Reliability of the RNA-DNA Filter Hybridization for the Detection of Oncornavirus-Specific DNA Sequences

Denatured DNA from leukemic myeloblasts or uninfected chicken embryos, immobilized on nitrocellulose filters, was hybridized to a vast excess of [(3)H]70S RNA from purified avian myeloblastosis virus. The viral RNA was eluted from the RNA-DNA hybrids, purified, and then rehybridized in solution to an excess of either leukemic or normal chicken embryonic DNA. This study revealed that all the slow and the fast hybridizing viral RNA sequences detectable by liquid hybridization in DNA excess had hybridized to the filter bound DNA. Both techniques also gave similar values for the number of 28S ribosomal RNA genes contained in a chicken cell genome: 210 by the liquid hybridization procedure and 218 by the filter hybridization technique. Therefore, filter hybridization can accurately detect DNA sequences present in relatively few numbers in the genome of higher organisms.     

Comparison of an avian osteopetrosis virus with an avian lymphomatosis virus by RNA-DNA hybridization.

Myeloblastosis-associated virus (MAV)-2(0), a virus which was derived from avian myeloblastosis virus and induced a high incidence of osteopetrosis, was compared with avian lymphomatosis virus 5938, a recent field isolate which induced a high incidence of lymphomatosis. The following information was obtained. (i) MAV-2(0) induced osteopetrosis, nephroblastoma, and a very low incidence of hepatocellular carcinoma. No difference was seen in the oncogenic spectrum of end point and plaque-purified MAV-2(0). (ii) 125I-labeled RNA sequences from MAV-2(0) formed hybrids with DNA extracted from osteopetrotic bone at a rate suggesting five proviral copies per haploid cell genome. The extent of hybridization of MAV-2(0) RNA with DNA from osteopetrotic tissue was more extensive (87%) than was observed in reactions with DNA from uninfected chicken embryos (52%). (iii) Competition of unlabeled viral RNA in hybridization reactions between the radioactive RNA from the two viruses and their respective proviral sequences present in tumor tissues showed that 15 to 20% of the viral sequences detected in these reactions were unshared. In contrast, no differences were detected in competition analyses of RNA sequences from the two viruses detected in DNA of normal chicken cells. (iv) MAV-2(0) 35S RNA was indistinguishable in size from avian lymphomatosis virus 5938 35S RNA by polyacrylamide gel electrophoresis.     

Comparison of an Avian Osteopetrosis Virus with an Avian Lymphomatosis Virus by RNA-DNA Hybridization

Myeloblastosis-associated virus (MAV)-2(0), a virus which was derived from avian myeloblastosis virus and induced a high incidence of osteopetrosis, was compared with avian lymphomatosis virus 5938, a recent field isolate which induced a high incidence of lymphomatosis. The following information was obtained. (i) MAV-2(0) induced osteopetrosis, nephroblastoma, and a very low incidence of hepatocellular carcinoma. No difference was seen in the oncogenic spectrum of end point and plaque-purified MAV-2(0). (ii) ¹²⁵I-labeled RNA sequences from MAV-2(0) formed hybrids with DNA extracted from osteopetrotic bone at a rate suggesting five proviral copies per haploid cell genome. The extent of hybridization of MAV-2(0) RNA with DNA from osteopetrotic tissue was more extensive (87%) than was observed in reactions with DNA from uninfected chicken embryos (52%). (iii) Competition of unlabeled viral RNA in hybridization reactions between the radioactive RNA from the two viruses and their respective proviral sequences present in tumor tissues showed that 15 to 20% of the viral sequences detected in these reactions were unshared. In contrast, no differences were detected in competition analyses of RNA sequences from the two viruses detected in DNA of normal chicken cells. (iv) MAV-2(0) 35S RNA was indistinguishable in size from avian lymphomatosis virus 5938 35S RNA by polyacrylamide gel electrophoresis.     

Characterization of reticuloendotheliosis virus strain T DNA and isolation of a novel variant of reticuloendotheliosis virus strain T by molecular cloning.

