Complementary DNAs (cDNAs) were synthesized from polyadenylated RNAs of myoblasts and myotubes and used to analyze changes in the sequence complexity and frequency distribution of messenger RNAs during myogenesis in vitro. cDNA . polyadenylated-RNA hybridization kinetics show the presence of messenger RNA sequences specific for myotubes in fully differentiated muscle cultures. These sequences ar,e accumulated just prior to fusion, as was shown by hybridizations of myotube cDNA and total cytoplasmic RNAs from cells at different stages of differentiation. The myotube cDNA can be enriched 10-fold in myotube-specific RNA species by a hybridization with cytoplasmic RNAs from myoblasts and subsequent removal of these hybridized sequences by hydroxyapatite.After a period of cell division, mononucleated myoblasts from the Yaffe Lg line, when cultivated in vitro, undergo a specific differentiation characterized by the formation of myotubes [1,2] and by a strong increase in the synthesis of muscle-specific proteins [3,5]. This increase could result from changes in the transcriptional pattern during cell differentiation or/ and from modifications in the stability of specific messenger RNAs.Changes in the messenger RNA populations have been studied by Paterson and Bishop [6] in primary chick embryo muscle culture. Changes in messenger RNA diversity have also been observed in committed myogenic cells when compared to undifferentiated embryonal carcinoma cells [7]. An increase in myosin heavy chain messenger RNA during myogenesis was also demonstrated [8].A general increase in the stability of polyadenylated RNA was observed in differentiating myogenic cells [9]. The putative messenger RNA of myosin heavy chain was shown to become stable just prior to cell fusion [lo].We have recently studied some chromatin events occurring during myogenesis. A strong decrease in Definition. rot values are given in mol I-' s; rotlp is that value of rot at which the half-maximal level of saturation is reached.Enzymes. S 1 nuclease, single-strand-specific endonuclease, from AspergiNus oryzae (EC 3.1.4.21); RNase TI (EC 3.1.4.8); RNase I (EC 3.1.4.22).protein kinase specific activities and characteristic changes in the pattern of non-histone protein phosphorylation were observed during the course of cell differentiation [ll]. It could be assumed that these changes are related to modifications in the transcriptional pattern.The work of Paterson and Bishop [6] showed accumulation of specific messenger RNA species during differentiation of chick myoblasts in primary cultures, but their conclusions were hampered because of the use of primary cultures contaminated by 20% fibroblasts and of bromodeoxyuridine for preventing differentiation. This inhibitor could interfere with normal gene expression.In this study we have used pure myoblast cultures and we have followed changes in the messenger RNA populations during the successive steps of myogenic differentiation.In the first part of this work we have performed hybridizations and cross-hybridizations...
We previously found that a minor fraction of single-stranded DNA (ss-DNA) isolated from native nuclear DNA of normal chicken embryonic cells and cells of other species hybridized with bulk nuclear DNA or cellular RNA in great excess. At least one-third of ss-DNA belonging to the nonrepetitious part of the cell genome could be hybridized to homologous RNAs. In the present work, similar results were obtained with ss-DNA from cells of chickens infected by avian myeloblastosis virus (AMV). To investigate whether this enrichment of ss-DNA in transcribed DNA sequences involves provirus DNA, radioactive AMV RNA and cDNA copies of AMV RNA were used. Most of the 70S AMV RNA hybridized much faster to ss-DNA from productively infected leukemic cells than to bulk DNA. cDNA, either double-stranded or single-stranded, made in the presence of actinomycin D hybridized to total nuclear DNA with similar kinetics. In contrast, about half of the double-stranded cDNA molecules hybridized 40-50 times faster to ss-DNA than to total DNA, indicating that only one of the provirus DNA strands seems to be present in ss-DNA. This was confirmed by the fact that relatively insignificant amounts of the ss-cDNA molecules made in the presence of actinomycin D could be annealed to ss-DNA as compared with bulk DNA. These results indicate that actively transcribed DNA sequences can be selectively distributed in the ss-DNA fraction, probably because of single strand breaks in the vicinity of transcription sites.The provirus theory of Temin (1), which postulates that the replication of RNA tumor viruses proceeds via DNA copies of viral RNA integrated in the host genome, has been substantiated by the discovery of successful transfection mediated by reverse transcriptase (2, 3), through an intermediate of viral DNA isolated from oncornavirus-transformed cells (4-6) and molecular hybridization between the viral genome and cell nucleic acids (7,8). In productively infected cells, the integrated provirus DNA appears to be more actively transcribed than most of the cellular genes (9).We have isolated, from the nDNA of various species, a minor fraction of single-stranded DNA (ss-DNA) and demonstrated that about one-third of the ss-DNA from cultured normal embryonic chicken cells (10) The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be-hereby marked "advertsement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
Single stranded DNA (s.s.DNA) comprising 1-2% of the total nuclear DNA was isolated by an improved method of hydroxyapatite chromatography from native nuclear DNA3 of embryonic chick cells, labeled for several cell generations with 3H-thymidine. Small quantities of 3H-DNA were annealed with a large excess of unlabeled DNA or polysomal RNA from chick embryos. Hybridization kinetics (monitored by the use of SI nuclease digestion, hydroxyapatite chromatography and thermalfusion), indicated that s.s.DNA belongs to the non repetitious fraction of the cell genome. One third represents DNA sequences engaged in the transcription of messenger RNA's.
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