Repair Dna Synthesis in Differentiated Embryonic Muscle Cells

Stockdale, Frank E.; O'Neill, Michael C.

doi:10.1083/jcb.52.3.589

Cited by 40 publications

(7 citation statements)

References 25 publications

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“…To remove the unintegrated viral DNA, Hirt fractionation was employed (Hirt, 1967). Chromosomal DNA prepared as described above was subjected to CsCi centrifugation (5.73 g CSC1/4.4 ml) in an SW65 rotor at 45000 r.p.m, for 48 h (Stockdale & O'Neill, 1972). Positions corresponding to LL DNA (myotube DNA) and HL DNA (fibroblast DNA) were determined by measuring the radioactivity of each fraction.…”

Section: Preparation Of Myotube Chromosomal Dna (Budr Method)mentioning

confidence: 99%

“…1961 ; Pullman & Yeoh, 1978;Lim & Hauschka, 1984;Kaufman & Robert-Nicoud, 1985), and has also been confirmed in our laboratory (Kobayashi & Kaji, 1978). In this experiment, the myotube cultures were incubated with the nucleoside analogue BUdR for 48 h starting 2 days post-infection, and the extracted chromosomal DNA was subjected to CsCI density gradient centrifugation (Stockdale & O'Neill, 1972;Varmus et al, 1977). The fibroblast DNA which replicated in the culture and incorporated BUdR should be of higher density than the myotube DNA which had not replicated.…”

Section: Absence Of Integrated Viral Dna In Late-infected Myotubesmentioning

confidence: 99%

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Infection of terminally differentiated myotubes with Rous sarcoma virus (RSV): lack of DNA integration but presence of RSV mRNA

Tanaka¹,

Hara

Park

et al. 1992

Journal of General Virology

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We re-examined the generally accepted concept that replication of Rous sarcoma virus (RSV) requires host DNA synthesis. We used terminally differentiated chicken myotubes as the host because chromosomal DNA replication is completely abolished by the natural differentiation process. Southern blot analysis detected unintegrated viral DNA in both the nucleus and cytoplasm of infected myotubes. This indicated that reverse transcription of the infecting viral RNA and transport of the newly synthesized viral DNA into the nucleus proceeded normally in myotubes. However, restriction enzyme digestion of high Mr DNA prepared from infected myotubes produced none of the fragments specific for RSV, indicating that the viral DNA had failed to integrate into the myotube chromosomal DNA. In these infected myotubes, viral RNA was detected by in situ hybridization. Northern blot analysis showed the presence of all three RSV mRNAs (38S, 28S and 21S). The amount of these viral RNAs in infected myotubes was comparable with that found in infected fibroblasts. We conclude that host DNA synthesis is required for RSV integration, but, in contrast to the generally accepted concept, viral DNA integration is not an absolute requirement for transcription of the RSV genome.

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Section: Preparation Of Myotube Chromosomal Dna (Budr Method)mentioning

confidence: 99%

Section: Absence Of Integrated Viral Dna In Late-infected Myotubesmentioning

confidence: 99%

Infection of terminally differentiated myotubes with Rous sarcoma virus (RSV): lack of DNA integration but presence of RSV mRNA

Tanaka¹,

Hara

Park

et al. 1992

Journal of General Virology

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“…The lower concentration of proviral DNA in the supernatant than in the pellet could result from the presence in the supernatant of proviral DNA fragments, which are much smaller than the provirus and may not attach as well as larger fragments and/or may elute more easily from filters during hybridization. DISCUSSION The induction of various types of DNA repair by UV irradiation of cultured chicken embryonic cells has been well documented (13,15,31,35,36,37,46,56). The simplest and most efficient mechanism, enzymatic photoreactivation at light of wavelength 320 to 500 nm, requires a single enzyme that directly monomerizes pyrimidine dimers to individual bases in situ.…”

Section: R Was Obtained Visually Inmentioning

confidence: 99%

Integration of Proviral DNA in Chicken Cells Infected with Schmidt-Ruppin Rous Sarcoma Virus Is Not Enhanced by DNA Repair

