PubMed Health⌕ Search

Biomedical subjects

B M Sutherland

Publications and source records attributed to B M Sutherland.

At least 19 recordsLinked to original sources
PubMed2000

Clustered damages and total lesions induced in DNA by ionizing radiation: oxidized bases and strand breaks.

Ionizing radiation induces both isolated DNA lesions and clustered damages-multiple closely spaced lesions (strand breaks, oxidized purines, oxidized pyrimidines, or abasic sites within a few helical turns). Such clusters are postulated to be difficult to repair and thus potentially lethal or mutagenic lesions. Using highly purified enzymes that cleave DNA at specific classes of damage and electrophoretic assays developed for quantifying isolated and clustered damages in high molecular length genomic DNAs, we determined the relative frequencies of total lesions and of clustered damages involving both strands, and the composition and origin of such clusters. The relative frequency of isolated vs clustered damages depends on the identity of the lesion, with approximately 15-18% of oxidized purines, pyrimidines, or abasic sites in clusters recognized by Fpg, Nth, or Nfo proteins, respectively, but only about half that level of frank single strand breaks in double strand breaks. Oxidized base clusters and abasic site clusters constitute about 80% of complex damages, while double strand breaks comprise only approximately 20% of the total. The data also show that each cluster results from a single radiation (track) event, and thus clusters will be formed at low as well as high radiation doses.

DNA↗
PubMed2000

Clustered DNA damages induced in isolated DNA and in human cells by low doses of ionizing radiation.

Clustered DNA damages-two or more closely spaced damages (strand breaks, abasic sites, or oxidized bases) on opposing strands-are suspects as critical lesions producing lethal and mutagenic effects of ionizing radiation. However, as a result of the lack of methods for measuring damage clusters induced by ionizing radiation in genomic DNA, neither the frequencies of their production by physiological doses of radiation, nor their repairability, nor their biological effects are known. On the basis of methods that we developed for quantitating damages in large DNAs, we have devised and validated a way of measuring ionizing radiation-induced clustered lesions in genomic DNA, including DNA from human cells. DNA is treated with an endonuclease that induces a single-strand cleavage at an oxidized base or abasic site. If there are two closely spaced damages on opposing strands, such cleavage will reduce the size of the DNA on a nondenaturing gel. We show that ionizing radiation does induce clustered DNA damages containing abasic sites, oxidized purines, or oxidized pyrimidines. Further, the frequency of each of these cluster classes is comparable to that of frank double-strand breaks; among all complex damages induced by ionizing radiation, double-strand breaks are only about 20%, with other clustered damage constituting some 80%. We also show that even low doses (0.1-1 Gy) of high linear energy transfer ionizing radiation induce clustered damages in human cells.

Bacteriophage T7↗
PubMed2000

UV radiation-sensitive norin 1 rice contains defective cyclobutane pyrimidine dimer photolyase.

Norin 1, a progenitor of many economically important Japanese rice strains, is highly sensitive to the damaging effects of UVB radiation (wavelengths 290 to 320 nm). Norin 1 seedlings are deficient in photorepair of cyclobutane pyrimidine dimers. However, the molecular origin of this deficiency was not known and, because rice photolyase genes have not been cloned and sequenced, could not be determined by examining photolyase structural genes or upstream regulatory elements for mutations. We therefore used a photoflash approach, which showed that the deficiency in photorepair in vivo resulted from a functionally altered photolyase. These results were confirmed by studies with extracts, which showed that the Norin 1 photolyase-dimer complex was highly thermolabile relative to the wild-type Sasanishiki photolyase. This deficiency results from a structure/function alteration of photolyase rather than of nonspecific repair, photolytic, or regulatory elements. Thus, the molecular origin of this plant DNA repair deficiency, resulting from a spontaneously occurring mutation to UV radiation sensitivity, is defective photolyase.

DNA Repair↗
PubMed1998

A robust, inexpensive filter for blocking UVC radiation in broad-spectrum 'UVB' lamps.

Accurate studies of the biological effects of UBV radiation require suitable laboratory sources. Lamps labeled as UVB sources often emit UVC radiation that contributes significantly to the levels of DNA damage. The UVC from an unfiltered UVB source produced more pyrimidine dimers in soybean DNA than a lamp filtered by a Pyrex dish that removes wavelengths of < 280 nm. Calculations based on action spectra and on the emission spectra of unfiltered lamps indicate that UVC contributes approximately 13%, 4% and approximately 1% of the total dimers induced in unshielded cells or DNA, alfalfa cotyledons, and human skin, respectively. Further, relative to a Pyrex dish-filtered lamp, an unfiltered lamp would produce approximately 7-, 2.4- or 2.8-fold more dimers in these three biological systems. We report here that a Pyrex dish provides an effective, stable, robust and inexpensive filter for reducing or excluding the contribution of UVC to damage induced by broad-spectrum 'UVB' lamps.

DNA↗
PubMed1998

UV-DNA damage in mouse and human cells induces the expression of tumor necrosis factor alpha.

