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Biomedical subjects

B M Sutherland

Publications and source records attributed to B M Sutherland.

At least 73 records · Page 4Linked to original sources
PubMed1981

Photoreactivation in bacteria and in skin.

In many procaryotic and eucaryotic cells, photoreactivating enzyme mediates light-dependent repair of UV-induced damage: the enzyme binds to a pyrimidine dimer in DNA, and, on absorption of a photon (300-600 nm), specifically monomerizes the dimer, thus repairing the DNA. Photoreactivating enzyme has been found in human tissues and human cells in culture; human cells in culture can photoreactivate cellular dimers, and can mediate photoreactivation of Herpes (human fibroblasts) and Epstein-Barr virus (human leukocytes). Measurements of pyrimidine dimer formation and repair in human skin indicate that detectable numbers of dimers are formed at 1 minimal erythemal dose, that the dimers are rapidly removed in skin kept in the absence of light, and they are more rapidly removed when the skin is exposed to visible light.

Bacteria↗
PubMed1981

Ability of Bacillus subtilis protoplasts to repair irradiated bacteriophage deoxyribonucleic acid via acquired and natural enzymatic systems.

A novel form of "enzyme therapy" was achieved by utilizing protoplasts of Bacillus subtilis. Photoreactivating enzyme of Escherichia coli was successfully inserted into the protoplasts of B. subtilis treated with polyethylene glycol. This enzyme was used to photoreactivate ultraviolet-damaged bacteriophage deoxyribonucleic acid (DNA). Furthermore, in polyethylene glycol-treated protoplasts, ultraviolet-irradiated transfecting bacteriophage DNA was shown to be a functional substrate for the host DNA excision repair system. Previous results (R. E. Yasbin, J. D. Fernwalt, and P. I. Fields, J. Bacteriol. 137:391-396, 1979) showed that ultraviolet-irradiated bacteriophage DNA could not be repaired via the excision repair system of competent cells. Therefore, the processing of bacteriophage DNA by protoplasts and by competent cells must be different. This sensitive protoplast assay can be used to identify and to isolate various types of DNA repair enzymes.

Bacillus subtilis↗
PubMed1981

Action spectra for ultraviolet light-induced transformation of human cells to anchorage-independent growth.

We have determined action spectra for transformation of human embryonic skin and muscle fibroblasts to anchorage-independent growth. Tests under our experimental conditions indicate that reciprocity holds for photon rate and exposure time and that all the dose-effect curves in the wavelength range of 248 to 297 nm are smaller. These data can be used to construct action spectra with a maximum at about 265 nm, which do not implicate moieties other than nucleic acids as absorbers in the transformation process.

Cell Transformation, Neoplastic↗
PubMed1980

Escherichia coli photoreactivating enzyme: purification and properties.

We have purified large quantities of Escherichia coli photoreactivating enzyme (EC 4.1.99.3) to apparent homogeneity and have studied its physical and chemical properties. The enzyme has a molecular weight of 36 800 and a S020,W of 3.72 S. Amino acid analysis revealed an apparent absence of tryptophan, a low content of aromatic residues, and the presence of no unusual amino acids. The N terminus is arginine. The purified enzyme contained up to 13% carbohydrate by weight. The carbohydrate was composed of mannose, galactose, glucose, and N-acetylglucosamine. The enzyme is also associated with RNA (approximately 10 nucleotides/enzyme molecule) containing uracil, adenine, guanine, and cytosine with no unusual bases detected.

Amino Acids↗
PubMed1980

Insertion of E. coli photoreactivating enzyme into V79 hamster cells.

Photoreactivating enzyme mediates the specific repair of UV light [220-300 nm, (UV)]-induced cyclobutyl pyrimidine dimers in DNA. It binds to dimer-containing DNA, and on absorption of light in the wavelength range 300-500 nm monomerizes the dimer, restoring biological activity to DNA. The specificity of the enzyme for pyrimidine dimers in DNA allows its use as an analytical tool. If UV-induced biological damage is photoreactivable, dimers are probably a major cause of that damage. Thus dimers have been implicated in producing death and mutation in prokaryotes and in simple eukaryotes. Although this photoreactivation test has great potential value in assessing the role of dimers in UV-induced damage in mammalian cells, its use in cultured mammalian cells has been limited by the dependence of photoreactivating enzyme levels on the cell species, genotype and culture medium. We have developed a method for insertion of Escherichia coli photoreactivating enzyme into mammalian cells, and show here that the inserted bacterial enzyme can mediate photoreactivation of pyrimidine dimers in V79 rodent cells.

Animals↗
PubMed1980

Pyrimidine dimer formation and repair in human skin.

Cyclobutyl pyrimidine dimers have been detected in the DNA of human skin following in vivo irradiation with suberythemal doses of ultraviolet (UV) radiation from FS-20 sun lamp fluorescent tubes. Dimers were assayed by treatment of extracted DNA with Micrococcus luteus UV-specific endonuclease, alkaline agarose electrophoresis, and ethidium bromide staining. This technique, in contrast to conventional dimer assays, can be used with nonradioactive DNA and is optimal at low UV light doses. M. luteus endonuclease-sensitive sites were determined after exposure of untanned skin in two volunteers to UV light (0.97, 1.94, or 3.88 X 10(3) J/sq m; lambda, 290 to 360 nm). At 20 min postirradiation (dose, 1.94 X 10(3) J/sq m), fewer M. luteus endonuclease-sensitive sites were found in the DNA than immediately after the irradiation. Even fewer endonuclease-sensitive sites were found at 20 min when the UV-irradiated skin was subsequently irradiated with visible light than when the area was kept in the dark. These data suggest that some dimer disappearance by excision repair occurs within 20 min of UV irradiation and that photoreactivation of dimers can make a contribution to the total repair process.

