RAPID DNA SEQUENCING WITH LASER BASED MASS SPECTROMETRY

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The functional analysis of the yeast genome is currently focused on large open reading frames (ORFs). However, approximately one third of the genome is comprised of "intergenic regions," including small ORFs and other important non-coding elements. The overall goal of this proposed research is to develop a new means of random mutagenesis in yeast that can be applied on either the whole genome, on individual chromosomes, or on defined regions of individual chromosomes. We will test the usefulness of this new technology by analyzing intergenic regions of yeast chromosome VIII. Random insertion mutagenesis (RIM) involves the construction of DNA libraries in new integration vectors (pHITs) that allows linearization within the insert sequences via digestion with a unique type III restriction enzyme and subsequent integration into the yeast genome via homologous recombination. In the first phase of this project, we will construct these new integration vectors that will permit: (I) random mutagenesis directly in yeast and also allow control of the mutation frequency; (ii) selection of mutations in yeast; (iii) screening for in-frame fusions; (iv) recovery of mutations and amplification of mutagenized DNA sequences in E. Coli or via PCR; (v) removal of integrated sequences; (vi) recycling of selective yeast markers; (vii) repeated mutagenesis with the same library (e.g. to search for suppressors or synthetic lethals); (viii) unique tagging of newly identified open reading frames (ORFs) with a gene-specific oligonucleotide; and most importantly, (ix) reporter gene shuffling in vivo. This latter technique allows a rapid and easy exchange of reporter genes on a large scale to optimize conditions for determination of the cellular localization or for gene expression studies, secretion studies, two- hybrid analysis, etc. Furthermore, we will determine the integration efficiency, target specifically mutagenize defined areas in chromosome VIII of S288C diploid yeast. This will allow a specific search for novel small open reading frames and non- coding genetic elements. We will characterize selected phenotypes of RIM-induced mutations. Mutagenized DNA sequences will be recovered and sequenced. Disruption strains and plasmids will be made available to the scientific community.