Tsuruo¹,

Baluda²

1977

J Virol

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The effect DNA repair might have on the integration of exogenous proviral DNA into host cell DNA was investigated by comparing the efficiency ofproviral DNA integration in normal chicken embryonic fibroblasts and in chicken embryonic fibroblasts treated with UV or 4-nitroquinoline-1-oxide. The cells were treated with UV or 4-nitroquinoline-1-oxide at various time intervals ranging from 6 h before to 24 h after infection with Schmidt-Ruppin strain A of Rous sarcoma virus. The chicken embryonic fibroblasts were subsequently cultured for 18 to 21 days to ensure maximal integration and elimination ofnonintegrated exogenous proviral DNA before DNA was extracted. Integration of proviral DNA into the cellular genome was quantitated by hybridization of denatured cellular DNA on filters with an excess of 3H-labeled 35S viral RNA. The copy number ofthe integrated proviruses in normal cells and in infected cells was also determined from the kinetics of liquid RNA-DNA hybridization in DNA excess. Both RNA excess and DNA excess methods of hybridization indicate that two to three copies of the endogenous provirus appear to be present per haploid normal chicken cell genome and that two to three copies of the provirus of Schmidt-Ruppin strain A of Rous sarcoma virus become integrated per haploid cell genome after infection. The copy number of viral genome equivalents integrated per cell treated with UV or 4-nitroquinoline-1-oxide at different time intervals before or after infection did not differ from the copy number in untreated but infected cells. This finding supports our previous report that the integration of oncornavirus proviral DNA is restricted to specific sites in the host cell DNA and suggests a specific mechanism for integration.

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“…The former were two to four times more active. In a subsequent paper, Stockdale and O'Neill (11) showed that the unscheduled DNA synthesis was due to repair synthesis. The isolation by Yaffe (15) of LG cells, an established line of myoblasts which has retained the ability to fuse and form myotubes, has provided a promising system for studying the differentiation of muscle cells.…”

mentioning

confidence: 98%

Reduced DNA repair during differentiation of a myogenic cell line.

Chan¹,

Walker²

1976

The Journal of Cell Biology

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Repair synthesis induced by 4-nitroquinoline-l-oxide (4NQO) in L6 myoblasts before and after cellular fusion was measured by [3H]thymidine incorporation into unreplicated DNA. The level of repair synthesis was reduced after the cells had fused into myotubes. The terminal addition of radioactive nucleotides into DNA strands occurred only to a minor extent, and the dilution of [3H]thymidine by intracellular nucleotide pools was shown not to be responsible for the observed difference in repair synthesis. Both the initial rate and the overall incorporation of [3H]thymidine were found to be 50% lower in the myotubes.4NQO treatment of myoblasts and myotubes induced modifications in the DNA which were observed as single-strand breaks during alkaline sucrose sedimentation. After the myoblasts were allowed a post-treatment incubation, most of the single-strand breaks were no longer apparent. In contrast, a post-treatment incubation of myotubes did not change the extent of single-strand breakage seen. Both myoblasts and myotubes were equally effective in repairing single-strand breaks induced by X radiation. It would appear that when myoblasts fuse, a repair enzyme activity is lost, probably an endonuclease that recognizes one of the 4 NQO modifications of DNA. The result observed is a partial loss of repair synthetic ability and a complete loss of ability to remove the modification that appears as a single-strand break in alkali.During the differentiation of muscle cells, a number of enzyme activities rise and others fall (8). In the latter category is an enzyme or enzymes associated with the repair of DNA. Thus, Hahn et al. (4) reported that methyl methanesulfonate-stimulated unscheduled DNA synthesis was readily apparent in freshly cultured rat embryo myoblasts, but this activity declined considerably as the myoblasts fused. Stockdale (10) compared ultraviolet light-stimulated unscheduled DNA synthesis in cloned chick embryo myoblasts and the multinucleated myotubes derived from them. The former were two to four times more active. In a subsequent paper, Stockdale and O'Neill (11) showed that the unscheduled DNA synthesis was due to repair synthesis. The isolation by Yaffe (15) of LG cells, an established line of myoblasts which has retained the ability to fuse and form myotubes, has provided a promising system for studying the differentiation of muscle cells. These cells display the same characteristic changes in biochemistry as freshly explanted embryonic muscle cells in culture, (8) but are more amenable to experimental manipulation. We have therefore chosen to examine the ability of cultured L6 muscle cells, in the undifferentiated myoblast form and the differentiated myotube form, to repair DNA. Repair synthesis was measured by the incorporation of

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Repair Dna Synthesis in Differentiated Embryonic Muscle Cells

Abstract: INTRODUCTION

Cited by 40 publications

References 25 publications

Infection of terminally differentiated myotubes with Rous sarcoma virus (RSV): lack of DNA integration but presence of RSV mRNA

Infection of terminally differentiated myotubes with Rous sarcoma virus (RSV): lack of DNA integration but presence of RSV mRNA

Integration of Proviral DNA in Chicken Cells Infected with Schmidt-Ruppin Rous Sarcoma Virus Is Not Enhanced by DNA Repair

Reduced DNA repair during differentiation of a myogenic cell line.

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