SEQUENCING THE 500-KB GC-RICH SYMBIOTIC REPLICON OF RHIZOBIUM SP NGR234 USING DYE TERMINATORS AND A THERMOSTABLE SEQUENASE - A BEGINNING

Genomes of the soil-borne nitrogen-fixing symbionts of legumes [Azo(Br ady)Rhizobium species] typically have GC contents of 59-65 mol%. As a consequence, compressions (up to 400 per cosmid) are common using auto mated dye primer shotgun sequencing methods. To overcome this difficul ty, we have exclusively applied dye terminators in combination with a thermostable ''sequenase'' for shotgun sequencing GC-rich cosmids from pNGR234a, the 500-kbp symbiotic replicon of Rhizobium sp. NGR234. A t hermostable sequenase incorporates dye terminators into DNA more effic iently than Taq DNA polymerase, thus reducing the concentrations neede d (20- to 250-fold). Unincorporated dye terminators can simply be remo ved by ethanol precipitation. Here, we present data of pXB296, one of 23 overlapping cosmids representing pNGR234a. We demonstrate that the greatly reduced number of compressions results in a much Faster assemb ly of cosmid sequence data by comparing assembly of the shotgun data f rom pXB296 and the data from another pNGR234a cosmid (pXB11O) sequence d using dye primer methods. Within the 34,010-bp sequence from pXB296, 28 coding regions were predicted. All of them showed significant homo logies to known proteins, including oligopeptide permeases, an essenti al cluster for nitrogen fixation, and the C-4-dicarboxylate transporte r DctA.