The proposed experiments are designed to demonstrate the power of our cosmid pDUAL """"""""in vivo deletion factory"""""""" vector for large scale genomic mapping and sequencing. We will (1) construct a high-redundancy cosmid library of the approximately 760 kb Ureaplasma parvum (U. urealyticum """"""""parvo"""""""") genome in the pDUAL-3 vector; (2) identify contigs by colony hybridization, probing primarily for genes that encode pathogenicity determinants; (3) sequence at least 100 kb in one approximately 70 kb contig, plus at least 30 kb from other contigs using pDUAL nested deletions; and, (4) construct a high resolution genomic restriction/gene/feature map, again taking advantage of pDUAL nested deletions. Ureaplasma parvum, the model organism to be used in this study, is a close relative of other well studied mycoplasmas, and is an important human pathogen, implicated in urogenital inflammation and spontaneous abortions. Our pDUAL deletion factory vector is used to generate very useful nested deletions that extend from a gamma-delta transposon end in the vector in either direction to random sites in cloned DNA, simply by intramolecular transposition; the desired deletions are selected by plating on simple selective bacteriological media. The vector contains a replication origin that permits cloned DNA to be maintained in single copy during growth, but amplified many-fold by temperature shift for plasmid preparation. This study will demonstrate the value of the very efficient pDUAL approach for genome analysis, including easy DNA sequencing, restriction mapping, gene localization and contig alignment. It will develop the biological substrates for efficient sequencing of the complete genome, and for comparing the genome organization of this interesting and important human pathogen with the genomes of other pathogenic mycoplasmas.
