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A Kleinhofs

Publications and source records attributed to A Kleinhofs.

At least 19 recordsLinked to original sources
PubMed1992

Cloning and mapping of telomere-associated sequences from Hordeum vulgare L.

We present a novel approach to the efficient cloning of telomere-associated sequences and demonstrate its application to the cloning and mapping of these sequences from barley. The method is a modification of the Vectorette PCR technique and allows specific amplification and cloning of subtelomeric sequences. Telomere-associated sequences isolated from barley include hypervariable, repetitive sequences. Polymorphisms detected by subtelomeric markers behaved as Mendelian factors and were mapped to the most distal positions of barley chromosomes 1, 2, and 4.

Base Sequence↗
PubMed1991

Characterization and sequence of a novel nitrate reductase from barley.

Barley (Hordeum vulgare L.) has both NADH-specific and NAD(P)H-bispecific nitrate reductases. Genomic and cDNA clones of the NADH nitrate reductase have been sequenced. In this study, a genomic clone (pMJ4.1) of a second type of nitrate reductase was isolated from barley by homology to a partial-length NADH nitrate reductase cDNA and the sequence determined. The open reading frame encodes a polypeptide of 891 amino acids and its interrupted by two small introns. The deduced amino acid sequence has 70% identity to the barley NADH-specific nitrate reductase. The non-coding regions of the pMJ4.1 gene have low homology (ca. 40%) to the corresponding regions of the NADH nitrate reductase gene. Expression of the pMJ4.1 nitrate reductase gene is induced by nitrate in root tissues which corresponds to the induction of NAD(P)H nitrate reductase activity. The pMJ4.1 nitrate reductase gene is sufficiently different from all previously reported higher plant nitrate reductase genes to suggest that it encodes the barley NAD(P)H-bispecific nitrate reductase.

Amino Acid Sequence↗
PubMed1991

Analysis of barley nitrate reductase cDNA and genomic clones.

Barley nitrate reductase cDNA and genomic clones were isolated by homology with the barley nitrate reductase cDNA clone bNRp10 and sequenced. This is the first reported analysis of a full-length nitrate reductase gene and its corresponding cDNA in the same species. The longest cDNA clone extends to within 9 bp of the ATG start codon and the sequence is similar to that reported for the higher plant NR sequences. As expected, the amino acid sequence of barley nitrate reductase is more related closely to the rice (84% homology) than to the Arabidopsis (62%) sequence. Four different polyA addition sites were identified from sequence analysis of nine barley NR cDNA clones. A 7.3 kb region of a genomic recombinant lambda clone was subcloned as two contiguous BamHI fragments into p Bluescript, designated pMJ7 and pMJ8, and sequenced. These clones include the entire nitrate reductase coding region, one large intron, 2.7 kb of untranslated sequence 5' to the translation start codon and 0.25 kb 3' to the translation termination codon. The mRNA cap site was identified as a cytosine, 111 bases upstream of the ATG translation start codon. The putative CAAT and TATA boxes were identified at -115 and -33 bp, respectively, with the mRNA cap site designated as +1. The barley nitrate reductase gene coding region strongly favors G or C in the third codon position.

Base Sequence↗
PubMed1988

Metabolic activation of the mutagen azide in biological systems.

Inorganic azide (N3-) mutagenicity is mediated through a metabolically synthesized organic azide, L-azidoalanine (N3-CH2-CH(-NH2)-COOH). L-Azidoalanine appears to be formed by the action of O-acetylserine (thiol)-Lyase (EC 4.2.99.8) using O-acetylserine and azide as substrates. In both plants and bacteria tested, azide substitutes for the natural substrate sulfide (S2-) in this reaction. Azide (L-azidoalanine) mutagenesis is highly attenuated by a deficiency in the excision of UV-like DNA damage (uvr-). Thus a premutation lesion recognizable by the bacterial excision-repair enzymes must be formed. Mutagenesis appears to proceed from this by 'direct mispairing' pathway. Azide (L-azidoalanine) mutagenicity is highly specific and involves a stereoselective process, but the molecular nature of the specificity has not been determined.

Alanine↗
PubMed1987

Cloning of seven differently complementing DNA fragments with chl functions from Escherichia coli K12.

Seven genomic libraries of chromosomal Escherichia coli K12 wild-type DNA were constructed in plasmid vectors. These were used to transform chl insertion mutants. Selection for growth on nitrate under anaerobic conditions yielded four plasmids which complemented mutants of the chlA, B, E and G types. The chromosomal fragments were mapped with restriction enzymes and subcloned. Three complementation groups were observed among the chlA mutants and two among the chlE mutants. The established complementation groups plus mutants of the chlD type represent eight distinct functions, which are all believed to be required for the molybdenum cofactor activity in the reduction of nitrate to nitrite by E. coli.

Chlorates↗
PubMed1987

Interaction of the mutagenic metabolite of sodium azide, synthesized in vitro, with DNA of barley embryos.

