Technology development at the interface of proteome research and genomics: mapping nonpolymorphic proteins on the physical map of mouse chromosomes.

Abstract

Data obtained from protein spots by peptide mass fingerprinting are used to identify the corresponding genes in sequence databases. The relevant cDNAs are obtained as clones from the Integrated Molecular Analysis of Genome Expression (I.M.A.G.E.) consortium. Mapping of I.M.A.G.E. clones is performed in two steps: first, cDNA clones are hybridized against a 10-hit genomic mouse bacterial artificial chromosome (BAC) library. Second, interspersed repetitive sequence polymerase chain reaction (IRS-PCR) using a single primer directed against the mouse B1 repeat element is performed on BACs. As each cDNA detects several BACs, and each individual BAC has a 50% chance to recover an IRS-PCR fragment, the majority of cDNAs produce at least a single IRS-PCR fragment. Individual IRS fragments are hybridized against high-density spotted filter grids containing the three-dimensional permutated pools of yeast artificial chromosome (YAC) library resources that are currently being used to construct a physical map of the mouse genome. IRS fragments that hybridize to YAC clones already placed into contigs immediately provide highly precise map positions. This technology therefore is able to draw links between proteins detected by 2-D gel electrophoresis and the corresponding gene loci in the mouse genome.