scholarly journals CHARACTERIZATION OF POSTREPLICATION REPAIR IN MUTAGEN-SENSITIVE STRAINS OF DROSOPHILA MELANOGASTER

Genetics ◽  
1976 ◽  
Vol 84 (3) ◽  
pp. 507-526
Author(s):  
J B Boyd ◽  
R B Setlow
Keyword(s):  
Drosophila Melanogaster ◽  
Uv Radiation ◽  
Specific Activity ◽  
Gradient Centrifugation ◽  
High Specific Activity ◽  
X Rays ◽  
Postreplication Repair ◽  
Strand Breaks ◽  
Single Strand Breaks ◽  
Complementation Groups

ABSTRACT Mutants of Drosophila melanogaster, with suspected repair deficiencies, were analyzed for their capacity to repair damage induced by X-rays and UV radiation. Analysis was performed on cell cultures derived from embryos of homozygous mutant stocks. Postreplication repair following UV radiation has been analyzed in mutant stocks derived from a total of ten complementation groups. Cultures were irradiated, pulse-labeled, and incubated in the dark prior to analysis by alkaline sucrose gradient centrifugation. Kinetics of the molecular weight increase in newly synthesized DNA were assayed after cells had been incubated in the presence or absence of caffeine. Two separate pathways of postreplication repair have been tentatively identified by mutants derived from four complementation groups. The proposed caffeine sensitive pathway (CAS) is defined by mutants which also disrupt meiosis. The second pathway (CIS) is caffeine insensitive and is not yet associated with meiotic functions. All mutants deficient in postreplication repair are also sensitive to nitrogen mustard. The mutants investigated display a normal capacity to repair single-strand breaks induced in DNA by X-rays, although two may possess a reduced capacity to repair damage caused by localized incorporation of high specific activity thymidine-3H. The data have been employed to construct a model for repair of UV-induced damage in Drosophila DNA. Implications of the model for DNA repair in mammals are discussed.

SINGLE-STRAND BREAKS IN SUPERCOILED DNA INDUCED BY VACUUM-UV RADIATION IN AQUEOUS SOLUTION

1986 ◽  
Vol 44 (3) ◽  
pp. 397-400 ◽  
Author(s):  
Kaoru Takakura ◽  
Mitsuo Ishikawa ◽  
Kotaro Hidea ◽  
Katsumi Kobayashi ◽  
Atsushi Ito ◽  
...  
Keyword(s):  
Aqueous Solution ◽  
Uv Radiation ◽  
Single Strand ◽  
Supercoiled Dna ◽  
Strand Breaks ◽  
Single Strand Breaks ◽  
Vacuum Uv Radiation ◽  
Vacuum Uv

scholarly journals Purification of the enzyme NADPH: protochlorophyllide oxidoreductase

1981 ◽  
Vol 195 (1) ◽  
pp. 83-92 ◽  
Author(s):  
N S Beer ◽  
W T Griffiths
Keyword(s):  
Specific Activity ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
High Specific Activity ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Triton X ◽  
Etioplast Membranes

A procedure for the purification of the enzyme NADPH:protochlorophyllide oxidoreductase is described. This involves fractionation of sonicated oat etioplast membranes by discontinuous-sucrose-density-gradient centrifugation, which gives membranes in which the enzyme is present at a high specific activity. The enzyme is solubilized from the membranes with Triton X-100, followed by gel filtration of the extract; enzyme activity is eluted in fractions corresponding to a mol.wt of approx. 35000. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the enzyme-containing fractions from gel filtration shows two peptides, of mol.wts. approx. 35000 and 37000.

The Formation of Single-Strand Breaks in Intracellular DNA by X-Rays

1971 ◽  
Vol 48 (3) ◽  
pp. 599 ◽  
Author(s):  
Ivar Johansen ◽  
Ingmund Gurvin ◽  
W. Dean Rupp
Keyword(s):  
Single Strand ◽  
X Rays ◽  
Strand Breaks ◽  
Single Strand Breaks

scholarly journals The random primer labeling technique applied to in situ hybridization on tissue sections.

