Acquisition of new DNA sequences after infection of chicken cells with avian myeloblastosis virus

DNA-RNA hybridization studies between 70S RNA from avian myeloblastosis virus (AMV) and an excess of DNA from (i) AMV-induced leukemic chicken myeloblasts or (ii) a mixture of normal and of congenitally infected K-137 chicken embryos producing avian leukosis viruses revealed the presence of fast- and slow-hybridizing virus-specific DNA sequences. However, the leukemic cells contained twice the level of AMV-specific DNA sequences observed in normal chicken embryonic cells. The fast-reacting sequences were two to three times more numerous in leukemic DNA than in DNA from the mixed embryos. The slow-reacting sequences had a reiteration frequency of approximately 9 and 6, in the two respective systems. Both the fast- and the slow-reacting DNA sequences in leukemic cells exhibited a higher T_m (2 C) than the respective DNA sequences in normal cells. In normal and leukemic cells the slow hybrid sequences appeared to have a T_m which was 2 C higher than that of the fast hybrid sequences. Individual non-virus-producing chicken embryos, either group-specific antigen positive or negative, contained 40 to 100 copies of the fast sequences and 2 to 6 copies of the slowly hybridizing sequences per cell genome. Normal rat cells did not contain DNA that hybridized with AMV RNA, whereas non-virus-producing rat cells transformed by B-77 avian sarcoma virus contained only the slowly reacting sequences. The results demonstrate that leukemic cells transformed by AMV contain new AMV-specific DNA sequences which were not present before infection.     

Organization of chicken DNA sequences homologous to the transforming gene of avian myeloblastosis virus. II. Isolation and characterization of lambda proto-amv DNA recombinant clones from a library of leukemic chicken DNA.

Several lambda proto-amv recombinants isolated from a lambda Charon 4A library of leukemic chicken DNA were analyzed by using various restriction endonucleases and hybridization with specific probes representing different regions of the transforming gene of avian myeloblastosis virus. The position of 30 sites for 11 different restriction endonucleases was established in the proto-amv region of chicken DNA. Identical restriction endonuclease maps were obtained for the normal and leukemic DNAs in the proto-amv domain, which covers 8 to 9 kilobases of DNA. The cellular genetic elements homologous to the cellular sequence (amv) inserted into the avian myeloblastosis virus genome are contained within six major proto-amv segments which are interrupted by at least five large DNA regions lacking homology with amv.     

Isolation of the proviral coding strand of avian myeloblastosis virus from leukemic chicken myeloblast by affinity chromatography.

The coding strand of the integrated proviral DNA of avian myeloblastosis virus (AMV) was isolated from the DNA of leukemic chicken myeloblast. The three-step isolation procedure employed a combination of affinity chromatography with Sepharose-linked RNA, nucleic acid hybridization, and hydroxypatite chromatography techniques. At each step of purification the product was analyzed for the enrichment of AMV coding strand by hybridization with AMV RNA. The final product was the coding strand of the AMV DNA (90% pure). These results show that such a procedure can be used for the isolation and analysis of a specific structural gens of eukaryotic cells.     

Expression of endogenous avian myeloblastosis virus information in different chicken cells.

Uninfected chicken cells were found to contain endogenous avian myeloblastosis virus (AMV)-specific information. Different tissues from chicken embryos and chickens expressed different amounts of the AMV-specific information. The endogenous AMV-related RNA was most abundant in bone marrow cells, which contained about 20 copies per cell. About 5 to 10 copies of AMV endogenous RNA per cell were found in embryonic yolk sac cells and bursa cells. The spleen, muscle, liver, and kidney cells of chickens and the fibroblasts of chicken embryos contained about two copies per cell. The amounts of AMV endogenous RNA in bone marrow, yolk sac, and bursa varied with age. From 19-day-old embryos to 2-week-old chickens, the bone marrow contained 20 copies of AMV RNA per cell. Bone marrow cells from 2-year-old chickens contained five copies per cell. Yolk sac cells of 10-day-old embryos and 1-day-old chickens were found to contain two copies per cell, whereas in 15- to 17-day-old embryos, these cells contained 5 to 10 copies. These results indicate that the level of endogenous AMV expression correlates with the development of granulopoiesis of the chicken hemopoietic system. The results of experiments on the thermostability of RNA-DNA hybrids indicated that the endogenous AMV RNA is closely related to viral AMV RNA. The expression of endogenous AMV information is independent of the activity of the chick helper factor. This endogenous AMV information is expressed as 20 to 21S RNA in both bone marrow and yolk sac cells.     

