title: The Pichia pastoris HIS4 gene: nucleotide sequence, creation of a non-reverting his4 deletion mutant, and development of HIS4-based replicating and integrating plasmids.
abstract: We have obtained a clone of the Pichia pastoris HIS4 gene and have determined its nucleotide sequence. Based upon its deduced amino-acid sequence, the product of the P. pastoris HIS4 gene has the same structural organization as the Saccharomyces cerevisiae His4 protein and appears to encode a trifunctional enzyme catalyzing the second (phosphoribosyl-ATP pyrophosphohydrolase), third (phosphoribosyl-AMP cyclohydrolase), and tenth (histidinol dehydrogenase) steps in histidine biosynthesis. The chromosomal copy of the HIS4 gene was disrupted by homologous recombination, creating the strain SGY58. The his4 delta deletion mutation in this strain lacks the entire coding region of this gene and has a reversion rate that is undetectable. A set of complementary plasmids that carry the HIS4 gene was also developed. Among these are nine E. coli-P. pastoris shuttle vectors that transform the his4 delta deletion mutant at high efficiency and an integration vector for creating site-specific alterations of the P. pastoris genome.
authors.affiliation: Kennedy Krieger Research Institute, Baltimore, MD 21205.
authors.forename: D I
authors.initials: DI
authors.lastname: Crane
authors.forename: S J
authors.initials: SJ
authors.lastname: Gould
mesh_terms: D000429:Alcohol Oxidoreductases; D000595:Amino Acid Sequence; D000619:Aminohydrolases; D001483:Base Sequence; D003062:Codon; D004261:DNA Replication; D005656:Fungal Proteins / Q000235:genetics*; D017353:Gene Deletion; D005800:Genes, Fungal; D016681:Genome, Fungal; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics*; D016366:Open Reading Frames; D010843:Pichia / Q000201:enzymology / Q000235:genetics*; D010957:Plasmids / Q000378:metabolism*; D011755:Pyrophosphatases; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000235:genetics*
chemical_list: D003062:Codon; D005656:Fungal Proteins; D009097:Multienzyme Complexes; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D000429:Alcohol Oxidoreductases; C092116:HIS4 protein, S cerevisiae; D000619:Aminohydrolases; D011755:Pyrophosphatases