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Biomedical subjects

B M Sutherland

Publications and source records attributed to B M Sutherland.

At least 55 records · Page 3Linked to original sources
PubMed1986

Substrate range of the 40,000-dalton DNA-photoreactivating enzyme from Escherichia coli.

We determined the ability of the 40 000-dalton Escherichia coli photoreactivating enzyme to act on a variety of pyrimidine-pyrimidine photoproduct substrates in nucleic acids. The enzyme is at least as active on cis-syn-cyclobutylpyrimidine dimers in supercoiled DNA as in linear DNA, but inactive on dimers in RNA. Both the phosphodiester bond internal to the deoxyriboses of the pyrimidines of the dimer and the N-glycosyl bond joining the pyrimidine to deoxyribose must be intact for enzyme action. The enzyme has no activity toward (6-4) pyrimidine-cytosine products in DNA.

Base Sequence↗
PubMed1986

Quantitation of radiation-, chemical-, or enzyme-induced single strand breaks in nonradioactive DNA by alkaline gel electrophoresis: application to pyrimidine dimers.

We have developed an alkaline agarose gel method for quantitating single strand breaks in nanogram quantities of nonradioactive DNA. After electrophoresis together with molecular length standards, the DNA is neutralized, stained with ethidium bromide, photographed, and the density profiles recorded with a computer controlled scanner. The median lengths, number average molecular lengths, and length average molecular lengths of the DNAs can be computed by using the mobilities of the molecular length standards. The frequency of single strand breaks can then be determined by comparison of the corresponding average molecular lengths of DNAs treated and not treated with single strand break-inducing agents (radiation, chemicals, or lesion-specific endonuclease). Single strand break yields (induced at pyrimidine dimer sites in uv-irradiated human fibroblasts DNA by the dimer-specific endonuclease from Micrococcus luteus) from our method agree with values obtained for the same DNAs from alkaline sucrose gradient analysis. The method has been used to determine pyrimidine dimer yields in DNA from biopsies of human skin irradiated in situ. It will be especially useful in determining the frequency of single strand breaks (or lesions convertible to single strand breaks by specific cleaving reagents or enzymes) in small quantities of DNA from cells or tissues not amenable to radioactive labeling.

DNA Damage↗
PubMed1986

Higher pyrimidine dimer yields in skin of normal humans with higher UVB sensitivity.

We have measured UVB (280-320 nm)-induced DNA damage in skin of individuals with different sensitivities to UVB irradiation as measured by minimal erythema dose (MED). The DNA damage was susceptible to cleavage by Micrococcus luteus UV endonuclease, which recognizes pyrimidine dimers in DNA. An alkaline agarose gel electrophoresis method was used to quantitate the number of M. luteus UV endonuclease-sensitive sites in nonradioactive DNA from skin biopsies of 7 individuals irradiated with UVB (0-180 mJ X cm-2). The production of sites correlated well with MED (correlation coefficient = 0.78). The slope of the dose response curve for the most UVB-sensitive individual (MED = 24 mJ X cm-2) and for the least UVB-sensitive individual (MED = 146 mJ X cm-2) were 11.5 X 10(-4) and 2.6 X 10(-4) sites per 1000 bases per mJ X cm-2, respectively. The UVB-induced DNA damage was determined to be pyrimidine dimers by its susceptibility to cleavage by M. luteus UV endonuclease and its photoreactivability by Escherichia coli photoreactivating enzyme.

Adult↗
PubMed1985

A densitometric nondestructive microassay for DNA quantitation.

A nondestructive assay for small quantities of nonradioactive DNA has been developed. Submicroliter volumes of DNA samples of concentrations as low as 10 micrograms/ml in 10 mM Tris, pH 8, containing 10 micrograms/ml ethidium bromide are photographed in 1-microliter microcapillary tubes on a near-uv transilluminator. The negatives are scanned with a densitometer, producing a sharp peak for each capillary; samples whose peak heights show apparent linearity with DNA content are compared to the linear portion of a standard curve constructed from DNA samples of known concentrations. The samples may be recovered undamaged, as introduction into capillaries and illumination for photography do not introduce nicks into the DNA. The DNA samples may be of heterogeneous molecular weight, but all DNAs should be of similar base composition and in the same buffer. Traces of phenol do not interfere with the determination.

Animals↗
PubMed1985

Transformation of human cells by DNAs ineffective in transformation of NIH 3T3 cells.