Primer recognition by avian myeloblastosis virus RNA-directed DNA polymerase.

Tryptophanyl-tRNA was specifically labeled at the 3' end with [3H]tryptophan and cleaved in half with RNase under denaturing conditions, and the 3' half was shown to hybridize exclusively at the 5' end of avian myeloblastosis virus RNA. The RNA-dependent DNA polymerase of avian myeloblastosis virus is capable of efficiently binding the 3' half of the primer molecule.     

Identification of DNA in the core component of avian myeloblastosis virus.

Avian myeloblastosis virus (AMV) was found to contain DNA associated with the virion. The viral envelope was removed by treating the virus with a nonionic detergent and the DNA was found in the core fraction. These experiments indicate that the DNA associated with tumor virus is not contaminant associated with the viral envelope and suggest that the DNA is part of the internal core component. The DNA from avian myeloblastosis virus has a density of 1.70 g/cm3.     

Utilization of mismatched initiator termini by avian myeloblastosis virus DNA polymerase.

DNA synthesis by avian myeloblastosis virus was studied using poly(C) as template and modified oligo(dG) as primer. The addition of one noncomplementary base to the 3'-end of the primer has no important effect on synthesis. The mispaired base is incorporated into the product and the apparent Km (for primer) and the V of the reaction remain unchanged. This confirms the absence of a 3' leads to 5'-exodeoxynuclease activity using a template that is transcribed faithfully rather than one that can undergo a slippage reaction.     

Endonuclease activity of purified RNA-directed DNA polymerase from avian myeloblastosis virus.

Highly purified preparations of RNA-directed DNA polymerase from avian myeloblastosis virus (AMV) contain a Mn2+-activated endonuclease activity capable of nicking supercoiled DNA. This endonuclease activity co-sediments in glycerol gradients with the alphabeta form of AMV DNA polymerase, and co-chromatographs with DNA polymerase activity on DEAE-cellulose, phosphocellulose, and heparin-Sepharose. It is also present in AMV alphabeta-DNA polymerase purified by electrophoresis through nondenaturing polyacrylamide gels and subsequently chromatographed on poly(C)-agarose. alphabeta-associated endonuclease is co-immunoprecipitated with DNA polymerase activity by antiserum directed against alphabeta holoenzyme. The alpha form of AMV DNA polymerase lacks this activity. In its enzymatic properties, alphabeta-associated endonuclease resembles the endodeoxyribonuclease activity associated with the AMV p32 protein, which has been shown to be structurally related to the beta (but not the alpha) subunit of AMV DNA polymerase.     

The binding of polynucleotides to the DNA polymerase of avian myeloblastosis virus.

The interaction between avian myeloblastosis virus DNA polymerase and synthetic nucleic acids was studied by an adaptation of the membrane filter binding technique. Bacillus subtilis DNA was used as a substrate for the binding reaction and was retained on the filters in the presence of the viral polymerase. The polymerase activity was demonstrated to be retained on the filter in either the presence or absence of the bacterial DNA. Characterization of the polymerase-DNA interaction demonstrated a marked similarity to previous data regarding the binding of Escherichia coli DNA-dependent RNA polymerase to nucleic acids when studied using related techniques. In contrast, the association between methylated bovine serum albumin and the B. subtilis DNA was found to differ significantly in both reaction stoichiometry and stability. Synthetic polynucleotides were shown to inhibit the binding of the bacterial DNA to the viral DNA polymerase and poly 2′-fluoro-2′-deoxyuridylic acid was found to be the most potent inhibitor of this reaction. Results from the binding-inhibition studies correlated well with studies concerning the inhibition of enzyme activity and it is concluded that the inhibitory polynucleotides act by interfering with binding of nucleic acid template to the viral enzyme.     

Concerted integration of viral DNA termini by purified avian myeloblastosis virus integrase.

Concerted integration of retroviral DNA termini, which produces a characteristic duplication of sequences at the integration site and formation of the proviral state, is a necessary step of the retroviral life cycle. We investigated the pairwise integration reaction catalyzed by purified avian retrovirus integrase by measuring the response to solution parameters and how the sequences of the viral termini, which comprise the avian imperfect inverted repeat, affect the reaction. When we optimized the reaction, an efficiency was achieved which approached that measured in systems using cytoplasmic extracts from virus-infected cells. The response of purified avian integrase to solution parameters was similar to that of the integration activity derived from cellular extracts. For strand transfer, the U3 viral terminal sequences were preferred to those of the U5 termini, a result we previously showed for the trimming reaction. That the sequence preference was the same for trimming and strand transfer may be further evidence that only one catalytic site is used for both reactions. A significant number of integration sites were sequenced. Interesting trends were found for the fidelity of the host duplications to the avian 6-bp duplication size, the clustering of the integration sites in the nonessential region of the lambda host DNA, and the sequence characteristics of the duplication sites.     

