Homogeneity and complexity of avian oncornavirus proviral DNA determined by molecular hybridization

Abstract

The homogeneity of DNA complementary to the 35S RNA subunit of avian myeloblastosis virus (AMV) has been demonstrated by single or multistep hybridization. For multistep hybridizations, 35S AMV RNA was preselected for its ability to hybridize either to unfractionated leukemic DNA or to leukemic DNA enriched for unique or for reiterated sequences. These experiments indicate that the viral genome is complementary to DNA sequences with a low reiteration frequency. Competition experiments confirm the absence of fasthybridizing sequences in viral DNA. Computer analyses of the data reveal that there are two to four copies of viral DNA in infected cells.

Measurement of proviral genes in uninfected and avian myeloblastosis virus-infected cells by hybridization with 3H-labeled complementary DNA probe excess

“…Downloaded from DISCUSSION There have been three major procedures for determining the number of viral genes integrated into avian cell genomes. They are: (i) hybridizing [5H]vRNA or annealing singlestrand [5H]cDNA probes with excess cell DNA in a liquid reaction mixture (8,13,15,19,21,24,25,28); (ii) hybridizing [3H]vRNA to cell DNA bound to filters and using a standard DNA previously calibrated by saturation with excess [3H]-vRNA to calculate the proviral DNA content (2, 3, 20, 22); (iii) acceleration of doublestranded, virus-specific [3H]cDNA reassociation with excess cell DNA (25,26).…”

“…Formation ofhybrids between 3H-labeled viral RNA ([3H]vRNA) or 3H-labeled complementary DNA ([3H]cDNA) and cellular DNA has proven to be a useful means for establishing the presence of virus-specific DNA sequences in uninfected chick cells and the acquisition of new viral genes by cells after infection (2,4,19). Estimates of 1 to 10 proviral DNA copies in the genome of uninfected chicken cells have been made (2,8,13,19,24,25,28), although as many as 10 to 22 copies per cell have been reported (2,3,13,26). Virus-specific DNA in the cell after virus infection has been found to increase twoto three-fold (2,8,17,28), although in one case no increase occurred (26).…”

“…Estimates of 1 to 10 proviral DNA copies in the genome of uninfected chicken cells have been made (2,8,13,19,24,25,28), although as many as 10 to 22 copies per cell have been reported (2,3,13,26). Virus-specific DNA in the cell after virus infection has been found to increase twoto three-fold (2,8,17,28), although in one case no increase occurred (26). These differences in results between laboratories can be caused, in part, by biological variation (2), but there are several uncertain parameters in the measurement of the kinetics of hybridizing [3H]cDNA or [3H]vRNA to an excess of cell DNA that may lead to a limitation in the accuracy of kinetic data (6,12,16).…”

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

“…UV irradiation or 4NQO treatment at various time intervals, from 6 h before infection to 24 h after infection, should have induced DNA repair sometime during the period of maximal proviral DNA synthesis. Provirus synthesis starts within 1 h after infection, and a relatively large concentration of proviral DNA accumulates intracellularly between 20 and 60 h later (1,19,51). In normal infection, provirus integration starts within 8 h. Also, extraction of cellular DNA 18 to 21 days after infection allowed for maximal integration of the exogenous proviruses (two to four per cell) and for the elimination of free proviral DNA (1, 6, 33, 42; Baluda et al, in press).…”

“…In leukemic cells induced by avian myeloblastosis virus (AMV), the AMV provirus appears to be integrated in tandem with the endogenous proviral DNA between contiguous cellular DNA sequences, which are repeated approximately 1,200 times per haploid cell genome (17, 18,42). There are one to two AMV genomes in addition to one to two endogenous proviruses integrated per haploid leukemic chicken cell genome (19,42). In cells infected with AMV, Rous sarcoma virus (RSV), or Rous-associated virus-type 2 (RAV-2), the number of proviruses that eventually become integrated per chicken cell genome is much smaller than the number of proviruses synthesized early after infection (1, 6, 30).…”