Neonatal human foreskin fibroblasts can be transformed to anchorage-independent growth by transfection with DNAs inefficient in transforming NIH 3T3 cells. Human cells transfected with DNA from GM 1312, a multiple myeloma cell line, or MOLT-4, a permanent lymphoblast line, grow without anchorage at a much higher frequency than do the parental cells and their DNAs can transform human cell recipients to anchorage-independent growth; they have extended but not indefinite life spans and are nontumorigenic. Human fibroblasts are also transformed by DNAs from two multiple myeloma lines that also transform 3T3 cells; however, restriction analysis suggests that different transforming genes in this DNA are acting in the human and murine systems. These results indicate that the human cell transfection system allows detection of transforming genes not effective in the 3T3 system and points out the possibility of detection of additional transforming sequences even in DNAs that do transform murine cells.

Animals↗
PubMed1985

Comparative protection efficiency of UVA- and UVB-induced tans against erythema and formation of endonuclease-sensitive sites in DNA by UVB in human skin.

UVA- and UVB-induced tans which were visually identical with each other were induced in separate sites on the lower back of 5 normal human volunteers of good tanning ability. Tanning was achieved by 4 exposures to UVA and UVB administered over an 8-day period. One week after the last exposure the protection afforded by the two types of tan against UVB-induced erythema and against UVB-induced DNA damage was measured. Protection against erythema was measured by comparison of the minimal erythema doses of UVB in tanned and untanned skin. Protection against DNA damage was assessed by comparing the numbers of endonuclease-sensitive sites in epidermal DNA extracted from biopsies taken from tanned and untanned sites exposed to the same dose of UVB. The UVB tans conferred significant protection (mean 2.98-fold) against UVB-induced erythema. UVA tans were not associated with significant protection (mean 1.4-fold). In contrast, both UVA- and UVB-induced tans were associated with a similar reduction in yield of endonuclease-sensitive sites in epidermal DNA (in UVA tan to 47% and in UVB tan to 45% of the yield in untanned skin). Protection conferred by the tans against erythema was therefore not paralleled by protection against DNA damage.

Adult↗
PubMed1985

Human cell transfection with skin cancer DNAs.

Spectacular progress towards understanding the molecular changes in DNA of many human tumors has been gained using NIH 3T3s as cellular recipients of transfecting DNA (1). Similar advances in analyzing human skin cancers have not been possible, as DNAs of most skin cancers or skin cancer cell lines--including most malignant melanomas--do not transform 3T3s (2). We have developed a DNA transfection system using human cells as recipients (3): normal human cells are transformed to anchorage-independent growth by transfection with a variety of skin cancer DNAs. This method may allow identification of the genes and specific sequences altered in sunlight-induced human skin cancers.

Animals↗
PubMed1984

Transformation of human cells by DNA transfection.

Normal human cells can be transformed to anchorage-independent growth by transfection with DNA from MOLT-4 lymphoblasts, derived from a patient with acute lymphocytic leukemia. Cells were treated with polyethylene glycol 6000, and DNA was administered as a calcium phosphate coprecipitate . The transforming activity of MOLT DNA was inactivated by treatment with DNase or by the restriction enzymes Bgll or BamHl but not by EcoRV . DNA from transfected colonies can transform human recipient cells to anchorage-independent growth; the transforming ability and anchorage independence are maintained stably for at least 30 generations.

Animals↗
PubMed1983

Quantitation of pyrimidine dimer contents of nonradioactive deoxyribonucleic acid by electrophoresis in alkaline agarose gels.

We have developed a method of quantitating the pyrimidine dimer content of nonradioactive DNAs. DNA samples are treated with the UV-endonuclease from Micrococcus luteus and then separated according to molecular weight by electrophoresis on alkaline agarose gels. From their migration relative to known molecular weight standards, their median molecular weights and thus the number of dimers per DNA molecule in each sample can be calculated. Results of action spectra for dimer formation in T7 bacteriophage measured by this method agree well with action spectra for T7 killing. In addition, the method gives dimer yields in good agreement with those obtained by others using alkaline sucrose gradient sedimentation.

DNA, Bacterial↗
PubMed1983

Action spectra for ultraviolet photosensitized killing of mammalian cells by misonidazole and para-nitroacetophenone.

V-79 Chinese hamster fibroblasts in monolayer culture were exposed to ultraviolet radiation at 313, 334, 365, 380, and 405 nm in the presence of either misonidazole or para-nitroacetophenone, drugs which act as both photosensitizers and radiosensitizers of cell killing. Survival was measured by a colony-forming assay. The resulting action spectra for cell death photosensitized by the drugs (the reciprocals of the exposures required at each wavelength to reduce cell survival to a given level) closely match their absorption spectra over a range of three orders of magnitude. These results demonstrate that cells can be killed upon excitation of misonidazole or para-nitroacetophenone in the absence of any other types of energy deposition or biomolecular damage.

Acetophenones↗