Reticuloendotheliosis virus strain T (REV-T) is a highly oncogenic avian retrovirus which causes a rapid neoplastic disease of the lymphoreticular system. Upon infection, this virus gives rise to two species of unintegrated linear viral DNA, which are 8.3 and 5.5 kilobase pairs long and represent the helper virus (REV-A) and the oncogenic component (REV-T), respectively. Restriction endonuclease cleavage maps of these two DNA components indicate that REV-T DNA has a large portion of the genome deleted with respect to REV-A DNA and a substitution about 0.8 to 1.5 kilobase pairs long that is unrelated to REV-A DNA. These additional sequences comprise the putative transforming region of REV-T (rel). A chicken spleen cell line transformed by REV-T produced virus which upon infection gives rise to three species of unintegrated linear viral DNA (8.3, 5.5, and 3,3 kilobase pairs). We isolated the proviruses of the 8.3- and 3.3-kilobase pair species from this cell line by cloning in the phage vector Charon 4A. Restriction enzyme mapping showed that the two proviral clones are proviruses of REV-A and a variant of REV-T, respectively. A subclone of the variant REV-T provirus specific for the rel sequences of REV-T was used as a hybridization probe to demonstrate that the rel sequences are different from the putative transforming sequences of Schmidt-Ruppin Rous sarcoma virus strain A, avain myelocytomatosis virus, avian myeloblastosis virus, avian erythroblastosis virus, Abelson murine leukemia virus, and Friend erythroleukemia virus. In addition, the rel-specific hybridization probe was used to identify a specific set of sequences which are present in uninfected avian DNAs digested with several restriction enzymes. The corresponding cell sequences are not arranged like rel in REV-T.     

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

The effect DNA repair might have on the integration of exogenous proviral DNA into host cell DNA was investigated by comparing the efficiency of proviral 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 of nonintegrated 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 (3)H-labeled 35S viral RNA. The copy number of the 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.     

Presence in Leukemic Cells of Avian Myeloblastosis Virus-Specific DNA Sequences Absent in Normal Chicken Cells

(3)H-labeled 35S RNA from purified avian myeloblastosis virus (AMV) was exhaustively hybridized with an excess of normal chicken DNA to remove all viral RNA sequences which are complementary to DNA from uninfected cells. The [(3)H]RNA which failed to hybridize was isolated by hydroxylapatite column chromatography which separates DNA-RNA hybrids from single-stranded [(3)H]RNA. The residual RNA hybridized to leukemic chicken DNA but did not rehybridize with normal chicken DNA. This demonstrates conclusively that DNA from AMV-induced leukemic cells contain viral-specific sequences which are absent in DNA from normal cells.     

Titration of integrated simian virus 40 DNA sequences, using highly radioactive, single-stranded DNA probes.

Nick-translated simian virus 40 (SV40) [32P]DNA fragments (greater than 2 X 10(8) cpm/micrograms) were resolved into early- and late-strand nucleic acid sequences by hybridization with asymmetric SV40 complementary RNA. Both single-stranded DNA fractions contained less than 0.5% self-complementary sequences; both included [32P]-DNA sequences that derived from all regions of the SV40 genome. In contrast to asymmetric SV40 complementary RNA, both single-stranded [32P]DNAs annealed to viral [3H]DNA at a rate characteristic of SV40 DNA reassociation. Kinetics of reassociation between the single-stranded [32P]DNAs indicated that the two fractions contain greater than 90% of the total nucleotide sequences comprising the SV40 genome. These preparations were used as hybridization probes to detect small amounts of viral DNA integrated into the chromosomes of Chinese hamster cells transformed by SV40. Under the conditions used for hybridization titrations in solution (i.e., 10- to 50-fold excess of radioactive probe), as little as 1 pg of integrated SV40 DNA sequence was assayed quantitatively. Among the transformed cells analyzed, three clones contained approximately one viral genome equivalent of SV40 DNA per diploid cell DNA complement; three other clones contained between 1.2 and 1.6 viral genome equivalents of SV40 DNA; and one clone contained somewhat more than two viral genome equivalents of SV40 DNA. Preliminary restriction endonuclease maps of the integrated SV40 DNAs indicated that four clones contained viral DNA sequences located at a single, clone-specific chromosomal site. In three clones, the SV40 DNA sequences were located at two distinct chromosomal sites.     

