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D W Mount

Publications and source records attributed to D W Mount.

At least 19 recordsLinked to original sources
PubMed2001

Arabidopsis UVH3 gene is a homolog of the Saccharomyces cerevisiae RAD2 and human XPG DNA repair genes.

To identify mechanisms of DNA repair in Arabidopsis thaliana, we have analyzed a mutant (uvh3) which exhibits increased sensitivity to ultraviolet (UV) light, H2O2 and ionizing radiation and displays a premature senescence phenotype. The uvh3 locus was mapped within chromosome III to the GL1 locus. A cosmid contig of the GL1 region was constructed, and individual cosmids were used to transform uvh3 mutant plants. Cosmid N9 was found to confer UV-resistance, H2O2-resistance and a normal senescence phenotype following transformation, indicating that the UVH3 gene is located on this cosmid and that all three phenotypes are due to the same mutation. Analysis of cosmid N9 sequences identified a gene showing strong similarity to two homologous repair genes, RAD2 (Saccharomyces cerevisiae) and XPG (human), which encode an endonuclease required for nucleotide excision repair of UV-damage. The uvh3 mutant was shown to carry a nonsense mutation in the coding region of the AtRAD2/XPG gene, thus revealing that the UVH3 gene encodes the AtRAD2/XPG gene product. In humans, the homologous XPG protein is also involved in removal of oxygen-damaged nucleotides by base excision repair. We discuss the possibility that the increased sensitivity of the uvh3 mutant to H2O2 and the premature senescence phenotype might result from failure to repair oxygen damage in plant tissues. Finally, we show that the AtRAD2/XPG gene is expressed at moderate levels in all plant tissues.

Amino Acid Sequence↗
PubMed2000

Repair of UV damage in plants by nucleotide excision repair: Arabidopsis UVH1 DNA repair gene is a homolog of Saccharomyces cerevisiae Rad1.

To analyze plant mechanisms for resistance to UV radiation, mutants of Arabidopsis that are hypersensitive to UV radiation (designated uvh and uvr) have been isolated. UVR2 and UVR3 products were previously identified as photolyases that remove UV-induced pyrimidine dimers in the presence of visible light. Plants also remove dimers in the absence of light by an as yet unidentified dark repair mechanism and uvh1 mutants are defective in this mechanism. The UVH1 locus was mapped to chromosome 5 and the position of the UVH1 gene was further delineated by Agrobacterium-mediated transformation of the uvh1-1 mutant with cosmids from this location. Cosmid NC23 complemented the UV hypersensitive phenotype and restored dimer removal in the uvh1-1 mutant. The cosmid encodes a protein similar to the S. cerevisiae RAD1 and human XPF products, components of an endonuclease that excises dimers by nucleotide excision repair (NER). The uvh1-1 mutation creates a G to A transition in intron 5 of this gene, resulting in a new 3' splice site and introducing an in-frame termination codon. These results provide evidence that the Arabidopsis UVH1/AtRAD1 product is a subunit of a repair endonuclease. The previous discovery in Lilium longiflorum of a homolog of human ERCC1 protein that comprises the second subunit of the repair endonuclease provides additional evidence for the existence of the repair endonuclease in plants. The UVH1 gene is strongly expressed in flower tissue and also in other tissues, suggesting that the repair endonuclease is widely utilized for repair of DNA damage in plant tissues.

Amino Acid Sequence↗
PubMed1997

Evidence that heat and ultraviolet radiation activate a common stress-response program in plants that is altered in the uvh6 mutant of Arabidopsis thaliana.

The uvh6 mutant of Arabidopsis was previously isolated in a screen for increased sensitivity to ultraviolet (UV) radiation. uvh6 mutant plants were killed by incubation at 37 degrees C for 4 d, a treatment not lethal to wild-type plants. Furthermore, under permissive conditions, uvh6 plants were yellow-green with an approximately one-third lower chlorophyll content. Genetic analysis of the uvh6 mutant strongly suggested that all three mutant phenotypes were due to mutation at the same genetic locus. To understand UVH6 function more fully, the response of wild-type plants to growth at elevated temperatures and exposure to UV radiation was analyzed. Wild-type plants grown at 30 degrees C were as UV-hypersensitive and yellow-green as uvh6 mutant plants grown at 24 degrees C. Mutant uvh6 plants induced heat-shock protein HSP21 at a lower threshold temperature than wild-type plants, indicating that the uvh6 mutant was exhibiting signs of heat stress at a 4 to 5 degrees C lower temperature than wild-type plants. We propose the UV damage and heat induce a common stress response in plants that leads to tissue death and reduced chloroplast function, and that the UVH6 product is a negative regulator of this response.

