Avian Myeloblastosis Virus RNA Research Articles

Characterization of the cDNA synthesized by avian retrovirus reverse transcriptase using 35 S avian myeloblastosis virus RNA and an exogenous bovine primer tRNA

Bovine tRNA Trp can be partially hybridized to the avian myeloblastosis virus (AMV) 35 S RNA at 37°C, in the presence of AMV RNA-dependent DNA polymerase (reverse transcriptase). This template-primer complex is active in the synthesis of viral cDNA. The size of the cDNA products synthesized in the in vitro reconstituted AMV system was determined by urea-polyacrylamide gel electrophoresis using a tRNA labelled at the 3′-end by yeast tRNA nucleotidyl transferase. The synthesized cDNA has a size of about 100 nucleotides and was shown by Southern blotting to be complementary to a specific sequence of the 5′-end of the retroviral genome. These results indicate that reverse transcriptase is able to anneal the exogenous primer tRNA at the ‘primer-binding site’ near the 5′-end of the long terminal repeat (LTR) of AMV RNA.

RNase H-mediated release of the retrovirus RNA polyadenylate tail during reverse transcription.

By examining enzymatic reactions in vitro, we found that an early event during reverse transcription of the avian myeloblastosis virus RNA genome is the release of the 3' polyadenylate tail from the viral RNA template. By using specially constructed molecules containing minus-strand, strong-stop DNA and the 3' end of the viral RNA genome, we found that the reverse transcriptase-associated RNase H is responsible for the endonucleolytic release of polyadenylate.

Increase of cloning efficiencies by using high-performance liquid chromatography-purified vectors linkers

High-performance liquid chromatography (HPLC) was applied to the purification of vector linker DNAs used in molecular cloning experiments. The data clearly demonstrate that HPLC (Blue Column from LKB/TSK G 4000 SW) based on steric exclusion is a very rapid convenient method for the size separation of DNA fragments that are virtually free of any contamination. Application of vector and linker DNAs purified by this method enabled us to clone nanogram amounts of avian myeloblastosis virus RNA which was used for the evaluation of a recently described cloning procedure.

Studies on the function of the non-primer tRNAs associated with the 70 S RNA of avian myeloblastosis virus

Significant amounts of three tRNAs are associated with the 70 S RNA of avian myeloblastosis virus (AMV). The temperatures at which they are half dissociated from the 70 S RNA in 50 mM NaCl and their respective quantities relative to 35 S RNA are: tRNA Arg, 51°C, 1.6; tRNA Lys, 57°C, 0.7 and tRNA Trp, 76°C, 1.0. Possible functions for the non-primer tRNAs (tRNA Arg and tRNA Lys) were evaluated by determining the effect of their thermal dissociation on: (a) conversion of 70 S to 35 S RNA, (b) capacity of 70 S and/or 35 S RNA to be translated in vitro, and (c) capacity of 70 S and/or 35 S RNA to be reverse transcribed in vitro. Conversion of 70 S to 35 S RNA occurred with a t m of 56°C and is consistent with the hypothesis that tRNA Lys might be involved in joining two 35 S RNA subunits to form the 70 S RNA complex. There was no indication that the association of either tRNA Arg or tRNA Lys influenced the rate or quality of translation of 70 S or 35 S RNA. A decrease in the rate at which 70 S RNA is transcribed occurs in parallel with the dissociation of tRNA Arg and tRNA Lys.

In vitro translation of avian myeloblastosis virus RNA.