Molecular cloning of bovine leukemia virus DNA integrated into the bovine tumor cell genome

The bovine leukemia virus (BLV) DNA harbored in the bovine tumor cell genome was cloned in lambda Charon 4A phage. Using either representative or 3' half-enriched BLV cDNA as a blot hybridization probe, clone lambda BLV-1 was shown to carry 9 kb of the BLV genome, flanked by cellular sequences at both ends. Restriction mapping with twelve endonucleases and hybridization of the DNA fragments to BLV cDNA representing a 3'-end portion of the viral genome revealed the presence and precise location of two long terminal repeats (LTRs) and virus-cell junctions. Thus, lambda BLV-1 appears to contain the complete BLV genome and flanking tumor cellular sequences. The restriction map of the cloned BLV proviral DNA closely resembles that previously reported for unintegrated linear proviral DNA, but differs significantly from that of the integrated provirus of another BLV isolate, the difference occurring preferentially in the putative gag and pol genes.     

Detection of Avian Tumor Virus RNA in Uninfected Chicken Embryo Cells

Uninfected chicken embryo cells were analyzed for the presence of viral ribonucleic acid (RNA) by molecular hybridization with the single-stranded deoxyribonucleic acid (DNA) product of the RNA-dependent DNA polymerase contained in avian sarcoma-leukosis virions. Viral RNA was detected in all cells which contained the avian tumor virus group-specific antigen and the virus-related helper factor. The amounts of viral RNA in these cells ranged from approximately 3 to 40 copies of viral-specific sequences per cell. In general, the viral RNA content correlated with the level of helper activity in the cells. Cells infected with Rous-associated virus 2 contained 3,000 to 4,000 copies of viral RNA per cell. RNA from these infected cells hybridized with nearly 100% of the viral (3)H-DNA. By contrast, a maximum of less than 50% hybridization was obtained with RNA from the uninfected helper-positive cells, suggesting that not all of the viral RNA sequences were present in these cells. No viral RNA was detected in cells which lacked group-specific antigen and helper activity. Under the conditions used in these studies, less than 0.3 viral genome equivalents of RNA per cell would have been detected.     

Synethesis and integration of viral DNA in chicken cells at different time after infection with various multiplicities of avian oncornavirus.

To see if integration of the provirus resulting from RNA tumor virus infection is limited to specific sites in the cell DNA, the variation in the number of copies of virus-specific DNA produced and integrated in chicken embryo fibroblasts after RAV-2 infection with different multiplicities has been determined at short times, long times, and several transfers after infection. The number of copies of viral DNA in cells was determined by initial hybridization kinetics of single-stranded viral complementary DNA with a moderate excess of cell DNA. The approach took into account the different sizes of cell DNA and complementary DNA in the hybridization mixture. It was found that uninfected chicken embryo fibroblasts have approximately seven copies, part haploid genome of DNA sequences homologous to part of the Rous-association virus 2 (RAV-2) genome. Infection with RAV-2 adds additional copies, and different sequences, of RAV -2- specific DNA. By 13 h postinfection, there are 3 to 10 additional copies per haploid genome. This number can not be increased by increasing the multiplicity of infection, and stays relatively constant up to 20 h postinfection, when some of the additional viral DNA is integrated. Between 20 and 40 h postinfection, the cells accumulated up to 100 copies per haploid genome of viral DNA. Most of these are unintegrated. This number decreases with cell transfer, until cells are left with one to three copies of additional viral DNA sequences per haploid genome, of which most are integrated. The finding that viral infection causes the permanent addition of one to three copies of integrated viral DNA, despite the cells being confronted with up to 100 copies per haploid genome after infection, is consistent with a hypothesis that chicken cells contain a limited number of specific integration sites for the oncornavirus genome.     