Arabidopsis↗
PubMed1995

Arabidopsis mutants deficient in T-DNA integration.

Arabidopsis thaliana mutants originally isolated as hypersensitive to irradiation were screened for the ability to be transformed by Agrobacterium transferred DNA (T-DNA). One of four UV-hypersensitive mutants and one of two gamma-hypersensitive mutants tested showed a significant reduction in the frequency of stable transformants compared with radioresistant controls. In a transient assay for T-DNA transfer independent of genomic integration, both mutant lines took up and expressed T-DNA as efficiently as parental lines. These lines are therefore deficient specifically in stable T-DNA integration and thus provide direct evidence for the role of a plant function in that process. As radiation hypersensitivity suggests a deficiency in repair of DNA damage, that plant function may be one that is also involved in DNA repair, possibly, from other evidence, in repair of double-strand DNA breaks.

Agrobacterium tumefaciens↗
PubMed1995

Radiation-sensitive mutants of Arabidopsis thaliana.

Five Arabidopsis mutants have been isolated on the basis of hypersensitivity of leaf tissue to UV light. For each mutant, the UV-hypersensitive phenotype (uvh) was inherited as a single recessive Mendelian trait. In addition, each uvh mutant represented a separate complementation group. Three of the mutations producing the UV hypersensitive phenotype have been mapped relative to either genetic markers or physical microsatellite polymorphisms. Locus UVH1 is linked to nga76 on chromosome 5, UVH3 to GL1 on chromosome three, and UVH6 to nga59 on chromosome 1. Each uvh mutant has a characteristic pattern of sensitivity based on UV sensitivity of leaf tissue, UV sensitivity of root tissue, and ionizing radiation sensitivity of seeds. On the basis of these patterns, possible molecular defects in these mutants are discussed.

Arabidopsis↗
PubMed1994

Identification of high affinity binding sites for LexA which define new DNA damage-inducible genes in Escherichia coli.

A multi-step screening procedure was devised to identify new operators for the LexA repressor in the sequenced portions of the genomes of Escherichia coli and its plasmids and bacteriophages. Sequence analysis methods were employed initially to distinguish true LexA operators from "operator-like" sequences stored within the GenBank and EMBL databases. The affinity of purified LexA protein for cloned DNA fragments containing several of the prospective new sites was then assessed using quantitative electrophoretic mobility shift assays and site-directed mutagenesis. Calculated binding affinities were compared directly with values determined for known and mutant LexA operators in concurrent experiments. Three E. coli chromosomal segments (near pyrC, hsdS and ntrla) and two bacteriophage sequences (near the P1 cre and lambda oop genes) bound LexA protein specifically. These sites and most others identified in the screening are located immediately upstream of known genes and/or large open reading frames. These results and additional transcription data demonstrate that several of the sequences define new DNA damage-inducible (din) genes and include the previously uncharacterized dinD locus. Furthermore, the search identified an SOS gene within the genome of P1 which encodes a protein that is homologous to UmuD', the RecA-promoted cleavage product of the umuD gene. The success of the combinatorial approach described here suggests that analogous searches for new regulatory sequences within the E. coli genome and the genomes of other organisms will also yield favorable results.

Amino Acid Sequence↗
PubMed1994

Isolation of uvh1, an Arabidopsis mutant hypersensitive to ultraviolet light and ionizing radiation.

A genetic screen for mutants of Arabidopsis that are hypersensitive to UV light was developed and used to isolate a new mutant designated uvh1. UV hypersensitivity in uvh1 was due to a single recessive trait that is probably located on chromosome 3. Although isolated as hypersensitive to an acute exposure to UV-C light, uvh1 was also hypersensitive to UV-B wavelengths, which are present in sunlight that reaches the earth's surface. UV-B damage to both wild-type and uvh1 plants could be significantly reduced by subsequent exposure of UV-irradiated plants to photoreactivating light, showing that photoreactivation of UV-B damage is important for plant viability and that uvh1 plants are not defective in photoreactivation. A new assay for DNA damage, the Dral assay, was developed and used to show that exposure of wild-type and uvh1 plants to a given dose of UV light induces the same amount of damage in chloroplast and nuclear DNA. Thus, uvh1 is not defective in a UV protective mechanism. uvh1 plants were also found to be hypersensitive to ionizing radiation. These results suggest that uvh1 is defective in a repair or tolerance mechanism that normally provides plants with resistance to several types of DNA damage.

