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Biomedical subjects

B M Sutherland

Publications and source records attributed to B M Sutherland.

At least 37 records · Page 2Linked to original sources
PubMed1994

Isolation of high-molecular-weight plant DNA for DNA damage quantitation: relative effects of solar 297 nm UVB and 365 nm radiation.

Quantitation of UV-induced DNA damages in nanogram quantities of non-radioactive DNA from irradiated plants by gel electrophoresis requires a prompt, efficient, high-yield method of isolating DNA yielding high-molecular-weight, enzymatically digestible DNA. To meet these criteria we devised a high-yield method for isolating from plant tissue, DNA whose single-strand molecular length is greater than about 170 kb. Leaf tissue is embedded in agarose plugs, digested with Proteinase K in the presence of detergent, and treated with phenylmethylsulfonyl fluoride (PMSF). The agarose plugs are then soaked with buffer appropriate to the desired enzyme treatment. Evaluation of the DNA on neutral and alkaline gels indicates its high molecular length and low frequency of single-strand breaks. The DNA can be digested with damage-specific and other endonucleases. The method is especially suitable for DNA damage quantitation, as tissue processing is carried out immediately after harvesting (allowing DNA lesion measurement at precisely known times after irradiation), and many samples can be easily handled at once. It should also be useful for molecular analysis of large numbers of plant samples available only in small quantities. We here use this method to quantitate DNA damage induced by 297 and 365 nm radiation, and calculate the relative damaging effects of these wavebands in today's solar spectrum.

DNA↗
PubMed1993

Promoter-specific synthetic photoendonuclease: rose bengal-labeled T7 RNA polymerase.

We have constructed a synthetic photoendonuclease composed of T7 RNA polymerase linked to rose bengal. The promoter-specific polymerase confers site-specific binding, and the photosensitizer rose bengal allows light-induced DNA cleavage. Using a gentle labeling procedure, we find that the polymerase can be labeled with 1-30 rose bengals. Polymerase labeled to about 8 rose bengals per molecule retains the same efficiency and specificity of binding to promoter-containing DNA as unlabeled polymerase. At this level of rose bengal substitution, the synthetic endonuclease, in the presence of visible light, specifically cleaves linear or supercoiled DNA containing a T7 promoter. It induces frank single-strand breaks, rather than labile sites convertible to breaks upon additional treatments. Neither the free rose bengal moiety not bonded to polymerase nor the free (not bound to DNA) rose bengal-substituted polymerase cleaves DNA. Although rose bengal is an efficient generator of singlet oxygen, depletion of oxygen from reaction mixtures increases the cleavage rate. This indicates that singlet oxygen cleavage is not a major mechanism of DNA nicking by the synthetic endonuclease. At higher levels of rose bengal substitution, the labeled polymerase shows decreased binding efficiency and increased nonspecific binding to DNA without a T7 promoter; the specificity of DNA cleavage also decreases. These results indicate that the site specificity of rose bengal photocleavage by the synthetic endonuclease results from specific binding of the polymerase, and thus rose bengal photonicking reflects polymerase binding.

Binding Sites↗
PubMed1993

Quantitative detection of single-copy genes in nanogram samples of human genomic DNA.

We have developed methods for Southern hybridization analysis of single-copy genes in as little as 10 ng of mammalian DNA electrophoresed under either neutral or alkaline conditions. The signal on the radiogram is proportional to the quantity of DNA. These methods permit Southern hybridization analysis of DNA from about 2 x 10(3) cultured human cells. The use of minimal cell numbers greatly reduces the costs of culturing cells for hybridization analysis and makes feasible analysis of DNA from tissues or cell types available only in limited quantities.

Blotting, Southern↗
PubMed1992

Quantitation of supercoiled DNA cleavage in nonradioactive DNA: application to ionizing radiation and synthetic endonuclease cleavage.

