The Saccharomyces cerevisiae HIS7 gene was cloned by its location immediately downstream of the previously isolated and characterized ARO4 gene. The two genes have the same orientation with a distance of only 416 bp between the two open reading frames. The yeast HIS7 gene represents the first isolated eukaryotic gene encoding the enzymatic activities which catalyze the fifth and sixth step in histidine biosynthesis. The open reading frame of the HIS7 gene has a length of 1,656 bp resulting in a gene product of 552 amino acids with a calculated molecular weight of 61,082. Two findings implicate a bifunctional nature of the HIS7 gene product. First, the N-terminal and C-terminal segments of the deduced HIS7 amino acid sequence show significant homology to prokaryotic monofunctional glutamine amidotransferases and cyclases, respectively, involved in histidine biosynthesis. Second, the yeast HIS7 gene is able to suppress His auxotrophy of corresponding Escherichia coli hisH and hisF mutants. HIS7 gene expression is regulated by the general control system of amino acid biosynthesis. GCN4-dependent and GCN4-independent (basal) transcription use different initiator elements in the HIS7 promoter.
A novel method for molecular typing of organisms, amplified fragment length polymorphism analysis, was tested for its suitability in epidemiological studies in medical microbiology. Amplified fragment length polymorphism analysis, originally developed for typing crop plants, consists of a simple restriction-ligation reaction and a subsequent PCR amplification. In a single-step reaction, the genomic DNA is digested and the restriction fragments are ligated to specially constructed adapters. PCR amplification of such tagged restriction fragments with primers complementary to the adapters allows the detection of restriction fragment length polymorphisms upon resolution on agarose gels. The method is fast, efficient, and reproducible for typing strains of Legionella pneumophila isolated from both humans and the environment. The accuracy of the method was tested by comparison with standard restriction fragment length polymorphism typing performed with both a ribosomal and a genomic probe.
The HIS7 gene of Saccharomyces cerevisiae encodes a bifunctional glutamine amidotransferase:cyclase catalyzing two reactions that lead to the formation of biosynthetic intermediates of the amino acid histidine and the purine adenine. The HIS7 gene is activated by GCN4p under environmental conditions of amino acid starvation through two synergistic upstream sites GCRE1 and GCRE2. The BAS1p-BAS2p complex activates the HIS7 gene in response to adenine limitation. For this activation the proximal GCN4p-binding site GCRE2 is required. GCN4p and BAS1p bind to GCRE2 in vitro. Under conditions of simultaneous amino acid starvation and adenine limitation the effects of GCN4p and BAS1/2p are additive and both factors are necessary for maximal HIS7 transcription. These results suggest that GCN4p and BAS1/2p are able to act simultaneously through the same DNA sequence in vivo and use this site independently from each other in a non-exclusive manner.
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