This proposal outlines work on the annotation and elucidation of several bacterial genomes, including that of Rhodobacter capsulatus, now being sequenced at the University of Chicago in the laboratory in the laboratory of Dr. Robert Hasselkorn. The general aim of this work is to create a dictionary of gene functions that will be used to find commercially important genes in the genomes of pathogenic and industrially important organisms and attempt functional assignments in the human genome. Unusual DNA sequence symmetries, often implicated in the regulation of transcription, replication and recombination, will be mapped as part of a global annotation effort. These sequences, including inverted, mirror, and direct repeats, are statistically excluded from coding regions and will be used to eliminate spurious open reading frames (ORFs) and to identify important regulatory regions. Annotation of these degenerate sequences will be included in an automatic annotation system, called MAGPIE, being developed at the University of Chicago. ORF boundaries will be confirmed by direct transcriptional assays. Important regulatory elements can be tested for function by destroying the symmetries in these sequences and assaying for transcriptional anomalies in vitro. Parallel sequencing of the genomes of other strains of R. capsulatus will allow for the comparison of degenerate sequence motifs and recombination points. Suggestive degenerate sequences will be tested for recombinagenic activity directly in a simple in vivo system involving restoration of an antibiotic resistance marker. Insights into ORF determination and predicting recombination may be relevant to similar tasks in much more complicated sequences like the human genome.
