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W M Barnes

Publications and source records attributed to W M Barnes.

At least 19 recordsLinked to original sources
PubMed1995

Crystal structure of the large fragment of Thermus aquaticus DNA polymerase I at 2.5-A resolution: structural basis for thermostability.

The crystal structure of the large fragment of the Thermus aquaticus DNA polymerase (Klentaq1), determined at 2.5-A resolution, demonstrates a compact two-domain architecture. The C-terminal domain is identical in fold to the equivalent region of the Klenow fragment of Escherichia coli DNA polymerase I (Klenow pol I). Although the N-terminal domain of Klentaq1 differs greatly in sequence from its counterpart in Klenow pol I, it has clearly evolved from a common ancestor. The structure of Klentaq1 reveals the strategy utilized by this protein to maintain activity at high temperatures and provides the structural basis for future improvements of the enzyme.

Amino Acid Sequence↗
PubMed1994

Effective amplification of long targets from cloned inserts and human genomic DNA.

We have used the polymerase chain reaction (PCR) to amplify up to 22 kb of the beta-globin gene cluster from human genomic DNA and up to 42 kb from phaga lambda DNA. We have also amplified 91 human genomic inserts of 9-23 kb directly from recombinant lambda plaques. To do this, we increased pH, added glycerol and dimethyl sulfoxide, decreased denaturation times, increased extension times, and used a secondary thermostable DNA polymerase that possesses a 3'-to 5'-exonuclease, or "proofreading," activity. Our "long PCR" protocols maintain the specificity required for targets in genomic DNA by using lower levels of polymerase and temperature and salt conditions for specific primer annealing. The ability to amplify DNA sequences of 10-40 kb will bring the speed and simplicity of PCR to genomic mapping and sequencing and facilitate studies in molecular genetics.

Bacteriophage lambda↗
PubMed1994

PCR amplification of up to 35-kb DNA with high fidelity and high yield from lambda bacteriophage templates.

A target length limitation to PCR amplification of DNA has been identified and addressed. Concomitantly, the base-pair fidelity, the ability to use PCR products as primers, and the maximum yield of target fragment were increased. These improvements were achieved by the combination of a high level of an exonuclease-free, N-terminal deletion mutant of Taq DNA polymerase, Klentaq1, with a very low level of a thermostable DNA polymerase exhibiting a 3'-exonuclease activity (Pfu, Vent, or Deep Vent). At least 35 kb can be amplified to high yields from 1 ng of lambda DNA template.

Bacteriophage lambda↗
PubMed1992

The fidelity of Taq polymerase catalyzing PCR is improved by an N-terminal deletion.

KlenTaq DNA polymerase is an N-terminally truncated Thermus aquaticus (Taq) DNA polymerase I. As expressed from a gene construct in Escherichia coli, translation initiates at Met236, bypassing the 5'----3' exonuclease domain of the DNA polymerase-encoding gene. A sensitive forward mutation assay was used to measure the relative number of mutations introduced into the entire lacZ gene by the polymerase chain reaction (PCR) under various conditions which allow the amplification of such a large DNA span. Two selectable markers, one at each end of the test lacZ fragment, were employed to avoid the plating and scoring of PCR artefacts such as primer initiation in the midst of the lacZ gene, and cloning artefacts such as empty vector plasmid. The measured relative mutation rate was twofold lower for KlenTaq as compared to the full-length Taq DNA polymerase.

Base Sequence↗
PubMed1990

Variable patterns of expression of luciferase in transgenic tobacco leaves.

A carboxyl-terminally modified firefly luciferase, encoded as a gene fusion to the neomycin phosphotransferase gene (which confers kanamycin resistance), was found to be enzymatically active for both enzymes when expressed in bacteria and in transgenic plants. A military-type starlight vision system was used to conveniently analyze the pattern of gene expression in transgenic tobacco plant leaves. Transgenic tobacco plants which expressed luciferase uniformly in all areas of the leaf, and assays for luciferin, demonstrated that luciferin rapidly penetrates all regions of a tobacco leaf in at least two dimensions. Depending on the test gene structure or, presumably, on the transferred DNA (T-DNA) insertional context, other transgenic plants were obtained that expressed luciferase with a wide range of nonuniform patterns from nominally the same cauliflower mosaic virus 35S promoter. For instance, the veins can be dark, while only the interveinal regions of the leaf lamina glow, or only the small capillary veins glow, or only the major veins glow. Local and/or systemic induction in response to wounding was also demonstrated.

Animals↗
PubMed1989

Integration of multiple copies of a foreign sequence into the Ti plasmid of Agrobacterium tumefaciens.

A method for constructing Ti plasmids bearing multiple copies of a sequence integrated in tandem is described. A small plasmid that confers tetracycline resistance (TcR), contains homology to a Ti plasmid, and is unable to replicate in Agrobacterium tumefaciens, was mobilized from Escherichia coli to A. tumefaciens. Ti plasmids of exconjugants selected for resistance to 12-14 micrograms Tc/ml all contained multiple tandem repeats of the integrative plasmid. Tc-sensitive variants with fewer integrated copies arose spontaneously at low frequency in the absence of Tc selection, or could be enriched for by selection on Tc in combination with the bactericidal antibiotic augmentin. Variants having an increased number of integrated copies were obtained by growth on high Tc concentrations. Tandem repeats integrated between border sequences provide, in principle, a way to reproducibly introduce many linked copies of any foreign gene into plants.