DNA↗
PubMed1980

Ultraviolet light-induced transformation of human cells to anchorage-independent growth.

We have developed a system for ultraviolet light (UV) transformation of human embryonic cells to anchorage-independent growth. The procedure involves multiple UV irradiations, post irradiation growth, and plating in soft agar. Transformants are obtained at frequencies from 1 to 80 per 10(5) cells at UV exposures to 25 J/sq m. The resulting transformants can be subcultured on solid surfaces. The cells show crisscrossing and piling up; they reach 2- to 5-fold higher saturation densities than the parental cells. Some subcultures show increased plating efficiency in soft agar and increased life span. The susceptibility of the UV transformation process to apparent photoenzymatic reversal implies that purimidine dimers play a role in its induction.

Cell Adhesion↗
PubMed1979

Multiple loci affecting photoreactivation in Escherichia coli.

Sutherland et al. mapped a phr gene in Escherichia coli at 17 min and found that induction of an E. coli strain lysogenic for a lambda phage carrying this gene increased photoreactivating enzyme levels 2,000-fold. Recently, Smith and Youngs and Sancar and Rupert located a phr gene at 15.9 min. We have therefore investigated the properties of photoreactivating enzyme and cellular photoreactivation in cells containing deletions of the gene at 17 min. Cells with this deletion photoreactivated ultraviolet-induced killing at a rate 20% of normal; they also contained approximately 20% of the normal photoreactivating enzyme level. The residual enzyme in these cells was characterized to determine whether the reduced cellular photoreactivation rate and photoreactivating enzyme levels resulted from reduced numbers of normal enzymes or from an altered enzyme. Photoreactivating enzymes from strains carrying a deletion of the region at 17 min had an apparent Km about two- to threefold higher than normal enzyme and showed markedly increased heat lability. The gene at 17 min thus contains information determining the function of the E. coli photoreactivating enzyme rather than the quantity of the enzyme. It is proposed that the gene at 17 min be termed phrA and that located at 15.9 min be termed phrB.

DNA Repair↗
PubMed1978

Photoreactivation: evaluation of pyrimidine dimers in ultraviolet radiation-induced cell transformation.

The PRE repairs DNA by the specific and exclusive monomerization of pyrimidine dimers. Photoreactivation thus provides an analytical test for determining the role of pyrimidine dimers in UV damage to biologic systems. We have shown the first UV transformation of cultured human cells (judged by ability to grow in soft agar). Preliminary results indicate that 1) lesions leading to cell transformation are subject to photoreactivation and 2) pyrimidine dimers may well be important in solar carcinogenesis.

Animals↗
PubMed1976

Culture conditions affect photoreactivating enzyme levels in human fibroblasts.

Photoreactivation of pyrimidine dimers in mammalian cells occurs under our experimental conditions but has not been observed under conditions used by others. We have tested three possible differences in experimental procedures including dimer separation and analysis methods, illumination conditions and cell culture techniques. We show that out methods of dimer separation and analysis indeed measure cis-syn pyrimidine dimers and give results in quantitative agreement with the methods of others. We find that while light pre-illumination of fibroblasts from the xeroderma pigmentosum line XP12BE or of normal cells does not affect the cellular capacity for dimer photoreactivation. However, we show that cell culture conditions can affect photoreactivating enzyme levels and thus, cellular dimer photoreactivation capacity. Cells grown in Eagle's minimal essential medium (supplemented with 15% fetal bovine serum) contain very low levels of photoreactivating enzyme and cannot photoreactivate dimers in their DNA; however, companion cultures maintained in Dulbecco's modified Eagle's minimal medium do contain photoreactivating enzyme and can photoreactivate cellular dimers.

Cell Line↗
PubMed1976

Photoreactivation of pyrimidine dimers in the DNA of normal and xeroderma pigmentosum cells.

Photoproducts formed in the DNA of human cells irradiated with ultraviolet light (uv) were identified as cyclobuytl pyrimidine dimers by their chromatographic mobility, reversibility to monomers upon short wavelength uv irradiation, and comparison of the kinetics of this monomerization with that of authentic cis-syn thymine-thymine dimers prepared by irradiation of thymine in ice. The level of cellular photoreactivation of these dimers reflects the level of photoreactivating enzyme measured in cell extracts. Action spectra for cellular dimer photoreactivation in the xeroderma pigmentosum line XP12BE agree in range (300 nm to at least 577 nm) and maximum (near 400 nm) with that for photoreactivation by purified human photoreactivating enzyme. Normal human cells can also photoreactivate dimers in their DNA. The action spectrum for the cellular monomerization of dimers is similar to that for photoreactivation by the photoreactivating enzyme in extracts of normal human fibroblasts.

Binding Sites↗