Quantitation of the conversion of nonradioactive supercoiled DNA to its open circular or linear forms on ethidium-stained electrophoretic gels has been difficult because of differential binding of ethidium to supercoiled DNA vs other forms under different conditions and the nonlinear response of photographic film. We have developed methods for adding a linear DNA as an internal fluorescence standard to "normalize" the quantity of DNA loaded into each lane of a gel. Inclusion of a linear normalizing DNA in samples before partitioning for individual supercoil cleavage reactions allows the quantitation of the resultant species, is technically easy, and does not require quantitative application of the sample to the gel. If the presence of a normalizing DNA during supercoil cleavage is undesirable, the addition of a normalizing plasmid to each sample after supercoil cleavage (but before electrophoresis) or the quantitative application of samples containing test DNA alone to the gel gives similar data, but with increased variability. We use the normalizing DNA method in cleavage by a physical agent (ionizing radiation) and in a more complex situation, by a protein-based, light-dependent synthetic endonuclease. We show how the fraction of intact supercoiled DNA can be calculated from measurement of the cleaved and normalizing species only. The method also can be used in reactions involving the depletion of one DNA species, whether supercoiled or not, such as protein-DNA interactions as detected by gel retardation assays.

DNA, Superhelical↗
PubMed1992

Quantitation of pyrimidine dimers in DNA from UVB-irradiated alfalfa (Medicago sativa L.) seedlings.

Depletion of stratospheric ozone will increase the solar ultraviolet radiation in the range from 290-320 nm (UVB) that reaches the surface of the earth, placing an increased UV burden on exposed organisms. One consequence of increased UVB may be decreased productivity of crop plants. A principal lesion caused by UV in DNA is the cyclobutyl pyrimidine dimer. We have adapted a method for measuring these dimers in nanogram quantities of non-radioactive DNA for use in UV-irradiated plants. We find that biologically relevant doses of broad band UVB radiation induce easily detectable frequencies of pyrimidine dimers in the DNA of irradiated alfalfa sprout leaves and that the dose response for dimer formation is linear up to doses of at least 690 J m-2. We also find easily measurable frequencies of dimers in the leaves of seedlings grown in glass filtered sunlight but not exposed to additional UVB, suggesting that significant numbers of dimers are formed in plants exposed to normal sunlight.

DNA↗
PubMed1991

DNA damage induced by 193-nm radiation in mammalian cells.

The contribution of DNA damage to the effects of 193-nm excimer laser radiation on mammalian cells in culture was studied in order to evaluate the mutagenic potential of this UV wavelength in vivo. Two approaches were taken: measurement of pyrimidine dimer-specific endonuclease-sensitive sites/megabase and comparison of the 193-nm radiation-induced cytotoxicity in normal versus DNA repair-deficient cells. The formation of pyrimidine dimer-specific endonuclease-sensitive sites/megabase was inversely related to the thickness of the cytoplasm overlying the nuclei of normal human fibroblasts (NHF) and Chinese hamster ovary cells. The results of these measurements and a calculation of the absorption coefficient of cytoplasm indicate that each 1 micron of cytoplasm attenuates the incident radiation by greater than 90% and, therefore, the nuclear DNA in tissue will be highly protected from 193-nm radiation by overlying cytoplasm. The reduction in colony-forming ability induced by 254-nm, 193-nm, and X-ray radiation was measured in NHF, xeroderma pigmentosum (group A) cells, and ataxia telangiectasia cells. Xeroderma pigmentosum (group A) cells were 16.5 times more sensitive to 254-nm radiation but only 3.5 times more sensitive to 193-nm radiation than NHF cells, indicating that cyclobutylpyrimidine dimers were not the major lethal lesion formed at 193 nm. AT cells were 3.4 times more sensitive to X-rays than NHF cells, but these cell types were almost equally sensitive to 193-nm radiation, indicating that 193 nm did not induce the same type of lethal lesions as X-rays.

Animals↗
PubMed1991

Do pyrimidine dimer yields correlate with erythema induction in human skin irradiated in situ with ultraviolet light (275-365 nm)?

Ultraviolet radiation produces erythema in human skin, and damages the DNA of living cells in skin. Previous work showed that broad-band UV-B (290-320 nm) radiation produced higher levels of cyclobutyl pyrimidine dimers in DNA of individuals with high UV-B sensitivity (low minimal erythema dose) than in subjects of low UV-B sensitivity [Freeman et al. (1986) J. Invest. Dermatol., 86, 34-36]. We examined the relationship between erythema induction and dimer yields in DNA of human skin irradiated in situ with narrow band radiation spanning the wavelength range 275-365 nm. We find that, in general, higher dimer yields are produced per incident photon in volunteers with higher susceptibility to erythema induced by radiation of the same wavelength.

