Isolation of the gene encoding Pyrococcus furiosus ornithine carbamoyltransferase and study of its expression profile in vivo and in vitro

Abstract

The gene coding for ornithine carbamoyltransferase (OTCase, argF in the hyperthermophilic archaea Pyrococcus furiosus was cloned by complementation of an OTCase mutant of Escherichia coil. The cloned P. furiosus argF gene also complemented a similar mutant of Saccharomyces cerevisiae. Sequencing revealed an open reading frame of 314 amino acids homologous to known OTCases and preceded by a TATA box showing only limited similarity with the Euryarchaeota consensus sequence. This is in accordance with the comparatively low in vitro promoter activity observed in a cell-free purified transcription system. Transcription initiates in vivo as well as in vitro at a guanine, 22 nucleotides downstream of the TATA box. Upstream from argF is a putative gene for diphthine synthetase, a eukaryotic enzyme assumed to occur also in archaea but not in bacteria.