1988 ◽  
Vol 36 (10) ◽  
pp. 1335-1340 ◽  
Author(s):  
M Thomas-Cavallin ◽  
O Aït-Ahmed
Keyword(s):  
Drosophila Melanogaster ◽  
In Situ Hybridization ◽  
Specific Activity ◽  
High Specific Activity ◽  
High Sensitivity ◽  
Random Primer ◽  
Tissue Sections ◽  
In Situ Hybridizations ◽  
Labeling Method

We report an application of the random primer labeling technique to in situ hybridizations on tissue sections. The ease of the method and the high specific activity achieved make it valuable when a large number of probes must be analyzed and high sensitivity is needed. We have applied this technique to study the spatial expression of a cluster of maternally acting genes (the yema gene region of Drosophila melanogaster which encodes eleven transcripts, some of them having a very low level of expression) (Aït-Ahmed et al., 1978: Dev Biol 122:153; Aït-Ahmed et al., unpublished results). The results reported here concern one of the transcripts of the yema region, which displays a peculiar anterior localization in the oocyte. We demonstrate that the "oligo-labeling" method allows a far better level of detection of the transcript of interest.

scholarly journals INDUCTION OF THIOGUANINE- AND OUABAIN-RESISTANT MUTANTS AND SINGLE-STRAND BREAKS IN THE DNA OF CHINESE HAMSTER OVARY CELLS BY 3H-THYMIDINE

Genetics ◽  
1977 ◽  
Vol 87 (1) ◽  
pp. 129-138
Author(s):  
James E Cleaver
Keyword(s):  
Ultraviolet Light ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Single Strand ◽  
X Rays ◽  
Ovary Cells ◽  
Strand Breaks ◽  
Single Strand Breaks ◽  
Ultraviolet Light Irradiation ◽  
Resistant Mutants

ABSTRACT Cultured Chinese hamster cells were labeled with 6-3H-thymidine or 5-methyl-3H-thymidine and allowed to accumulate damage from 3H decays for various periods of time while frozen. The frequencies of cells resistant to 6-thioguanine or ouabain and the amount of DNA damage (i.e., number of single-strand breaks) were determined and compared with the mutation frequencies resulting from X and ultraviolet light irradiation. Whereas 3H decays and X rays made only 6-thioguanine-resistant mutants, ultraviolet light made both 6-thioguanine- and ouabain-resistant mutants. 3H decays originating at the 6 position were two to three times as effective as decays at the 5-methyl position in making drug-resistant mutants, but decays at both sites were equally effective in making single-strand breaks. Mutants and strand breaks produced by beta irradiation of the nucleus probably are the same irrespective of the site of the decay in thymine; these results indicate that the local transmutation effects of 3H decay produce more mutations when they occur at the 6 position than at the 5-methyl position.

scholarly journals THE mei-9a MUTANT OF DROSOPHILA MELANOGASTER INCREASES MUTAGEN SENSITIVITY AND DECREASES EXCISION REPAIR

Genetics ◽  
1976 ◽  
Vol 84 (3) ◽  
pp. 527-544
Author(s):  
J B Boyd ◽  
M D Golino ◽  
R B Setlow
Keyword(s):  
Dna Repair ◽  
Drosophila Melanogaster ◽  
Meiotic Recombination ◽  
Excision Repair ◽  
Primary Cultures ◽  
Postreplication Repair ◽  
Single Strand Breaks ◽  
Pyrimidine Dimers ◽  
Mutagen Sensitivity ◽  
Cellular Dna