Distribution of Deoxyribonucleic Acid Complementary to the Ribonucleic Acid of Avian Myeloblastosis Virus in Tissues of Normal and Tumor-Bearing Chickens

(3)H-labeled 70S ribonucleic acid (RNA) from purified avian myeloblastosis virus (AMV) was used as a probe in deoxyribonucleic acid (DNA)-RNA hybridization experiments to detect the presence of DNA complementary to the AMV genome in various tissues from noninfected normal chickens and from chickens infected with AMV. There was a remarkable constancy in the average cellular concentration of virus-specific DNA found in every tissue from the same uninfected chicken, and even in different chickens from the same strain. In contrast, different tissues from chickens bearing AMV-induced kidney tumors (embryonal nephromas) revealed an unequal distribution in the average virus-specific DNA content per cell. The increase was limited to tumor cells and to tissues that contain target cells for AMV, i.e., red blood cells, kidney cells, and possibly leukocytes. The red blood cells from AMV-infected chickens suffering from acute myeloblastic leukemia, although producing no virus, contained as many viral genome equivalents per cell as did leukemic myeloblasts known to produce large quantities of AMV. An increased viral DNA content was observed in the target cells of chickens that did not show any sign of tumor formation 6 months after infection with AMV. This study demonstrates that vertically transmitted viral DNA is uniformly and stably distributed among all tissues of the offspring, but that horizontal infection after hatching results in an increase in viral DNA content only in some dividing, target tissues that may or may not give rise to neoplasias.     

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

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 fast-hybridizing sequences in viral DNA. Computer analyses of the data reveal that there are two to four copies of viral DNA in infected cells.     

Single copies of HIV proviral DNA detected by fluorescent in situ hybridization

A fluorescent in situ DNA hybridization assay employing a biotinylated DNA probe was used to visualize single copies of human immunodeficiency virus (HIV) proviral DNA in the nuclei and metaphase chromosomes of infected cells. In clonal cell lines that contain either one or two copies of proviral DNA, the efficiency of detection of individual loci of proviral DNA was 57% to 78%. Only 1% of uninfected control cells exhibited a false-positive signal. HIV proviral DNA could be accurately identified in mixed populations comprised of only 5% infected cells. Thus, this assay could be used to identify cells that harbor HIV proviral DNA and to monitor the status of proviral DNA throughout the course of HIV infection.     

Use of DNA-DNA Annealing to Detect New Virus-Specific DNA Sequences in Chicken Embryo Fibroblasts After Infection by Avian Sarcoma Virus

Labeled, virus-specific DNA synthesized in vitro by the virion-associated polymerase of avian sarcoma virus (ASV) was used to measure virus-specific sequences in cell DNA in three ways: (i) by determining the effect of cell DNA upon the reassociation rate of double-stranded polymerase products; (ii) by measuring the kinetics of annealing of single-stranded polymerase product (cDNA) to cell DNA; or (iii) by measuring the amount of cDNA which anneals to a large excess of cell DNA. With these three assays and modifications of them, we show that fewer than five copies of ASV-specific DNA sequences are present per diploid cell in uninfected chicken embryos; that a two- to several-fold increase in copy number of viral DNA follows infection by ASV; that infection introduces to the cell viral sequences not present before infection; and that DNAs from uninfected Pekin duck and Japanese quail embryos show no homology with DNA synthesized by the ASV polymerase. Some of these results differ from data in a previous report from this laboratory (H. E. Varmus, R. A. Weiss, R. R. Friis, W. Levinson, and J. M. Bishop, 1972) and, in general, reconcile our observations with those from other laboratories.     

Avian myeloblastosis virus transforming gene is related to unique chicken DNA regions separated by at least one intervening sequence.