Avian myeloblastosis virus (AMV)-infected cells contain two viral mRNA's, a genome-sized 34S (7.5-kilobase) mRNA and a 21S (2.5-kilobase) subgenomic mRNA, which contains the AMV-specific sequences (myb sequences). We found that AMV virions packaged both the 7.5-kilobase full-length genomic RNA and the 2.5-kilobase subgenomic RNA. In vitro translation of AMV virion RNA sized by sucrose density gradient centrifugation yielded 76,000-, 56,000-, 48,500-, 47,000-, and 32,000-dalton products. The 76,000-dalton protein was coded for by RNA throughout the gradient, but the peak of activity was at 34S to 35S. The 56,000-, and 48,500-, and 32,000-dalton proteins were encoded in a 21S RNA, and 47,000-dalton protein was encoded in an RNA of approximately 24S. The 76,000-dalton protein was identified as Pr76gag, based upon immunoprecipitation with specific antiserum and the presence of the 19* dipeptide. 7-Methylguanosine triphosphate inhibited the syntheses of Pr76gag and the 56,000-, 48,500-, and 32,000-dalton proteins, but not the synthesis of the 47,000-dalton protein. The 56,000-, 48,500-, 47,000-, and 32,000-dalton proteins were not immunoprecipitated by anti-gag, anti-reverse transcriptase, or anti-gp85 antiserum. Two-dimensional peptide maps of the 56,000- and 48,500-dalton proteins indicated that they were unique. In vitro translational products of myeloblastosis-associated virus 1 were also analyzed to aid in the identification of the AMV myb gene product(s); the translational products analyzed included Pr76gag, p60env, and a 56,000-dalton polypeptide which apparently was not identical to the 56,000-dalton AMV translational product, as determined by two-dimensional peptide mapping. Our data indicated that one of these proteins (56,000, 48,500, or 32,000 daltons) may represent the product of the AMV myb gene and, therefore, the putative transforming protein(s) of AMV.

Size and genetic content of virus-specific RNA in myeloblasts transformed by Avian Myeloblastosis Virus (AMV)

Radiolabeled complementary DNA probes representing sequences specific for avian myeloblastosis virus (AMV) and sequences of different regions of avian leukosis virus (ALV) genome were used to analyze the gene content and size distribution of viral-specific RNA in AMV-transformed myeloblasts. The producer myeloblasts contained 8–10 times more copies per cell of virus-specific RNA than those of nonproducer (NP) myeloblasts. In the producer myeloblast, 35 S, 34 S, and 21 S virus-specific RNAs were detected. The 35 S RNA contained gag, pol, env, and c sequences; representing both the genomic and the messenger RNA of the helper virus. The 34 S RNA contained gag, pol, AMV, and c sequences, representing the genomic and the messenger RNA of AMV. The 21 S RNA of producer cells contained two different species: one contained env and c sequences and represented the glycoprotein messenger RNA of the helper virus; another contained AMV and c sequences and represented the subgenomic messenger RNA of AMV. In NP cells, two virus-specific RNAs were found; a 34 S RNA containing gag, pol, AMV, and c sequences and a 21 S RNA containing AMV and c sequences. Both RNAs hybridized to a probe specific for 5′ sequences of ALV. About 15% of pol sequences of virus-specific RNA in NP cells were found to be deleted.

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.

Isolation of complementary DNA unique to the genome of avian myeloblastosis virus (AMV)

Avian myeloblastosis virus (AMV) can transform avian cells of hemopoietic origin, such as bone marrow, embryonic yolk sac, and circulating macrophages. The AMV is defective in its replication and can only replicate in the presence of helper viruses. This defectiveness in replication is probably due to a deletion or substitution of nucleotide sequences in its genome. The AMV genome contains no sequences homologous to the src gene, which is responsible for the transforming function of the sarcoma viruses. We attempted to identify the AMV sequences that may contain sequences which are responsible for its transforming function. We isolated a complementary DNA (cDNA AMV) that hybridized preferentially to the RNA of the transforming AMV but not to the RNA of the helper virus. Using this cDNA AMV as a probe, we determined the size of the AMV genome to be 33–34 S with a molecular weight of 2.6 × 10 6. A similar molecular weight estimation of the AMV genome size was obtained by methylmercury-agarose gel electrophoresis of AMV RNA. In AMV-producer myeloblasts we can detect about 6000–7000 copies per cell of AMV-specific RNA, whereas fewer than 2 copies per cell of AMV RNA are found in helper virus-infected cells. In AMV nonproducer myeloblasts, about 2000 copies of AMV-specific RNA are detected. Furthermore, we find that RNA of AMV NP myeloblasts can only hybridize to 55% of cDNA complementary to helper virus genome. In uninfected hemopoietic cells, e.g., bone marrow cells, about 20 copies per cell of AMV-specific RNA are present, whereas in uninfected chick embryo fibroblasts less than 1 copy per cell is found.