The in vitro synthesized sodium azide mutagenic metabolite (azidoalanine) produced single-strand breaks and proteinase K-sensitive sites in isolated, germinating barley embryos. In contrast with sodium azide, the efficiency of DNA damage induction was lower, and both types of DNA lesions were totally or partially repaired in the course of subsequent 24 h incubation of the embryos. The mutagenic azide metabolite did not inhibit DNA replication, while azide did so even at doses which are not highly mutagenic. The metabolite labelled with 14C at the amino acid residue was taken up with a similar efficiency both into barley embryos germinating for 2 days and into cells of Salmonella typhimurium TA100. The majority of the radioactivity was incorporated into proteins, less into RNA and a negligible amount into DNA.

Alanine↗
PubMed1986

Synthesis and mutagenicity of the two stereoisomers of an azide metabolite (azidoalanine).

The L- and D-isomers of azidoalanine (azide metabolite) have been chemically synthesized with 60% yield using corresponding N-(tert-butoxycarbonyl)-serine as starting materials. The mutagenic properties of synthesized L-azidoalanine are very similar to those of azide and in vivo synthesized azidoalanine. Synthetic D-azidoalanine shows very low mutagenic activity on Salmonella typhimurium TA1530 strain compared to that of the L-isomer. Thus a stereoselective process is involved in azidoalanine mutagenicity. The data presented in this study suggest that further biochemical activation is required for L-azidoalanine to produce its mutagenic activity.

Alanine↗
PubMed1979

In vivo conversion of sodium azide to a stable mutagenic metabolite in Salmonella typhimurium.

Salmonella typhimurium TA1530 and G46 strains growing in minimal medium supplemented with sodium azide produce a stable mutagenic metabolite which is not azide. The production of this metabolite is restricted to the log phase of bacteria grown in the presence of azide. The metabolite is highly mutagenic in DNA-repair defective base-substitution strains TA1530 and TA1535, but ineffective in frameshift strains TA1538 and TA1537. The metabolite induces mutations in resting cells of the TA1530 strain.

Azides↗
PubMed1978

Mutagenic and chromosome-breaking effects of azide in barley and human leukocytes.

Azide (10-3 M, solution buffered at pH 3) is more effective in inducing mutations in embryonic shoots of seeds germinated between 8 and 16 h than in non-germinated seeds and in seeds germinated between 0 and 8 h and 16 to 28 h. This peak of chlorophyll-deficient seedling mutation frequency coincides with maximum frequencies of seeding lethals and DNA replication in the cells of the embryonic shoot. The mutation data suggest azide may only act on replicating DNA. Azide induced no chromosome-aberration frequencies significantly above controls in (1) embryonic shoots of barley seeds germinated for 8--12 h, (2) microspores of barley and (3) human leukocytes. It appears to be a point-mutation mutagen.

Azides↗
PubMed1978

Azide.

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Animals↗
PubMed1976

Effect of excision repair on azide-induced mutagenesis.

Azide mutagenesis was investigated in Salmonella typhimurium and Escherichia coli. Azide was highly effective in inducing his+ revertants in excision-repair deficient (uvrB) derivatives of S. typhimurium hisG46 and in inducing high frequencies of 5-fluorouracil resistant mutants in excision-repair deficient (uvrA) derivatives of E. coli B/r WP2. In excision-repair plus strains, azide was only a marginal or ineffective mutagen, demonstrating that the bacterial excision-repair system could repair nearly all azide-induced damage. This observation suggests that the initial azide-induced lesion causes a major DNA helix distortion recognizable by the excision-repair endonucleases. The presence of recombination deficient (recB or recC) genes in combination with uvrA increased E. coli sensitivity to azide killing, but depressed azide mutagenicity. These results are similar to those reported for UV-induced mutagenesis with the E. coli strains and suggest that post-replication repair might be the error-prone step in the repair process. Azide mutagenesis specificity is, however, unique and different from UV, as demonstrated by inability of azide to revert the ochre try locus in E. coli WP2s. These results show that the initial azide-induced DNA damage is highly specific but different from UV-induced DNA damage. Metabolic inhibitors, similar in action to azide, did not induce mutations in S. typhimurium strain TA1530, a strain highly susceptible to azide mutagenesis, thus ruling out the possibility that azide mutagenesis was due to peroxide accumulation. A mechanism based on in vivo activation of azide to the actual mutagen is proposed.

Azides↗
PubMed1975

On the question of the integration of exogenous bacterial DNA into plant DNA.

Extensive studies with pea, tomato, and barley failed to confirm the evidence presented by previous investigators for integration or replication of exogenously applied bacterial DNA in these plants. Labeled DNA of buoyant density in CsCl intermediate between that of high density donor bacterial DNA and of plant DNA was never observed with axenic plants. Intermediate peaks, similar to those used as evidence for recombination by earlier investigators, were observed only when the plants were contaminated with bacteria. Plant DNA prepared by a published procedure [Ledoux, L. & Huart, R. (1969) J. Mol. Biol. 43, 243-262] was found to be contaminated with unidentified impurities. Such DNA was partially protected from the action of DNase and produced aberrant banding patterns in CsCl after shearing. Much of the published evidence for integration of foreign DNA in plants is based upon experiments with plant DNA prepared by this procedure. We conclude that contamination is the likely explanation for what has been interpreted as evidence for integration.

Centrifugation, Density Gradient↗