Identification of several additional restriction endonuclease sites within the cellular substitution (amv) inserted into the avian myeloblastosis virus proviral genome has permitted us to isolate different regions of the amv sequence. These subsets of the avian myeloblastosis virus transforming gene have been cloned in the plasmid pBR322 and used as hybridization probes to investigate the topology of homologous (proto-amv) normal chicken DNA sequences. The results showed that the cellular proto-amv sequences in C/O chicken DNA are interrupted by at least one intervening sequence. A partial arrangement of the proto-amv sequences is presented.     

Analysis of cellular integration sites in avian sarcoma virus infected duck embryo cells.

The cellular sites of integration of avian sarcoma virus (ASV) have been examined in clones of duck embryo cells infected with the Bratislava 77 strain of ASV using restriction endonuclease digestion, agarose gel electrophoresis, Southern blotting, and hybridization with labeled ASV complementary DNA probes. DNA prepared from 11 clones of duck embryo cells infected with the Bratislava 77 strain of ASV was digested with the restriction enzymes HpaI, which cleaves once within the viral genome, and Hind III, which cleaves twice within the viral genome, and the virus-cell DNA juncture fragments were resolved by agarose gel electrophoresis. Analysis of the virus-cell junctures present in individual ASV-infected duck embryo clones revealed that all clones contain at least one copy of nondefective proviral DNA with some clones containing as many as 5 to 6 copies of proviral DNA. A comparison of the virus-cell juncture fragments present in different ASV-infected clones showed that each clone contains a unique set of virus-cell junctures. These data suggest that ASV DNA can integrate at multiple sites within the duck embryo cell genome and that these sites appear to be different as defined by digestion with the restriction enzymes HpaI and HindIII.     

Quantitative in situ hybridization using initial velocity measurements

In situ hybridization can be used to quantitate viral RNA at the single cell level by measuring levels of hybridization after saturation hybridization with an excess of cDNA probe has been achieved (1,2). In this paper we describe an alternative approach which consists in measuring the initial hybridization rate using a low concentration of cDNA probe and a short hybridization time. Under these conditions, we obtained a linear relationship between the number of autoradiographic grains and the number of viral genomes per cell in the range of 600 to 60,000 copies per cell of a 7-kb RNA genome. This approach allows an accurate measurement of copy number in a range for which saturation in situ hybridization is very difficult to achieve.     

Arrangement of Integrated Avian Sarcoma Virus DNA Sequences Within the Cellular Genomes of Transformed and Revertant Mammalian Cells

We have examined the arrangement of integrated avian sarcoma virus (ASV) DNA sequences in several different avian sarcoma virus transformed mammalian cell lines, in independently isolated clones of avian sarcoma virus transformed rat liver cells, and in morphologically normal revertants of avian sarcoma virus transformed rat embryo cells. By using restriction endonuclease digestion, agarose gel electrophoresis, Southern blotting, and hybridization with labeled avian sarcoma virus complementary DNA probes, we have compared the restriction enzyme cleavage maps of integrated viral DNA and adjacent cellular DNA sequences in four different mouse and rat cell lines transformed with either Bratislava 77 or Schmidt-Ruppin strains of avian sarcoma virus. The results of these experiments indicated that the integrated viral DNA resided at a different site within the host cell genome in each transformed cell line. A similar analysis of several independently derived clones of Schmidt-Ruppin transformed rat liver cells also revealed that each clone contained a unique cellular site for the integration of proviral DNA. Examination of several morphologically normal revertants and spontaneous retransformants of Schmidt-Ruppin transformed rat embryo cells revealed that the internal arrangement and cellular integration site of viral DNA sequences was identical with that of the transformed parent cell line. The loss of the transformed phenotype in these revertant cell lines, therefore, does not appear to be the result of rearrangement or deletions either within the viral genome or in adjacent cellular DNA sequences. The data presented support a model for ASV proviral DNA integration in which recombination can occur at multiple sites within the mammalian cell genome. The integration and maintenance of at least one complete copy of the viral genome appear to be required for continuous expression of the transformed phenotype in mammalian cells.