Nucleotide sequence and analysis of deletion mutants of the Escherichia coli gpt gene in plasmid pSV2gpt

Jagadeeswaran, Pudur; Ashman, Charles R.; Roberts, Savithri; Langenberg, John

doi:10.1016/0378-1119(84)90228-2

Cited by 12 publications

(5 citation statements)

References 10 publications

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“…One interesting feature of the deletions is that the great majority of them are relatively small. It should be possible to rescue vector sequences from Sgur A912 cell mutants that have deletions of the entire gpt gene (456 bp) (18)(19)(20) likely to occur, although the breakpoints of these deletions may not be exactly the same in each event.…”

Section: Methodsmentioning

confidence: 99%

Sequence analysis of spontaneous mutations in a shuttle vector gene integrated into mammalian chromosomal DNA.

Ashman¹,

Davidson²

1987

Proc. Natl. Acad. Sci. U.S.A.

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We have studied the molecular mechanisms of spontaneous mutations in mouse cells carrying a selectable bacterial gene. The mouse cells carry the Escherichia coli xanthine (guanine) phosphoribosyltransferase (gpt) gene in a retroviral shuttle vector integrated into chromosomal DNA in a proviral form. Cells with spontaneous mutations in the gpt gene were selected as resistant to 6-thioguanine and then were fused with COS cells for recovery of the mutant genes. Out of a total of 77 independent 6-thioguanine-resistant cell lines isolated in this study, vector sequences could be rescued from 43 of the mutant lines, and the base sequences were determined for the gpt genes in all 43 of these lines. There was a variety of mutational events among the mutant gpt genes sequenced. The most frequent mutational event was a deletion (in 29 of the 43 mutant genes), and the next most frequent event was a base substitution mutation (in 11 ofthe 43 mutant genes). Among the deletion mutants, the great majority represent deletions of less than 10 base pairs. In fact, 19 of the 29 deletion mutants had deletions of 3 base pairs, and among the mutants with 3-base. pair deletions, there was a very strong deletion hot spot appearing in 16 independent mutants. Afl 19 of the 3-base-pair deletions resulted in the "in frame" loss of an aspartic acid codon. Among the base substitution mutations, transitions and transversions occurred with approximately equal frequency. Our results raise the possibility that small deletions represent the predominant mechanisms by which spontaneous mutations occur in mammalian cells.Recently, several systems have been described that make possible the rapid and unambiguous determination of base sequence changes in genes that have undergone mutation in mammalian cells (1)(2)(3)(4)(5). In these systems, a target gene is introduced into the mammalian cells as part of a shuttle vector, which is capable of replication in both mammalian cells and bacteria. Following mutagenesis in the mammalian cell environment, the shuttle vector sequences are recovered from the mammalian cells and are introduced into bacteria, where large amounts of the mutant gene can be produced for sequence analysis. The major advantage of these shuttle vector systems is that large numbers of mutant genes can be rapidly isolated in a form that is convenient for DNA sequence analysis.We recently described the development of a system of this type, which utilizes a retroviral shuttle vector (4). As a target gene, this vector contains the Escherichia coli gpt gene, which codes for the enzyme xanthine (guanine) phosphoribosyltransferase (XPRTase). The vector was introduced by infection into the mouse A9 cell line (6), which is deficient in hypoxanithine (guanine) phosphoribosyltransferase. A stable cell line (A9I2), which contains a single copy of the vector integrated into chromosomal DNA in a proviral form, was isolated. Mutants resistant to 6-thioguanine (Sgu) were induced in A9I2 following treatment of the cells with either ethyl methanesu...

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Section: Methodsmentioning

confidence: 99%

Sequence analysis of spontaneous mutations in a shuttle vector gene integrated into mammalian chromosomal DNA.

Ashman¹,

Davidson²

1987

Proc. Natl. Acad. Sci. U.S.A.

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“…5A). The NueI site is located at nucleotide residue -66 of the rat HO gene [7] and the BstNI site is located at nucleotide residue 222 of pSV2gpt [17].…”

Section: Constructions Of Chimeric Fusion Genesmentioning

confidence: 99%

Structural organization of the human heme oxygenase gene and the function of its promoter