Arabidopsis↗
PubMed1993

Novel mechanism for UV sensitivity and apparent UV nonmutability of recA432 mutants: persistent LexA cleavage following SOS induction.

The recA432 mutant allele was isolated (T. Kato and Y. Shinoura, Mol. Gen. Genet. 156:121-131, 1977) by virtue of its defect in cellular mutagenesis (Mut-) and its hypersensitivity to damage by UV irradiation (UVs), which were phenotypes expected for a recA mutant. However, we found that in a different genetic background (lexA51 sulA211 uvrB+), recA432 mutants expressed certain mutant phenotypes but not the Mut- and UVs phenotypes (D.G. Ennis, N. Ossanna, and D.W. Mount, J. Bacteriol. 171:2533-2541, 1989). We present several lines of evidence that these differences resulted from the sulA genotype of the cell and that the apparent UVs and Mut- phenotypes of the sulA+ derivatives resulted from lethal filamentation of induced cells because of persistent derepression of sulA. First, transduction of sulA(Def) mutations into the recA432 strains restored cellular mutagenesis and resistance to UV. Second, recA432 sulA+ strains underwent filamentous death following SOS-inducing treatments. Third, cleavage of LexA repressor in a recA432 strain continued at a rapid rate long after UV induction, at a time when cleavage of the repressor in the recA+ parental strain had substantially declined. Fourth, we confirmed that a single mutation (recA432) conferring both the UVs and Mut- phenotypes mapped to the recA gene. These findings indicate that the RecA432 mutant protein is defective in making the transition back to the deactivated state following SOS induction; thus, the SOS-induced state of recA432 mutants is prolonged and can account for an excess of SulA protein, leading to filamentation. These results are discussed in the context of molecular models for RecA activation for LexA and UmuD cleavage and their roles in the control of mutagenesis and cell division in the SOS response.

Bacterial Proteins↗
PubMed1992

Genetic identification of the DNA binding domain of Escherichia coli LexA protein.

Two genetic approaches were taken to define the DNA binding domain of LexA protein, the repressor of the Escherichia coli SOS regulon. First, several dominant negative lexA mutants defective in DNA binding were isolated. The mutations altered amino acids in a region similar to the helix-turn-helix, a DNA binding domain of other repressors and DNA binding proteins. Second, the region encoding the predicted DNA recognition helix was subjected to oligonucleotide-directed mutagenesis and mutant LexA proteins with altered or relaxed specificity for several recA operator positions were isolated. By examining the effects of a series of amino acid substitutions on repressor specificity, it was shown that a glutamic acid residue at position 45 in LexA protein is important for recognition of the first base pair (G.C) in the recA operator.

Amino Acid Sequence↗
PubMed1992

Isolation of DNA damage-inducible promoters in Escherichia coli: regulation of polB (dinA), dinG, and dinH by LexA repressor.

A new genetic screening method has been developed to isolate Escherichia coli promoters which are components of the SOS regulon. Plasmids containing the regulatory regions of polB (dinA) and two new loci, dinG and dinH, were characterized. Galactokinase gene fusion experiments indicated that transcription of these genes is inducible by treatment with mitomycin and conforms to a classical model of SOS regulation involving simple LexA repression. Mapping studies using the E. coli DNA library of Kohara et al. (Y. Kohara, K. Akiyama, and K. Isono, Cell 50:495-508, 1987) revealed that dinG and dinH are located at 17.8 and 19.8 min on the chromosome, respectively. The nucleotide sequence of the dinH regulatory region contains a segment which is very similar to previously characterized binding sites for LexA protein. An asymmetric, noncanonical 20-bp LexA operator in the cloned dinG promoter region was identified. Additional experiments have revealed that the nucleotide sequence of the gene immediately downstream of the DNA damage-inducible polB locus encodes a polypeptide which has extensive sequence homology to several known and putative DNA and RNA helicase proteins. This gene, which is not regulated by the LexA repressor, has been designated hepA. The predicted amino acid sequence of the product of hepA contains several highly conserved sequence motifs that are also found in enzymes such as the RecQ and UvrB proteins of E. coli and the Rad3 protein of Saccharomyces cerevisiae.

Amino Acid Sequence↗
PubMed1992

Interaction of LexA repressor with the asymmetric dinG operator and complete nucleotide sequence of the gene.