Adult↗
PubMed1990

Does exposure of human skin in situ to 385 or 405 nm UV induce pyrimidine dimers in DNA?

A previous report [Freeman et al. (1986) Photochem. Photobiol. 43S, 93S] indicated that irradiation of human skin in situ with 385 or 405 nm radiation produced detectable levels of pyrimidine dimers in DNA. Since these wavelengths are absorbed poorly by DNA, these results suggested that DNA damage was sensitized by other absorbing molecules present in skin. Examination of two experimental aspects of the previous work indicates that (1) the static gel electrophoresis method for DNA dispersion used in lesion determination gave accurate values of the levels of induced dimers, and (2) the DNA damage apparently induced by 385 nm was actually induced by shorter wavelength UV present in the 20 nm bandpass beam of the monochromator. The current results indicate that monochromatic 385 and 405 nm radiation are ineffective in dimer production in human skin in situ.

Adult↗
PubMed1989

DNA photoreactivating enzyme from human tissues.

Photoreactivating enzyme activity has been quantitated in human fetal skin, kidney, lung, liver, brain and intestine, and in neonatal human foreskin. In all the tissues examined there were at least two activities: one nominally greater than 10,000 Da, and one nominally less than 10,000 Da. Both can photolyze pyrimidine dimers in DNA using only light of wavelengths greater than 320 nm, thus excluding tryptophan-mediated dimer splitting as an important mechanism for these activities. The activities are inactivated by digestion with trypsin or pronase, and decreased partially or totally by heating to 65 degrees C. The activities from all six tissues, as well as that from neonatal foreskin, act catalytically in dimer photolysis. The properties of macromolecular size, heat lability, protease sensitivity and catalytic pyrimidine dimer photolysis by a non-tryptophan-mediated mechanism correspond to those of a true photoreactivating enzyme.

Deoxyribodipyrimidine Photo-Lyase↗
PubMed1989

Wavelength dependence of pyrimidine dimer formation in DNA of human skin irradiated in situ with ultraviolet light.

The UV components of sunlight are believed to be a major cause of human skin cancer, and DNA is thought to be the principal molecular target. Alterations of the intensity and wavelength distribution of solar UV radiation reaching the surface of the earth, for example by depletion of stratospheric ozone, will change the effectiveness of solar radiation in damaging DNA in human skin. Evaluation of the magnitude of such effects requires knowledge of the altered sunlight spectrum and of the action spectrum for damaging DNA in human skin. We have determined an action spectrum for the frequency of pyrimidine dimer formation induced in the DNA of human skin per unit dose of UV incident on the skin surface. The peak of this action spectrum is near 300 nm and decreases rapidly at both longer and shorter wavelengths. The decrease in our action spectrum for wavelengths less than 300 nm is attributed to the absorption of the upper layers of the skin, an in situ effect that is inherently included in our measurements. Convolution of the dimer action spectrum with the solar spectra corresponding to a solar angle of 40 degrees under current levels of stratospheric ozone (0.32-cm O3 layer) and those for 50% ozone depletion (0.16-cm O3 layer), indicate about a 2.5-fold increase in dimer formation. If the action spectrum for DNA damage that results in skin cancer resembles that for dimer induction in skin, our results, combined with epidemiological data, suggest that a 50% decrease in stratospheric ozone would increase the incidence of nonmelanoma skin cancers among white males in Seattle, Washington, by 7.5- to 8-fold, to a higher incidence than is presently seen in the corresponding population of Albuquerque, New Mexico.

Adult↗
PubMed1988

Human cell transformation in the study of sunlight-induced cancers in the skin of man.

