Matthew Ashby

Role of Yeast Insulin-Degrading Enzyme Homologs in Propheromone Processing and Bud Site Selection

The Saccharomyces cerevisiae AXL1 gene product Axl1p shares homology with the insulin-degrading enzyme family of endoproteases. Yeast axl1 mutants showed a defect in a-factor pheromone secretion, and a probable site of processing by Axl1p was identified within the a-factor precursor. In addition, Axl1p appears to function as a morphogenetic determinant for axial bud site selection. Amino acid substitutions within the presumptive active site of Axl1p caused defects in propheromone processing but failed to perturb bud site selection. Thus, Axl1p has been shown to participate in the dual regulation of distinct signaling pathways, and a member of the insulinase family has been implicated in propeptide processing.

Serial Analysis of rRNA Genes and the Unexpected Dominance of Rare Members of Microbial Communities

ABSTRACT The accurate description of a microbial community is an important first step in understanding the roles of its components in ecosystem function. A method for surveying microbial communities termed serial analysis of rRNA genes (SARD) is described here. Through a series of molecular cloning steps, short DNA sequence tags are recovered from the fifth variable (V5) region of the prokaryotic 16S rRNA genes from microbial communities. These tags are ligated to form concatemers comprised of 20 to 40 tags which are cloned and identified by DNA sequencing. Four agricultural soil samples were profiled with SARD to assess the methods utility. A total of 37,008 SARD tags comprising 3,127 unique sequences were identified. A comparison of duplicate profiles from one soil genomic DNA preparation revealed that the method was highly reproducible. The large numbers of singleton tags, together with nonparametric richness estimates, indicated that a significant amount of sequence tag diversity remained undetected with this level of sampling. The abundance classes of the observed tags were scale-free and conformed to a power law distribution. Numerically, the majority of the total tags observed belonged to abundance classes that were each present at less than 1% of the community. Over 99% of the unique tags individually made up less than 1% of the community. Therefore, from either a numerical or diversity standpoint, taxa with low abundance comprised a significant proportion of the microbial communities examined and could potentially make a large contribution to ecosystem function. SARD may provide a means to explore the ecological roles of these rare members of microbial communities in qualitative and quantitative terms.

[20] Ras and a-factor converting enzyme

We have described several quantitative and qualitative assays that have been utilized to learn the basic properties of RACE and amphibian and mammalian counterparts. Owing to powerful genetic tractability, high specific activity, and an apparently well-conserved substrate specificity, yeast is an attractive organism in which to study RACE. Efforts are currently in progress to characterize the functional role of the endoproteolytic processing step of many essential proteins.