Isolation and dna sequence of the gene encoding thermus aquaticus yt-1 aspartate aminotransferase

The Aspartate Aminotransferase (AspAT) (E.C. 2.6.1.1) was purified to homogeneity from the gram negative, extremely thermophilic, bacteria Thermus aquaticus YT-1 and Thermus ruber Murrieta (isolated from Murrieta Hot Spring in California USA). The purified AspAT N-terminal amino acid equences were determined by automated Edman degradation using an ABI Procise 491 sequencer. The N-terminal amino acid sequence of the T. aquaticus AspAT was used to infer a nucleotide sequence which was used to construct PCR primers. These primers were then used to obtain a 120 bp product for probing Southern blots. T. aquaticus restriction enzyme digested DNA fragments, which hybridized with the PCR probe, were subsequently cloned, sequenced and analyzed. The full length T" aquaticus AspAT gene was determined to be 1146 base pairs. (The T. a uaticus native AspAT, like most bacterial AspATs, is made up of two identical subunits). An examination of the N-ferminal and the central portion of the nucleotide sequence of the T. aquaticus AspAT gene showed that there was a high degree of similarity with the AspAT gene sequence of Thermus thermophilus HB8 recently reported by Okamato et. al. (J. Biochem. 119:135-144 (1996). However the C-terminal end of the T. aquaticus AspAT gene showed a nucleotide and amino acid sequence that was quite different from that found with the T. thermophilus HB8 AspAT. This difference may tend to support the recent proposal of Williams et. al. (Int. J. Syst. Bacteriol. 45:495-499 (1995))that T.thermophilus be recognized as a distinct species of Thermus rather than be classified as a strain of T. aquaticus.

Duke Scholars

Author Eric James Benner Pediatrics, Neonatology