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F Hutchinson

Publications and source records attributed to F Hutchinson.

At least 19 recordsLinked to original sources
PubMed1992

Published data on mutagenesis by ionizing radiation of plasmids in solution probably reflect in part the specificity of adventitious transition metal ions complexed to the DNA.

A number of recent papers show that single base changes induced by mutagenesis with ionizing radiation of genes on plasmids in solution, followed by transfection into mammalian or bacterial cells for assay, are mostly at G:C base pairs, with mutagenic hot spots. Genes irradiated in mammalian or bacterial cells, on the other hand, have comparable numbers of base changes at all sites, with no evidence for hot spots. The differences are ascribed to induction of many base change mutations in vitro by reactions catalyzed by adventitious transition metal ions complexed to the DNA. Reasons are given why this process should play a much smaller role in vivo.

Base Composition↗
PubMed1991

Deletions induced by gamma rays in the genome of Escherichia coli.

An Escherichia coli lysogen was constructed with a lambda phage bearing a lacZ gene surrounded by about 100 x 10(3) base-pairs of dispensable DNA. The lacZ mutants induced by gamma rays in this lysogen were more than 10% large deletions, ranging in size from 0.6 x 10(-3) to 70 x 10(3) base-pairs. These deletions were centered, not on lacZ, but on a ColE1 origin of DNA replication located 1.2 x 10(3) bases downstream from lacZ. This suggested that this origin of replication was involved in the process by which the deletions were formed. In agreement with this hypothesis, a lysogen of the same phage without the ColE1 origin showed a very much lower percentage of radiation-induced deletions, as did a second lysogen of a lambda phage without any known plasmid origin of replication. Indirect evidence is presented for radiation-induced deletions centered on the lambda origin of DNA replication in a lysogen. It is suggested that high percentages of large deletions may occur among radiation-induced mutations in mammalian cells because deletions centered on some of the thousands of origins of replication in these genomes do not kill the cells.

Bacteriophage lambda↗
PubMed1991

Mutagenic specificity of N-methyl-N'-nitro-N-nitrosoguanidine in the gpt gene on a chromosome of Chinese hamster ovary cells and of Escherichia coli cells.

DNA base sequence changes induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) mutagenesis have been determined for the Escherichia coli gpt gene stably incorporated in a chromosome of Chinese hamster ovary cells and in the chromosome of both growing and starving E. coli cells, instead of on a plasmid as in most previous studies. In the three cases, nearly all mutations were G:C to A:T transitions, with a 2- to 4-fold higher mutation rate, compared to other sites, at guanines flanked on the 5' side by another guanine. Mutagenic hot spots in these experiments were less prominent than in published results for MNNG mutagenesis of gpt and of other genes. A suggested explanation involves repair of O6meG. At low levels of mutagenic products, most are repaired and even small differences in the repair rates leads to large differences in the relative amounts of residual O6meG at various sites; in contrast, at high levels of mutagenic products there is little effect of repair on the distribution.

Animals↗
PubMed1991

DNA sequence changes in mutations induced by ultraviolet light in the gpt gene on the chromosome of Escherichia coli uvr+ and urvA cells.

Sequence changes in mutations induced by ultraviolet light are reported for the chromosomal Escherichia coli gpt gene in almost isogenic E. coli uvr+ and excision-deficient uvrA cells. Differences between the mutagenic spectra are ascribed to preferential removal of photoproducts in the transcribed strand by excision repair in uvr+ cells. This conclusion is confirmed by analysis of published results for genes in both uvr+ and uvr- cells, showing a similar selective removal of mutagenic products from the transcribed strand of the E. coli lacI gene and of the lambda phage cI repressor gene. Comparison of these data with published results for ultraviolet mutagenesis of gpt on a chromosome in Chinese hamster ovary cells showed that a mutagenic hot spot in mammalian cells is not present in E. coli; the possibility is suggested that the hot spot might arise from localized lack of excision repair. Otherwise, mutagenesis in hamster cells appeared similar to that in E. coli uvr+ cells, except there appears to be a smaller fraction of single-base additions and deletions (frameshifts) in mammalian than in bacterial cells. Phenotypes of 6-thioguanine-resistant E. coli showed there is a gene (or genes) other than gpt involved in the utilization of thioguanine by bacteria.