Ultraviolet light induces the expression of tumor necrosis factor alpha (TNF alpha) in many mammalian cells. We have examined the signal for this induction in a human DNA repair-deficient cell line carrying a transgene composed of the murine TNF regulatory sequences fused to the chloramphenicol acetyltransferase (CAT) structural gene. When compared by fluence, UVC was a more efficient inducer of CAT than was UVB, but they were equivalent inducers when compared by the frequency of cyclobutyl pyrimidine dimers produced by each source. Further, treatment of UV-irradiated cells with the prokaryotic DNA repair enzyme T4 endonuclease V increased the level of repair of dimers and concomitantly reduced CAT gene expression. Membrane-bound TNF alpha expression was increased by UV and reduced by repair of dimers. Finally, in the TNFcat transgene system, DNA damage directly to the cell with the transgene was required as cocultivation of unirradiated TNFcat cells with UV-irradiated cells did not increase CAT activity. These results show that DNA damage is a signal for the induction of TNF alpha gene expression in mouse and human cells.

Animals↗
PubMed1997

Computer network for data acquisition, storage and analysis.

Modern scientific instruments can produce huge quantities of data, usually in digital form. However, data acquisition is only one of three important functions. To be useful, data must also be stored and analyzed. Fortunately, the same computer-based technologies that facilitate the generation of large data-sets provide tools to accomplish these tasks. We describe a data system based on computers connected to a network, developed for this purpose.

Computer Communication Networks↗
PubMed1997

UV effects in "the real world": problems of UV dosimetry in complex organisms.

Understanding the effects of UV radiation on higher plants and animals is essential for evaluating the consequences of environmental changes which could alter the UV levels in the biosphere. Although analytical approaches for quantifying UV exposures and resulting cellular damage levels in optically simple systems (DNA in dilute solution, viruses or microorganisms in dilute suspension and monolayers of cultured cells) are well known, these methods may not be applicable to optically complex systems, such as multilayered plants and animals, or microorganisms in absorbing milieu such as natural lakes or oceans. Understanding the problems, pitfalls and solutions to the quantification of UV exposures and cellular damage is critical for evaluating the effects of environmental UV on complex organisms.

Animals↗
PubMed1996

Double strand breaks induced by low doses of gamma rays or heavy ions: quantitation in nonradioactive human DNA.

We have developed a method of quantitating low frequencies (0-30 sites/10(9) base pairs) of double strand breaks in approximately 1 microgram of nonradioactive human DNA. Unirradiated or irradiated DNA is digested with the restriction endonuclease NotI, producing cleavage fragments that include a major group centered at approximately 1.2-1.3 Mbp. The DNA molecules are separated as a function of size by transverse alternating field electrophoresis. The frequency of double strand breaks is computed directly from the decrease in number average molecular length induced in the 1.2- to 1. 3-Mbp cleavage fragment group by 137Cs gamma or Fe26+ (1.1 GeV/nucleon) irradiation vs the corresponding unirradiated DNA samples. The double strand break frequency can be quantitated easily in the dose range of 0-10 cGy of gamma rays. The frequency of breaks per unit dose calculated for gamma irradiation of DNA in human cells (approximately 4.6 double strand breaks/10(9) bp/Gy) is within the range of values obtained by others (2-8 sites/10(9) bp/Gy) who used methods requiring higher doses.

Cell Line, Transformed↗
PubMed1996

Isolation of high-molecular-length DNA from human skin.

An agarose plug method for isolating high-molecular-length DNA from mammalian tissues has been developed, including from those that are difficult, such as skin. It gives high yields of DNA that contain a minimum of single-strand breaks and is readily digested by restriction and other nucleases. The method requires only simple equipment and is readily adaptable to field or clinical studies.

Adult↗
PubMed1995

Human white blood cells contain cyclobutyl pyrimidine dimer photolyase.

Although enzymatic photoreactivation of cyclobutyl pyrimidine dimers in DNA is present in almost all organisms, its presence in placental mammals is controversial. We tested human white blood cells for photolyase by using three defined DNAs (supercoiled pET-2, nonsupercoiled bacteriophage lambda, and a defined-sequence 287-bp oligonucleotide), two dimer-specific endonucleases (T4 endonuclease V and UV endonuclease from Micrococcus luteus), and three assay methods. We show that human white blood cells contain photolyase that can photorepair pyrimidine dimers in defined supercoiled and linear DNAs and in a 287-bp oligonucleotide and that human photolyase is active on genomic DNA in intact human cells.

Bacteriophage lambda↗
PubMed1995

Action spectroscopy in complex organisms: potentials and pitfalls in predicting the impact of increased environmental UVB.

Evaluating the effects of broad spectrum sources, including sunlight, on humans and their environment requires knowledge both of the effects of various wavelengths of radiation in those sources on biological systems and of the kinds and magnitude of changes in those sources. Action spectroscopy-the determination of the effect of radiation on an organism as a function of wavelength-provides a major method for evaluating the effects of a source, including that of changes in its intensity and wavelength distribution. Although action spectra for UV radiation damage to higher organisms can give useful information, inappropriate analyses can lead to misleading or even incorrect results. It is thus essential to consider carefully methods of analyzing action spectra and their consequences for evaluating the effects of changes in the solar spectrum.

DNA↗
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↗