Human cell transformation provides a powerful approach to understanding--at the cellular and molecular levels--induction of cancers in the skin of man. A principal approach to this problem is the direct transformation of human skin cells by exposure to ultraviolet and/or near-UV radiation. The frequency of human cells transformed to anchorage independence increases with radiation exposure; the relative transforming efficiencies of different wavelengths implies that direct absorption by nucleic acids is a primary initial event. Partial reversal of potential transforming lesions by photoreactivation suggests that pyrimidine dimers, as well as other lesions, are important in UV transformation of human cells. Human cells can also be transformed by transfection with cloned oncogenes, or with DNAs from tumors or tumor cell lines. Cells treated by the transfection procedure (but without DNA) or cells transfected with DNAs from normal mammalian cells or tissues show only background levels of transformation. Human cells can be transformed to anchorage-independent growth by DNAs ineffective in transformation of NIH 3T3 cells (including most human skin cancers), permitting the analysis of oncogenic molecular changes even in tumor DNAs difficult or impossible to analyze in rodent cell systems.

Cell Division↗
PubMed1987

Electronic imaging system for direct and rapid quantitation of fluorescence from electrophoretic gels: application to ethidium bromide-stained DNA.

We have built an electronic imaging system based on a modified charge-coupled-device television camera that directly quantitates the distribution of fluorescence from electrophoretic gels, chromatograms, and other stationary sources. Exposure times can exceed 1 min. Unlike the photographic system that it replaces, the response of the camera is directly proportional to the intensity of incident fluorescence, and image data are digitized and stored in computer memory ready for analysis immediately upon completion of an exposure. We describe procedures for the display, normalization, and archival storage of image data and programs that use images of ethidium bromide-stained DNA in alkaline agarose gels to quantitate single-strand breaks in DNA.

DNA↗
PubMed1987

Production of pyrimidine dimers in DNA of human skin exposed in situ to UVA radiation.

Cyclobutyl pyrimidine dimers, measured as sites recognized by the dimer-specific ultraviolet (UV) endonuclease from Micrococcus luteus, were produced in DNA of human skin exposed in situ to UVA (320-400 nm) radiation. The dimer yields produced by a broadband UVA source, by broadband UVA filtered to remove all light of wavelength less than 340 nm, and by narrow band radiation centered at 365 nm were similar, indicating that UVA radiation, and not stray shorter wavelength radiation, was responsible for dimer production. The identity of the UVA-induced DNA lesions was confirmed as pyrimidine dimers by photoreactivation of approximately 100% of the endonuclease-sensitive sites in vitro with the 40,000 dalton Escherichia coli photoreactivating enzyme.

Adult↗
PubMed1987

Alteration of DNA tertiary structure by physical and chemical carcinogens: involvement in DNA repair processes.

Parameters defining the topological state of DNA seem extremely important for describing the reactive state of the same DNA molecules. We have shown that physical and chemical DNA modifying agents alter the tertiary structure of DNA molecules. Variations in the tertiary structure of DNA were studied by one dimensional electrophoresis on an agarose gel of partially relaxed plasmid DNA topoisomers, a technique allowing the measurement of alterations in the degree of supercoiling equivalent to fractions of superhelical turns. Unwinding angles of -10.1 degrees or -8.7 degrees per pyrimidine or thymine dimer respectively, of -12 degrees per apurinic site, and of -3.4 degrees per methylated purine were obtained by titrating the number of damaged sites necessary to reduce the number of superhelical turns by one in each topoisomer. On the contrary, enzymatic methylation of the C-5 position of cytosine (a modified base present in prokaryotic and eukaryotic DNAs) did not alter the DNA tertiary structure. We have also shown that local alterations in DNA structure caused by UV-irradiation inhibit bacterial DNA topoisomerase I and DNA methylase, and that the topological state of DNA substrate influences the mode of methylation of Hpa II DNA methylase. These findings suggest that the natural topological state of DNA substrate (linear, relaxed, or covalently closed duplex DNA with varying degrees of supercoiling) influences the mode of action of enzymes possibly involved in DNA repair processes, while DNA structural alterations caused by DNA modifying agents might influence DNA repair processes in two ways: either by driving the interaction between repair enzymes and the modified sites of DNA, or by inhibiting or changing the mode of action of enzymes normally acting on unmodified DNA.

Animals↗