Animals↗
PubMed1989

DNA base sequence changes induced by ultraviolet light mutagenesis of a gene on a chromosome in Chinese hamster ovary cells.

The DNA base sequence changes induced by mutagenesis with ultraviolet light have been determined in a gene on a chromosome of cultured Chinese hamster ovary (CHO) cells. The gene was the Escherichia coli gpt gene, of which a single copy was stably incorporated and expressed in the CHO cell genome. The cells were irradiated with ultraviolet light and gpt- colonies were selected by resistance to 6-thioguanine. The gpt gene was amplified from chromosomal DNA by use of the polymerase chain reaction (PCR), and the amplified DNA sequenced directly by the dideoxy method. Of the 58 sequenced mutants of independent origin 53 were base change mutations. Forty-one base substitutions were single base changes, ten had two adjacent (or tandem) base changes, and one had two base changes separated by a single base-pair. Only one mutant had a multiple base change mutation with two or more well separated base changes. In contrast much higher levels of such mutations were reported in ultraviolet mutagenesis of genes on a shuttle vector in primate cells. Two deletions of a single base-pair were observed and three deletions ranging from 6 to 37 base-pairs. The mutation spectrum in the gpt gene had similarities to the ultraviolet mutation spectra for several genes in prokaryotes, which suggests similarities in mutational mechanisms in prokaryotes and eukaryotes.

Animals↗
PubMed1989

Prosthetic usage following major lower extremity amputation.

There were 157 patients following major lower extremity amputation who were evaluated to determine functional prosthetic ambulation. Twenty-eight patients were evaluated in the amputee clinic and found not to be candidates for prosthetic fitting. Forty-one patients were fit with a prosthesis but did not become functional prosthetic ambulators. Eighty-eight patients became functional prosthetic ambulators. Of all above-the-knee amputees, 46% became functional prosthetic ambulators. Only 19% of bilateral lower extremity amputees became functional prosthetic ambulators. Of all below-the-knee amputees, 66% became functional ambulators. The presence of coronary artery disease decreased the ambulatory potential in above-the-knee or bilateral amputees but not in below-the-knee amputees. A screening method for elderly, dysvascular amputees should be instituted prior to prosthetic fitting.

Aged↗
PubMed1988

Effect of photoreactivation on mutagenesis of lambda phage by ultraviolet light.

There is disagreement in the literature as to whether the major mutagenic photoproduct induced in DNA by ultraviolet light is the cyclobutane dipyrimidine dimer, the most common product, or the [6-4] photoproduct, the next most frequent. In the experiments reported here, cyclobutane dimers were removed from irradiated lambda phage DNA by enzymatic photoreactivation, a process thought to affect no other photoproduct. Photoreactivation of lambda phage in host cells and of lambda DNA in solution reduced clear plaque mutants per plaque-forming unit by two-thirds, in host cells with a constant and near-maximal expression of the SOS functions required for mutagenesis. This result is interpreted to mean that removal of cyclobutane dimers in or near the mutated gene reduces mutation induced by ultraviolet light by two-thirds; therefore, cyclobutane dimers in the phage DNA are responsible for most observed mutations. DNA sequences of mutations in photoreactivated phage showed a smaller fraction of G.C to A.T transitions and a larger fraction of A.T to G.C transitions, compared to phage that were not photoreactivated. This suggests that cyclobutane dimers at TC and CC sites are particularly mutagenic.

Bacteriophage lambda↗
PubMed1988

Changes in DNA base sequence induced by gamma-ray mutagenesis of lambda phage and prophage.