RNA primer used in synthesis of anticomplementary DNA by reverse transcriptase of avian myeloblastosis virus.

When either the homologous RNA (avian myeloblastosis virus RNA) or a heterologous RNA (poliovirus RNA) was used as a template, the anticomplementary DNA synthesized in vitro by avian myeloblastosis virus reverse transcriptase (RNA-directed DNA nucleotidyltransferase, EC 2.7.7.7) was primed by fragments of the original RNA template that usually had adenosine at their 3' ends. When we used phage T/ RNA ligase (EC 6.5.1.3) to label the 3' end of the RNA template fragments contained in the RNA . cDNA hybrid intermediate, adenosine was found to be the principal nucleoside carrying the label. We infer from these results that the ribonuclease H (hybrid nuclease) activity of the reverse transcriptase creates fragments of the original RNA template with adenosine as the principal 3' terminus and that these fragments serve as primers for the synthesis of anticomplementary DNA.

Inhibition of RNA-dependent DNA polymerase activity of oncornaviruses by caffeine

Caffeine was found to inhibit RNA-dependent DNA polymerase activity of Rauscher leukemia virus when endogenous viral RNA and poly(rA)·(dT) 12–18 were used as templates. Similar results were also obtained with purified RNA-dependent DNA polymerase (deoxynucleoside triphosphate; DNA nucleotidyl transferase; EC 2.7.7.7) from avian myeloblastosis virus (AMV) utilizing 70S and 35S RNA of AMV, poly(rA)·(dT) 12–18, globin mRNA and activated calf thymus DNA as templates. The “caffeine effect” was evident only when it was present during the initiation of polymerization reaction. Increasing the template concentration in the reaction mixture partly reversed the effect of caffeine. Of the analogs of caffeine tested, only theophylline inhibited AMV DNA polymerase, whereas aminophylline showed no effect.

Inhibition of reverse transcription of 70S and 35S avian myeloblastosis RNAs by nonprimer tRNA's.

We studied the kinetics of the reverse transcription of 70S and 35S RNA of avian myeloblastosis virus in the presence and absence of various tRNA's. All tRNA's inhibited synthesis. tRNA's from Escherichia coli and yeast exhibited a noncompetitive type of inhibition, i.e., they bound reversibly and randomly and did not alter the affinity of the viral RNA for the polymerase. Nonprimer tRNA's obtained from 70S RNA molecules produced a complex pattern of inhibition. The results show that the nonprimer tRNA's which bound to the reverse transcriptase decreased the affinity of the viral RNA for the enzyme. The maximum rate of synthesis with 70S RNA as the template was less than that with 35S RNA, presumably because the former contains nonprimer tRNA's which can interact with the polymerase.

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.

Template-specific requirements for DNA synthesis by the Mason-Pfizer monkey virus DNA polymerase: Unique aspects

The biochemical properties of DNA polymerase purified from Mason-Pfizer monkey virus were studied, with respect to synthetic and natural template-primer utilization. These studies revealed the following new information about the Mason-Pfizer monkey virus enzyme: (a) Mason-Pfizer monkey virus polymerase was found to prefer template : primer molar nucleotide ratios of 2.5–5 : 1 for optimal rates of synthesis with poly(C) · (dG) 12–18 as template-primer. (b) Poly(A)-directed synthesis was stimulated by the addition of low concentrations of inorganic phosphate to the reaction mixture. (c) Poly(2′- O-methylcytidylate), poly(rCm), was the only template studied for which Mn 2+ proved the preferred divalent cation. Combinations of divalent cations stimulated rather than inhibited poly(rCm)-directed poly(dG) synthesis by the Mason-Pfizer monkey virus enzyme. (d) Heteropolymeric regions of rabbit globin mRNA and avian myeloblastosis virus 70 S RNA could be copied by the Mason-Pfizer monkey virus polymerase with oligo(dT), oligo(U) or in the case of avian myeloblastosis virus RNA, endogenous primers. In all such studies, Mg 2+ was the preferred divalent cation and a distinct preference for the DNA primer in the reverse transcription of natural RNAs was observed. These new findings necessitated comparative studies with the DNA polymerases from Rauscher murine leukemia virus and murine mammary tumor virus, as representative type C and type B retroviruses. Although the Mason-Pfizer monkey virus enzyme was found to share some properties in common with both type C and type B mammalian viral enzymes, certain of the above properties rendered it unique among the polymerases examined.