ABSTRACT The mei-9a mutant of Drosophila melanogaster, which reduces meiotic recombination in females (Baker and Carpenter 1972), is deficient in the excision of UV-induced pyrimidine dimers in both sexes. Assays were performed in primary cultures and established cell lines derived from embryos. An endonuclease preparation from M. luteus, which is specific for pyrimidine dimers, was employed to monitor UV-induced dimers in cellular DNA. The rate of disappearance of endonuclease-sensitive sites from DNA of control cells is 10-20 times faster than that from mei-9a cells. The mutant mei-218, which is also deficient in meiotic recombination, removes nuclease-sensitive sites at control rates. The mei-9a cells exhibit control levels of photorepair, postreplication repair and repair of single strand breaks. In mei-9 cells DNA synthesis and possibly postreplication repair are weakly sensitive to caffeine. Larvae which are hemizygous for either of the two mutants that define the mei-9 locus are hypersensitive to killing by the mutagens methyl methanesulfonate, nitrogen mustard and 2-acetylaminofluorene. Larvae hemizygous for the mei-218 mutant are insensitive to each of these reagents. These data demonstrate that the mei-9 locus is active in DNA repair of somatic cells. Thus functions involved in meiotic recombination are also active in DNA repair in this higher eukaryote. The results are consistent with the earlier suggestions (Baker and Carpenter 1972; Carpenter and Sandler 1974) that the mei-9 locus functions in the exchange events of meiosis. The mei-218 mutation behaves differently in genetic tests and our data suggest its function may be restricted to meiosis. These studies demonstrate that currently recognized modes of DNA repair can be efficiently detected in primary cell cultures derived from Drosophila embryos.

scholarly journals Radiosensitivities of DNA molecules in lymphocytes from the circulating blood of man

Blood ◽  
1975 ◽  
Vol 45 (4) ◽  
pp. 503-509
Author(s):  
Y Hashimoto ◽  
T Ono ◽  
S Okada
Keyword(s):  
Sucrose Gradient ◽  
Gradient Centrifugation ◽  
Single Strand ◽  
Alkaline Condition ◽  
Transformed Cells ◽  
Dna Molecules ◽  
Strand Breaks ◽  
Sucrose Gradient Centrifugation ◽  
Single Strand Breaks ◽  
Radiation Induced

The radiosensitivities of DNA molecules from lymphocytes of human circulating blood were examined by alkaline sucrose gradient centrifugation. The single-strand breaks of DNA per rad, including the breaks formed under the present alkaline condition, were 1.2 plus or minus 0.1 breaks per 10–12 daltons DNA per rad. When the cells were transformed, the number of breaks was found to increase to 1.8 plus or minus 0.2 breaks per 10–12 daltons DNA per rad. The lymphocytes are capable of rejoining radiation-induced single-strand breaks of DNA. The rate of rejoining was dependent upon types of the suspending medium. The rate increased to ten times of that of the non-transformed cells upon transformation.

scholarly journals The Incorporation of Leucine-C14 into Microsomal Particles and other Subcellular Components of the Pea Epicotyl

1959 ◽  
Vol 5 (1) ◽  
pp. 59-68 ◽  
Author(s):  
Paul O. P. Ts'o ◽  
Clifford S. Sato
Keyword(s):  
Specific Activity ◽  
Gradient Centrifugation ◽  
High Specific Activity ◽  
Insoluble Fraction ◽  
Supernatant Fraction ◽  
Group Method ◽  
Pea Seedlings ◽  
Pea Epicotyl ◽  
Hydrolysis Of ◽  
End Group

Incorporation of leucine-C14 into subcellular fractions of the apical section of pea seedlings has been studied as a function of the length of incubation. The specific activity of the microsomes was higher than that of the supernatant for short but not for long incubations, in agreement with observations on other systems. In this developing tissue the nuclei and especially the mitochondria appear to incorporate amino acid very rapidly. An insoluble fraction of the microsome pellet, which is presumably a liponucleoprotein complex, was found to possess, after 1 hour of incubation, a specific activity much greater than that of the purified microsomal particles or the supernatant fraction. Ninety-eight per cent of the leucine-C14 in the purified microsomal particles has been shown to possess bound amino groups, presumably in peptide linkages, by the DNP-end group method. These particles liberate but little peptide or protein of very high specific activity when they are destroyed by removal of Mg or by hydrolysis of RNA. Microsomal particles were fractionated into an RNA fraction and five protein fractions by means of density gradient centrifugation. By this method 95 per cent of the RNA can be separated from 90 per cent of the protein of the particle. Furthermore, the RNA fraction has been shown to contain very little protein of high specific activity. A particular protein fraction which contains the remaining 5 per cent of the RNA, possessed after 1 hour of incubation a specific activity 2 to 9 times higher than the protein of the other fractions.

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