Abstract
This chapter describes the purification of RNA-dependent DNA polymerase from avian myeloblastosis virus (AMV). The reverse transcriptase has been purified from AMV approximately 80- to 100-fold over the activity detected in crude ammonium sulfate fractions, yielding preparations capable of incorporating into DNA more than 3.1 μmoles of deoxynucleotide per milligram of protein in 30 minutes. During the course of this purification, the yield of DNA synthesized in reactions primed with AMV RNA decreased with each purification step. A small molecular weight protein, referred to as stimulatory protein, can be isolated during the phosphocellulose chromatography step. When added to purified polymerase preparations, this protein increases the yield of RNA-primed DNA synthesis. The stimulatory protein increases the rate and yield of DNA synthesized in reactions containing viral RNA and purified viral polymerase. The amount of stimulatory protein giving optimal stimulation must be determined with each system because high concentrations are inhibitory.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 143-150 |
| Number of pages | 8 |
| Journal | Methods in enzymology |
| Volume | 29 |
| Issue number | C |
| DOIs | |
| State | Published - Jan 1 1974 |
Funding
scribing viral RNA into DNA. a,4 In the presence of four deoxynucleoside triphosphates and magnesium, these polymerases catalyze repair-type reactions on RNA, DNA, or RNA-DNA hybrids as shown above. Deoxy-nucleotide incorporation occurs from the 3'-hydroxyl end of primer strands attached to template strands which direct DNA synthesis yielding DNA products covalently linked to primer strands. 5-9 The reverse transcriptase has now been purified from avian myeloblastosis virus, 6,7,1° Rous sarcoma virus, n,12 and Rauscher leukemia virus. 5 1 This study was conducted under Public Health Service Contract 71-2251 within the Special Virus Cancer Program of the National Cancer Institute, Research Grant from the National Institute of General Medical Sciences GM-13344, and the American Cancer Society Grant P561-B. Postdoctoral FeUow of the Damon Runyon Cancer Foundation (DRF-659). 3 D. Baltimore, Nature (London) 226, 1209 (1970). 4 H. M. Temin and S. N\[izutani, Nature (London) 226, 1211 (1970). J. Hurwitz and J. Leis, J. Virol. 9, 116 (1972). e j. Leis and J. Hurwitz, J. Virol. 9, 130 (1972). P. Duesberg, E. Canaani, and K. V. D. Helm, Proc. Nat. Acad. Sci. U.S. 68, 2505 (1971). s D. Smoler, I. Molineux, and D. Baltimore, J. Biol. Chem. 246, 7697 (1971). 9 I. M. Verma, N. L. Meuth, E. Bromfeld, K. F. Manly, and D. Baltimore, Nature (London) 233, 131 (1971). 10 D. L. Kacian, K. F. Watson, A. Burny, and S. Spiegelman, Biochim. Biophys. Acta 246, 365 (1971).
ASJC Scopus subject areas
- Molecular Biology
- Biochemistry