Single-stranded DNA from oncornavirus-infected cells enriched in virus-specific DNA sequences

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.

Avian myeloblastosis virus RNA is terminally redundant: Implications for the mechanism of retrovirus replication

We have determined the terminal heteropolymeric sequences of AMV RNA by the following procedures: first, RNA sequence determination on the 5′ terminal and the poly(A)-linked 3′ terminal T1 oligonucleotides, and second, analysis by the Maxam and Gilbert (1977) method of AMV strong stop DNA and of DNA complementary to the poly(A)-linked T1 oligonucleotide, synthesized with reverse transcriptase and (pdT) 13 as primer. The structure deduced for the 5′ terminal region is (5′)7mGpppG mCCAUUCUACCUCUCACCACAUUG-GUGUGCACCUGGGUUGAUGGCCGGACCGUCGA-UUCCCUGACGACUACGAGCACCUGCAUGAAGC- AGAAGGCUUCAU … Two distinct 3′ terminal sequences were deduced: GCCAUUCUACCUCU-CAAA … A OH and GCCAUUCUACCUCUCAC-CAAA … A OH. The two termini, differing by a C-C-A sequence, may reflect genetic heterogeneity of the AMV stock or, more probably, may be generated at or after RNA transcription. These results demonstrate a terminal redundancy of the hetero polymeric sequence of 16 and 19 nucleotides, respectively. The terminal redundancy allows for mechanisms which involve transfer of the DNA segment synthesized on the 5′ terminal redundant sequence to the 3′ terminal redundant sequence.

Frog oocytes synthesize and completely process the precursor polypeptide to virion structural proteins after microinjection of avian myeloblastosis virus RNA.

After microinjection of Xenopus laevis oocytes with RNA from avian myeloblastosis virus, viral structural proteins p27, p19, p15, and p12 are formed by a sequence of posttranslational cleavages of a high-molecular-weight precursor polypeptide. The 60-70S RNA aggregate or its 30-40S RNA subunits obtained by heat or formamide treatment possess the same ability to serve as template in X. laevis oocytes. The processing pattern of virus-specific precursor polypeptides is the same in X. laevis oocytes as in chick embryo fibroblasts infected with avian myeloblastosis virus, but the processing takes place at a much slower rate.

Synthesis of full-length DNA copies of avian myeloblastosis virus RNA in high yields.

The synthesis of full-length complementary DNA copies of avian myeloblastosis virus RNA is described. The cDNA is estimated by electrophoresis in agarose-methylmercury gels to have a molecular weight of 2.6 X 10(6), equivalent in size to that of the RNA template. Most (60-70%) of the reaction product is the full-length material and yields correspond to 60-70% of the input RNA.

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.

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.

RNA-dependent DNA polymerase activity of RNA tumor virus. VI. Processive mode of action of avian myeloblastosis virus polymerase.

Purified avian myeloblastosis virus (AMV) polymerase consisting of alpha,beta subunits has been shown to act processively in catalyzing DNA synthesis primed with 34S AMV RNA oligo(dT), poly(A)-poly(dT), and poly(I)-poly(dC). DNA transcripts prepared with 34S AMV RNA-oligo(dT)14 and AMV polymerase (alphabeta) have been shown to have a molecular weight of 1.05 X 10(6), or approximately one-third the size of the 34S RNA genome. Polymerase subunit alpha acts nonprocessively with the above templates.