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Journal of virology1979; 29(3); 907-914; doi: 10.1128/JVI.29.3.907-914.1979

Synthesis of long complementary DNA in the endogenous reaction by equine infectious anemia virus.

Abstract: In the endogenous reverse transcriptase reaction, equine infectious anemia virus is able to synthesize complementary DNA (cDNA) of 8,000 nucleotides in high yield. After 2 h in 50 muM dNTP, about 2.8 mug of cDNA per mg of protein is produced, almost 30% of which is long cDNA. The system thus compares favorably with the other two well-characterized endogenous reaction systems, Moloney murine leukemia virus and avian sarcoma virus. Elongation rates of 100 to 150 nucleotides per min have been observed; these rates are comparable to those seen with purified avian myeloblastosis virus reverse transcriptase and significantly higher than those observed in vivo. In the absence of actinomycin D, equine infectious anemia virus does not require high dNTP levels for either optimal incorporation or long cDNA synthesis. The amount of long cDNA synthesized is maximal at 2 h in 50 muM dNTP; neither longer time nor higher dNTP levels (through 1.8 mM) increased this yield. Half-maximum yield in 2 h was achieved at about 15 muM dNTP, which is very similar to the published K(M)'s for isolated avian and murine reverse transcriptases. Total incorporation, on the other hand, continues to rise slowly through 1 mM dNTP; the half-maximum was 30 to 50 muM dNTP. In the presence of 100 mug of actinomycin D per ml, however, higher dNTP levels are required for long cDNA synthesis. We conclude that equine infectious anemia virus is exceptionally well-suited to studies of the physical organization of the retrovirus genome and to investigations of the mechanism of synthesis of the double-standard cDNA endogenous reaction product.
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Publication Date: 1979-03-01 PubMed ID: 87522PubMed Central: PMC353250DOI: 10.1128/JVI.29.3.907-914.1979Google Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

This research investigates the ability of the equine infectious anemia virus to produce a substantial amount of long complementary DNA (cDNA) through the endogenous reverse transcriptase reaction. The research also compares the efficacy of this reaction with that of other well-known virus systems and investigates its potential for future studies related to retrovirus genome organization and mechanisms of cDNA synthesis.

Objective

  • The study explores the endogenous reverse transcriptase reaction by equine infectious anemia virus and its ability to synthesize long cDNA of about 8,000 nucleotides efficiently.

Methodology

  • Experiments were conducted observing the reaction over a period of 2 hours in 50 muM dNTP (deoxyribonucleotide triphosphate).
  • The produced cDNA was evaluated and compared with two other well-characterized endogenous reaction systems, the Moloney murine leukemia virus and the avian sarcoma virus.
  • The researchers also varied the levels of dNTP to analyze its impact on the yield of long cDNA and the total incorporation rates.
  • The impact of the presence of actinomycin D on the reaction was considered by adding 100 mug of it per ml in the process.

Findings

  • In the stipulated conditions, about 2.8 mug of cDNA per mg of protein was produced, of which almost 30% was long cDNA.
  • The elongation rates of the synthesized nucleotides were between 100 to 150 per minute, which were significantly higher than in vivo rates.
  • The absence of actinomycin D did not affect the optimal incorporation or the synthesis of long cDNA despite the elevated dNTP levels.
  • Maximum long cDNA was synthesized within 2 hours in 50 muM dNTP, and increasing the dNTP levels or time didn’t influence the yield.
  • However, the presence of actinomycin D demanded higher dNTP levels for effective long cDNA synthesis.

Implications

  • The research indicates that the equine infectious anemia virus is more efficient for studies related to retrovirus genome structure and the mechanism of synthesis of double-standard cDNA in the endogenous reaction.
  • It also suggests that the virus does not require high dNTP levels for optimal incorporation or long cDNA synthesis, unlike other well-characterized endogenous reaction systems.

Cite This Article

APA
Rice NR, Coggins L. (1979). Synthesis of long complementary DNA in the endogenous reaction by equine infectious anemia virus. J Virol, 29(3), 907-914. https://doi.org/10.1128/JVI.29.3.907-914.1979

Publication

Journal of virology
ISSN: 0022-538X
NlmUniqueID: 0113724
Country: United States
Language: English
Volume: 29
Issue: 3
Pages: 907-914

Researcher Affiliations

Rice, N R
    Coggins, L

      MeSH Terms

      • Cell-Free System
      • DNA, Viral / biosynthesis
      • Dactinomycin / pharmacology
      • Infectious Anemia Virus, Equine / metabolism
      • Nucleotides / metabolism
      • RNA-Directed DNA Polymerase / metabolism

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      Citations

      This article has been cited 3 times.
      1. Boone LR, Skalka AM. Viral DNA synthesized in vitro by avian retrovirus particles permeabilized with melittin. I. Kinetics of synthesis and size of minus- and plus-strand transcripts. J Virol 1981 Jan;37(1):109-16.
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