Blotting, Southern↗
PubMed1989

Influence of uvrB and pKM101 on the spectrum of spontaneous, UV- and gamma-ray-induced base substitutions that revert hisG46 in Salmonella typhimurium.

Oligonucleotide probes were used to identify base substitutions in 1089 revertants of hisG46 in Salmonella typhimurium that arose spontaneously or following irradiation with UV- or gamma-rays. The hisG46 allele, carrying a mutant CCC codon (Pro) in place of the wild-type codon CTC (Leu69) reverted via 6 distinguishable mutational events--C to T transitions at codon sites 1 or 2, C to A or C to G transversions at codon site 1, C to A at codon site 2, and an extragenic suppressor mutation. The distribution of hisG46 revertants differed among treatments and was influenced by the DNA-repair capacity of the bacteria. Plasmid pKM101 enhanced the frequencies of both spontaneous and induced mutations; transversion events were enhanced more efficiently by pKM101 than were transition events. Compared to Uvr+ bacteria, Uvr- bacteria had higher frequencies of spontaneous and induced mutations; transition mutations were enhanced more efficiently than were transversion mutations. The influence of DNA-repair activities on the mutational spectra provides some insights on the origins of spontaneous and UV-induced mutations.

DNA Damage↗
PubMed1988

Phenotypic and reversion analysis of a Salmonella typhimurium constructed to have an arginine codon at the hisG46 missense codon.

Of the 6 single-base mutations that would be predicted to change the missense mutation hisG46 away from a proline codon in the Salmonella/microsome mutagen selection assay for histidine-independent revertants, only 5 have been observed. We have used site-specific mutagenesis to make the unobserved mutant [CCC (proline)----CGC (arginine)] codon in the Salmonella genome. Experiments with this arginine mutant demonstrate that, like bacteria containing the hisG46 mutation, bacteria with the arginine missense mutation are histidine auxotrophs which are capable of reversion to histidine independence. However, unlike the ATP phosphoribosyltransferase coded by the hisG46 his G gene (with a proline), the arginine mutant enzyme is partially active. This is indicated by a histidine-independent phenotype when the arginine hisG gene is present in multiple copies.

ATP Phosphoribosyltransferase↗
PubMed1988

An analysis of the mutagenicity of 1,2-dibromoethane to Escherichia coli: influence of DNA repair activities and metabolic pathways.

The mutagenicity of 1,2-dibromoethane (EDB) to Escherichia coli was reduced by the UV light-induced excision repair system but unaffected by the loss of a major apurinic/apyrimidinic site repair function. At high doses, 70-90% of the EDB-induced mutations were independent of SOS-mutagenic processing and approximately 50% were independent of glutathione conjugation. The SOS-independent mutations induced by EDB were unaffected by the enzymes that repair alkylation-induced DNA lesions. EDB-induced base substitutions were dominated by GC to AT and AT to GC transitions. These results suggest that EDB-induced premutagenic lesions have some, but not all, of the characteristics of simple alkyl lesions.

Biotransformation↗
PubMed1986

Target sequences for mutagenesis in Salmonella histidine-requiring mutants.

Nucleotide target sequences involved in reversion to the wild type phenotype are diagrammed for Salmonella frameshift histidine-requiring mutants hisD3052, hisD3018, hisD6610, and hisD6580 and for base-substitution mutants hisG46 and hisG428. Frameshift strain hisC3076 probably reverts by nucleotide changes similar to those that occur during reversion of hisD3018 and hisD6610. Multiple modes of reversion characterize each strain. Each strain also has a particularly diagnostic mutagen-susceptible sequence. These highly mutagen-susceptible stretches are the hisD3052 GCGCGCGC sequence, the hisD6610 CCCCCC sequence, the hisD6580 AAAAA sequence, and the A/T containing codon of hisG428 and G/C containing codon of hisG46, respectively. Between them, hisG46 and hisG428 are reverted by all of the six possible base substitution transition and transversion mutations.

Base Sequence↗
PubMed1986

Colony probing as an alternative to standard sequencing as a means of direct analysis of chromosomal DNA to determine the spectrum of single-base changes in regions of known sequence.

We demonstrate here the means to directly analyze bacterial chromosomal DNA for all classes of single-base mutations in a specific codon in any region of known sequence through the use of DNA probing as a powerful substitute for standard sequencing techniques. With this method, chromosomal DNA from hundreds of mutants can be examined for single-base changes without any DNA cloning. This method can conveniently provide a large data base for the assessment of all classes of single-base mutations occurring spontaneously or induced by a known or suspected mutagen. The method is demonstrated for the analysis of histidine-independent (His+) revertants of hisG46, a missense mutation of Salmonella typhimurium that can revert in six or seven ways.

Azacitidine↗
PubMed1984

Spontaneous mutation frequencies in Salmonella: enhancement of G/C to A/T transitions and depression of deletion and frameshift mutation frequencies afforded by anoxic incubation.