Efficient concerted integration of retrovirus-like DNA in vitro by avian myeloblastosis virus integrase.

We report the efficient concerted integration of a linear virus-like DNA donor into a 2.8 kbp circular DNA target by integrase (IN) purified from avian myeloblastosis virus. The donor was 528 bp, contained recessed 3' OH ends, was 5' end labeled, and had a unique restriction site not found in the target. Analysis of concerted (full-site) and half-site integration events was accomplished by restriction enzyme analysis and agarose gel electrophoresis. The donor also contained the SupF gene that was used for genetic selection of individual full-site recombinants to determine the host duplication size. Two different pathways, involving either one donor or two donor molecules, were used to produce full-site recombinants. About 90% of the full-site recombinants were the result of using two donor molecules per target. These results imply that juxtapositioning an end from each of two donors by IN was more efficient than the juxtapositioning of two ends of a single donor for the full-site reaction. The formation of preintegration complexes containing integrase and donor on ice prior to the addition of target enhanced the full-site reaction. After a 30 min reaction at 37 degrees C, approximately 20-25% of all donor/target recombinants were the result of concerted integration events. The efficient production of full-site recombinants required Mg2+; Mn2+ was only efficient for the production of half-site recombinants. We suggest that these preintegration complexes can be used to investigate the relationships between the 3' OH trimming and strand transfer reactions.     

Vertebrate DNAs contain nucleotide sequences related to the transforming gene of avian myeloblastosis virus.

Avian myeloblastosis virus contains a continuous sequence of approximately 1,000 nucleotides which may represent a gene (amv) responsible for acute myeloblastic leukemia in chickens. This sequence appears to have been acquired from chicken DNA and to be substituted for the envelope gene in the viral genome. We used hybridization probes enriched for the amv sequences and conditions that facilitate annealing of partially homologous nucleotide sequences to show that cellular sequences related to amv are present in the genomes of all vertebrates ranging from amphibians to humans but were not detected in fish, sea urchins, or Escherichia coli. In contrast to the preceding findings, nontransforming endogenous proviral nucleotide sequences closely related to the remainder of the avian myeloblastosis virus genome and to the entire myeloblastosis-associated helper virus are present only in chicken DNA. The amv-related cellular sequences appear to be highly conserved during evolution and to be contained at only one or a few locations in the genome of vertebrates. Within closely related species, they appear to share common evolutionary genetic loci. These findings and similar ones obtained with other highly oncogenic retroviruses containing a transforming gene suggest a general mechanism for acquisition of viral oncogenic sequences and an essential role for these sequences in the normal cellular state.     

Interspersion of repetitive sequences in rat liver DNA

In rat liver DNA, which contains only 20% repetitive sequences, a close interspersion of repetitive and unique sequences is found in about 35 % of the total DNA. The mean length of repetitive and unique alternating sequences is respectively 230 and 400 base pairs.     

Identification of the avian myeloblastosis virus genome. II. Restriction endonuclease analysis of DNA from lambda proviral recombinants and leukemic myeoblast clones.

Two lambda proviral DNA recombinants were characterized with a number of restriction endonucleases. One recombinant contained a complete presumptive avian myeloblastosis virus (AMV) provirus flanked by cellular sequences on either side, and the second recombinant contained 85% of a myeloblastosis-associated virus type 1 (MAV-1)-like provirus with cellular sequences adjacent to the 5' end of the provirus. Comparing the restriction maps for the proviral DNAs contained in each lambda hybrid showed that the putative AMV and MAV-1-like genomes shared identical enzyme sites for 3.6 megadaltons beginning at the 5' termini of the proviruses with respect to viral RNA. Two enzyme sites near the 3'-end of the MAV-1-like provirus were not present in the putative AMV genome. We also examined a number of leukemic myeloblast clones for proviral content and cell-provirus integration sites. The presumptive AMV provirus was present in all the leukemic myeloblast clones regardless of the endogenous proviral content of the target cells or the AMV pseudotype used for conversion. Multiple cellular sites were suitable for integration of the putative AMV genome and the helper genomes. The proviral genomes were all integrated colinearly with respect to linear viral DNA.