Mutations in the cI (repressor) gene were induced by gamma-ray irradiation of lambda phage and of prophage, and 121 mutations were sequenced. Two-thirds of the mutations in irradiated phage assayed in recA host cells (no induction of the SOS response) were G:C to A:T transitions; it is hypothesized that these may arise during DNA replication from adenine mispairing with a cytosine product deaminated by irradiation. For irradiated phage assayed in host cells in which the SOS response had been induced, 85% of the mutations were base substitutions, and in 40 of the 41 base changes, a preexisting base pair had been replaced by an A:T pair; these might come from damaged bases acting as AP (apurinic or apyrimidinic) sites. The remaining mutations were 1 and 2 base deletions. In irradiated prophage, base change mutations involved the substitution of both A:T and of G:C pairs for the preexisting pairs; the substitution of G:C pairs shows that some base substitution mechanism acts on the cell genome but not on the phage. In the irradiated prophage, frameshifts and a significant number of gross rearrangements were also found.

Bacteriophage lambda↗
PubMed1987

Ultraviolet light-induced mutagenesis in the Escherichia coli chromosome. Sequences of mutants in the cI gene of a lambda lysogen.

DNA sequences were determined for 56 mutations induced by ultraviolet light in the lambda cI gene of an Escherichia coli uvr+ lysogen, which should reflect those occurring in the E. coli chromosome. The spectrum of mutagenesis was similar to that found in the cI gene of irradiated phase assayed in uvr- host cells, except that the fraction of transversions is about 35% in prophage and about 15% in phage. The cause of this difference is not known. Of 17 frameshifts in phage and prophage, six have an accompanying base substitution. These double mutational events are consistent with a model in which a photoproduct in a template can cause a DNA polymerase to insert a wrong base and destabilize the next few bases added, thus leading to a frameshift by a slippage mechanism.

Base Composition↗
PubMed1987

Effect of the direction of DNA replication on mutagenesis by N-methyl-N'-nitro-N-nitrosoguanidine in adapted cells of Escherichia coli.

The methylating agent N-methyl-N'-nitro-N-nitrosoguanidine preferentially induces G:C to A:T transitions at DNA base pairs with the G in one particular strand of the cI gene in a lambda prophage, in this case the nontranscribed strand, in Escherichia coli cells in which the adaptive response is induced. The same preference is found for the cI gene inserted in the genome in the inverse orientation, so the differential effect is not caused by the direction of motion of the DNA replicating fork.

Base Composition↗
PubMed1985

Sequence specificity of mutagenesis in the cI gene of bacteriophage lambda.

Studies of DNA base sequence alterations have shown that for every agent the mutagenic process is specific with respect to the types of base changes induced and the location of the changes in the DNA. Analysis of the types of mutations produced by mutagenic agents can provide insight into the mechanism of mutation and can suggest which DNA lesions may be involved in the actual mutagenic event. We have developed a system for the analysis of chemically induced base sequence alterations in the cI repressor gene of bacteriophage lambda using DNA sequencing techniques. To illustrate the utility of this type of analysis, we present the results obtained with ultraviolet light (UV). Irradiation of target DNA with UV alone, or UV followed by photoreactivating light (which removes dimers), produces mostly transitions at pyrimidine-pyrimidine sites. Conversely, irradiation with 313 nm light plus acetophenone (which produces only thymine dimers) produces mostly transversions at low efficiency. This and other evidence suggests that the actual premutagenic UV lesion in E. coli may not be pyrimidine-pyrimidine dimers, but rather pyr(6-4)pyo photoproducts.

Bacteriophage lambda↗
PubMed1979

Response to bleomycin of Escherichia coli mutants deficient in DNA repair.

The effect of bleomycin on the colony forming ability of Escherichia coli K12 strains in exponential growth at 37 degrees C was not affected by introducing recA13, lexA1, polA1 and uvrA6 mutations. For cells starved for amino acids, wild type strains became ten-fold more resistant to bleomycin, but again introducing lexA1, polA1 and uvrA6 strains did not change the effect on colony forming ability; however, starved recA13 cells were now four-fold more sensitive. Strains with recA13, lexA1 and polA1 mutations were always more sensitive than wild type to gamma rays under the same conditions as used for the bleomycin treatment. It is suggested that bleomycin-induced lesions may be concentrated in that part of the bacterial genomes at the cell wall, near the replication forks.

Bleomycin↗