The dinG gene was originally isolated during a search for Escherichia coli promoters which are components of the SOS regulon. The regulatory region of this gene contains a potential binding site for LexA repressor which is quite different from other known sites. All previously described chromosomal LexA operators are imperfect palindromes containing the sequence CTG(N10)CAG. The noncanonical dinG sequence breaks the symmetry and takes the form TTG(N10)CAG. In the present study, a search for mutations within dinGop::galK fusion plasmids which render transcription independent of intracellular levels of LexA has yielded mutations only within this 16-bp sequence. Electrophoretic mobility shift assays performed with purified mutant and wild-type operator fragments revealed that the affinity of LexA for each of the mutant sites is greatly reduced compared with that of the wild type. One of the mutants contained an alteration in the putative promoter of dinG which increased the similarity of the -35 region to the consensus sequence (TTGGCT----TTGACT); the apparent promoter activity of this construct was subsequently found to be approximately eight times higher than that of the wild type in vivo. Additional experiments have established the complete nucleotide sequence of the dinG gene. A long open reading frame located immediately downstream of the asymmetric operator segment which could potentially encode a 72.9-kDa DinG protein was identified.

Amino Acid Sequence↗
PubMed1991

Mixed oligo designer (MOD), a computer program to aid planning of automated, mixed oligodeoxyribonucleotide synthesis for mutagenesis experiments.

A computer program, MOD (mixed oligo designer), which aids in planning site-directed mutagenesis experiments using highly substituted oligodeoxyribonucleotides (oligos), is described. The program calculates the relationship between the degree of oligo substitution and the mutation frequency, in order to achieve an optimal level of mutagenesis. The program can be used on a wide variety of computers and runs under a number of different operating systems.

Algorithms↗
PubMed1991

Repression of the E coli recA gene requires at least two LexA protein monomers.

To analyze the DNA binding domain of E coli LexA repressor and to test whether the repressor binds as a dimer to DNA, negative dominant lexA mutations affecting the binding domain have been isolated. A large number of amino acid substitutions between amino acid positions 39 and 46 were introduced using cassette mutagenesis. Mutants defective in DNA binding were identified and then examined for dominance to lexA+. A number of substitutions weakened repressor function partially, whereas other substitutions led to a repressor with no demonstrable activity and a defective dominant phenotype. Since the LexA binding site has dyad symmetry, we infer that this dominance results from interaction of monomers of wild-type LexA protein with mutant monomers and that an oligomeric form of repressor binds to operator. The binding of LexA protein to operator DNA was investigated further using a mutant protein, LexA408, which recognizes a symmetrically altered operator mutant but not wild-type operator. A mixture of mutant LexA408 and LexA+ proteins, but neither individual protein, bound to a hybrid recA operator consisting of mutant and wild-type operator half sites. These results suggest that at least 1 LexA protein monomer interacts with each operator half site. We discuss the role of LexA oligomer formation in binding of LexA to operator DNA.

Bacterial Proteins↗
PubMed1990

SPLICE, a computer program for automated extraction of information from GenBank sequence entries.

SPLICE, a software tool for the extraction of sequences from files in GenBank tape format, has been developed. The program can analyze the features table in this format and use any of the information provided to write the corresponding sequences into a standard sequence file format suitable for use with sequence analysis programs. Sequences that are present as several subsequent fragments in a single GenBank file, such as those encoding a peptide, can be spliced together by the program. Further, sequences that are present in more than one Genbank file, such as an exon which spans several different files, can also be spliced into one sequence. SPLICE runs under the MS/DOS and Unix operating systems, can be called as a sub-process by other programs and can process batches of files.

Animals↗
PubMed1989

Mutations in uvrD induce the SOS response in Escherichia coli.

We have isolated three new mutations in uvrD that increase expression of the Escherichia coli SOS response in the absence of DNA damage. Like other uvrD (DNA helicase II) mutants, these strains are sensitive to UV irradiation and have high spontaneous mutation frequencies. Complementation studies with uvrD+ showed that UV sensitivity and spontaneous mutator activity were recessive in these new mutants. The SOS-induction phenotype, however, was not completely complemented, which indicated that the mutant proteins were functioning in some capacity. The viability of one of the mutants in combination with rep-5 suggests that the protein is functional in DNA replication. We suggest that these mutant proteins are deficient in DNA repair activities (since UV sensitivity is complemented) but are able to participate in DNA replication. We believe that defective DNA replication in these mutants increases SOS expression.

Adenosine Triphosphatases↗