosen to call the cDNA brp-1 for bypass of pheromone response pathway.

brp-1 could be inducing FUS1 gene expression indirectly by altering chromatin structure or it could be interacting specifically at the FUS1 UAS. We tested these possibilities by determining whether brp could induce transcription in the absence of a known FUS1 UAS DNA binding protein. The STE12 gene product is a DNA binding protein that is capable of binding to the FUS1 UAS and is required for FUS1 gene expression. The ability of brp-1 to induce FUS1 gene expression in the absence of STE12 would suggest that brp-1 'sability to induce FUS1 gene expression is not specific to the FUS1 UAS and may be caused by a general effect of brp, e.g. altering chromatin structure. However, if STE12 is required for brp's ability to induce FUS1 gene expression than brp may have a direct role in FUS1 expression. Using a ste12 deletion strain with a FUS1 UAS::HIS3 fusion construct we were able to show that brp requires STE12 to induce FUS1 expression. brp-1 'saction is specific for the pheromone response elements of the FUS1 UAS. In addition, brp was unable to induce transcription at two other yeast promoters (CYC1 and GAL1 ).

The brp-1 mRNA is extremely abundant ( ~0.04% of messages). Sequence analysis revealed no significant homology with other proteins in the Genebank databases. brp-1 has certain hallmark features of transcriptional activators. brp-1 maps to YAC Y56A3 (A. Coulson) located on LGIII near unc-45 .