Incubation of Salmonella typhimurium under anoxic conditions (0.1% oxygen or less) results in a substantial decrease in small (3-and 6-basepair) deletions in an A/T-rich region of the hisG gene in the hisG428 ochre mutant and also decreases the frequency of minus frameshift mutations in G/C-rich sequences in the his-D3052 and hisC3076 mutants. In contrast, the frequency of G/C-----A/T transition mutations increases substantially during anoxic growth of hisG46. Growth of revertants of strains carrying accessory deletions in the uvrB region of the Salmonella chromosome is drastically impaired on glucose minimal medium when oxygen partial pressures are below 0.1% oxygen.

Aerobiosis↗
PubMed1984

Mercuric ion-resistance operons of plasmid R100 and transposon Tn501: the beginning of the operon including the regulatory region and the first two structural genes.

The mercuric ion-resistance operons of plasmid R100 (originally from Shigella) and transposon Tn501 (originally from a plasmid isolated in Pseudomonas) have been compared by DNA sequence analysis. The sequences for the first 1340 base pairs of Tn501 are given with the best alignment with the comparable 1319 base pairs of R100. The homology between the two sequences starts at base 58 after the end of the insertion sequence IS-1 of R100. The sequences include the transcriptional regulatory region, and the homology is particularly strong in regions just upstream from potential transcriptional initiation sites. The trans-acting regulatory gene merR consists of 180 base pairs in both cases and codes for a highly basic polypeptide of 60 amino acids, which is also rich in serine. The Tn501 and R100 merR genes differ in 25 of the 180 base positions, and the resulting polypeptides differ in seven amino acids. The regulatory region before the major transcription initiation site contains potential -35 and -10 sequences and dyad symmetrical sequences, which may be the merR binding sites for transcriptional regulation. The first structural gene, merT, encodes a highly hydrophobic polypeptide of 116 amino acids. The R100 and Tn501 merT genes differ in 17% of their positions, leading to 14 (12%) amino acid changes. This region had previously been shown to encode a protein governing membrane transport of mercuric ions. The second structural gene, merC, would give a 91 amino acid polypeptide with a hydrophobic amino-terminal segment. The Tn501 and R100 merC genes differ at 37 base positions, leading to 10 amino acid changes.

Amino Acid Sequence↗
PubMed1983

DNA sequence changes of mutations in the histidine operon control region that decrease attenuation.

The DNA sequence changes of 18 (9 different) mutations in the control region of the histidine operon of Salmonella typhimurium are presented. All of these mutations increase the level of expression of the operon, presumably by decreasing transcription termination at the attenuator. Five of the mutations were previously isolated hisO mutations, and the other four were isolated here as His+ pseudorevertants of His- stop codon mutations in the leader peptide gene. Only two mutations, O1242 and O3154, directly affect the terminator stem of the leader RNA. One mutation, O1202, creates a strong new stem that would compete with the terminator stem. Most of the other mutations damage other RNA stems. Their effect can best be explained by, and they thus provide supporting evidence for, the prevailing model of attenuator regulation involving alternative, competing RNA stems in the leader RNA. Two mutations that do not appear to significantly affect an RNA stem directly, including a deletion of three of the seven consecutive histidine codons, are best explained as effects of a translating ribosome upon the RNA stem structures, even though the histidine codons are not translated in the pseudorevertants.

Base Sequence↗
PubMed1983

Kilo-sequencing: an ordered strategy for rapid DNA sequence data acquisition.

A strategy for rapid DNA sequence acquisition in an ordered, nonrandom manner, while retaining all of the conveniences of the dideoxy method with M13 transducing phage DNA template, is described. Target DNA 3 to 14 kb in size can be stably carried by our M13 vectors. Suitable targets are stretches of DNA which lack an enzyme recognition site which is unique on our cloning vectors and adjacent to the sequencing primer; current sites that are so useful when lacking are Pst, Xba, HindIII, BglII, EcoRI. By an in vitro procedure, we cut RF DNA once randomly and once specifically, to create thousands of deletions which start at the unique restriction site adjacent to the dideoxy sequencing primer and extend various distances across the target DNA. Phage carrying a desired size of deletions, whose DNA as template will give rise to DNA sequence data in a desired location along the target DNA, may be purified by electrophoresis alive on agarose gels. Phage running in the same location on the agarose gel thus conveniently give rise to nucleotide sequence data from the same kilobase of target DNA.

Base Sequence↗
PubMed1983

Structure and transcription of the nopaline synthase gene region of T-DNA.

We present the DNA sequence and plant-tumor transcription pattern of some 2400 base pairs from the right border region of pTi T37 DNA from the virulent Agrobacterium tumefaciens strain T37. This region includes the entire transcription unit encompassing the nopaline synthase gene, together with parts of other transcription units. The strategy used to determine the sequence also produced two opposing series of defined, asymmetric deletions across the target DNA region, some of which may serve future purposes in the exploitation of this sequence, which is known to be expressed in a wide variety of host plant tissues.

Amino Acid Oxidoreductases↗