Shibahara

Sato

Müller

et al. 1989

European Journal of Biochemistry

132

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Human heme oxygenase is induced by its substrate heme, but not induced by heat shock [Yoshida et al. (1988) Eur. J . Biochem. 1 7 / , 457-4611. In order to study the molecular mechanisms of heme-mediated induction of human hcmc oxygenase, we have isolated and characterized the genomic clones for heme oxygenase. The human heme oxygenase gene (HO gene) is about 14 kb long and organized into five exons. The transcription initiation site was identified by S1 nuclease mapping and primer-extension analyses. Using HeLa whole cell extracts, we confirmed that the transcription of the cloned HO gene is initiated accurately at the assigned initiation site. In its 5'-flanking region, a potential heat-shock element (HSE) is present 367 bp upstream from the initiation site, although, in contrast to rat heme oxygenase, human enzyme is not induced by heat shock. We therefore analyzed the effects of heat shock on the transient expression of chimeric fusion genes harboring the promoter of the human HO gene ligated to the Escherichia coli gene gpt in mouse amelanotic melanoma cells. The 5'-flanking region of the human HO gene bearing a potential HSE failed to confer the heat-inducibility of gpt RNA production, suggesting that the HSE of the human HO gene is not functional.Heme oxygenase, an essential enzyme in heme catabolism, cleaves heme to form biliverdin [l], which is subsequently converted to bilirubin by biliverdin reductase in mammals [2]. The activity of heme oxygenase is highest in the spleen, where senescent erythrocytes (nearly 1 YO of erythrocytes in man) are sequestrated and destroyed every day. Heme oxygenase also plays an important role in determining the availability of cellular heme for various hemoproteins, such as cytochrome P-450, catalase and tryptophan pyrrolase [3]. It is therefore of particular significance that the activity of heme oxygenase is increased by its substrate heme [4, 51 as well as by various non-heme substances [3]. The increased activity of heme oxygenase may result in the depletion of cellular free heme and cause functional disturbances of such hemoproteins (reviewed in [6]).Recently, we have cloned and characterized the rat heme oxygenase gene (HO gene) [7]. Its 5'-flanking region contains a potential heat-shock element (HSE), which was originally found in the 5'-flanking regions of heat-shock protein genes and conferred the transcriptional induction of these genes by environmental derangements (reviewed in [S, 91). We thus found that rat heme oxygenase is a heat-shock protein and hemin induces heme oxygenase by a different mechanism from heat shock [lo]. In contrast to rat enzyme, human heme oxygenase is not induced by heat shock, suggesting that human enzyme is not a heat-shock protein, although hemin increases both mRNA levels and activity of heme oxygenase in human macrophages and glioma cells [I 11.In order to study the regulation of its expression, we have cloned and characterized the human HO gene. In this report, we show the structural organization of the gene and present the nucl...

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“…Actually the presence of a potential heat-shock element led us to find that heme oxygenase is a heat-shock protein in rat glioma cells [12]. The 5'-flanking region of the rat heme oxygenase gene is sufficient t o confer the heat inducibility of the transient expression of Escherichia coli Correspondence to S. Shibahara [13], both in rat glioma cells and in mouse amelanotic melanoma cells [12].…”

mentioning

confidence: 99%

Human heme oxygenase cDNA and induction of its mRNA by hemin

Yoshida

Bíró

Cohen

et al. 1988

European Journal of Biochemistry

299

203

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Hemin treatment increased both activity and mRNA level of heme oxygenase in human macrophages. Using poly(A)‐rich RNA prepared from human macrophages treated with hemin, we have constructed a cDNA library in the Okayama‐Berg vector. The human heme oxygenase cDNA was isolated by screening this library with a rat cDNA and was subjected to nucleotide sequence analysis. The deduced human heme oxygenase is composed of 288 amino acids with a molecular mass of 32800 Da. The homology in amino acid sequences between rat and human heme oxygenase is 80%. Like rat heme oxygenase, human enzyme has a putative membrane segment at its carboxyl terminus, which is probably essential for the insertion of heme oxygenase into endoplasmic reticulum. Both rat and human heme oxygenase have no cysteine residues. Recently we have shown that rat heme oxygenase is a heat‐shock protein [J. Biol. Chem. 262, 12889–12892 (1987)], and therefore we examined the effects of heat treatment on the induction of heme oxygenase in human macrophages and glioma cells. In contrast to hemin treatment, heat treatment had no apparent effects in either human cell line on the activity of heme oxygenase and its mRNA levels. These results suggest that human heme oxygenase may not be a heat‐shock protein.

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Nucleotide sequence and analysis of deletion mutants of the Escherichia coli gpt gene in plasmid pSV2gpt

Cited by 12 publications

References 10 publications

Sequence analysis of spontaneous mutations in a shuttle vector gene integrated into mammalian chromosomal DNA.

Sequence analysis of spontaneous mutations in a shuttle vector gene integrated into mammalian chromosomal DNA.

Structural organization of the human heme oxygenase gene and the function of its promoter

Human heme oxygenase cDNA and induction of its mRNA by hemin

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