Skip to main content
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1977 Oct;4(10):3455–3471. doi: 10.1093/nar/4.10.3455

The syntheiss of high yields of full-length reverse transcripts of globin mRNA.

E Y Friedman, M Rosbash
PMCID: PMC342664  PMID: 73163

Abstract

Conditions have been determined under which reverse transcriptase catalyzes the synthesis of the high yields of full length complementary deoxyribonucleic acid (cDNA). These conditions depend not only on the cencentration of deoxynucleoside triphosphates (1) but also on the concentration of reverse transcriptase. An analysis of the kinetics of cDNA synthesis and the size of cDNA synthesized as a function of time under different conditions indicates that the mechanism of action of reverse transcriptase is partially distributive. This accounts for the necessity of a high enzyme concentration to obtain high yields of full length cDNA. Additional experiments indicate that the yield of cDNA is limited by the fact that the template mRNA is rapidly inactivated. This is most likely due to the fact that the product cDNA is hydrogen bonded to the template mRNA during synthesis.

Full text

PDF
3455

Images in this article

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Baltimore D., Smoler D. Primer requirement and template specificity of the DNA polymerase of RNA tumor viruses. Proc Natl Acad Sci U S A. 1971 Jul;68(7):1507–1511. doi: 10.1073/pnas.68.7.1507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bank A., Terada M., Metafora S., Dow L., Marks P. A. In vitro synthesis of DNA components of human genes for globins. Nat New Biol. 1972 Feb 9;235(58):167–169. doi: 10.1038/newbio235167a0. [DOI] [PubMed] [Google Scholar]
  3. Bishop J. O., Morton J. G., Rosbash M., Richardson M. Three abundance classes in HeLa cell messenger RNA. Nature. 1974 Jul 19;250(463):199–204. doi: 10.1038/250199a0. [DOI] [PubMed] [Google Scholar]
  4. Dube D. K., Loeb L. A. On the association of reverse transcriptase with polynucleotide templates during catalysis. Biochemistry. 1976 Aug 10;15(16):3605–3611. doi: 10.1021/bi00661a031. [DOI] [PubMed] [Google Scholar]
  5. Efstratiadis A., Maniatis T., Kafatos F. C., Jeffrey A., Vournakis J. N. Full length and discrete partial reverse transcripts of globin and chorion mRNAs. Cell. 1975 Apr;4(4):367–378. doi: 10.1016/0092-8674(75)90157-9. [DOI] [PubMed] [Google Scholar]
  6. Faust C. H., Jr, Diggelmann H., Mach B. Isolation of poly(adenylic acid)-rich ribonucleic acid from mouse myeloma and synthesis of complementary deoxyribonucleic acid. Biochemistry. 1973 Feb 27;12(5):925–931. doi: 10.1021/bi00729a021. [DOI] [PubMed] [Google Scholar]
  7. Harrison P. R., Birnie G. D., Hell A., Humphries S., Young B. D., Paul J. Kinetic studies of gene frequency. I. Use of a DNA copy of reticulocyte 9 S RNA to estimate globin gene dosage in mouse tissues. J Mol Biol. 1974 Apr 25;84(4):539–554. doi: 10.1016/0022-2836(74)90115-6. [DOI] [PubMed] [Google Scholar]
  8. Hereford L. M., Rosbash M. Number and distribution of polyadenylated RNA sequences in yeast. Cell. 1977 Mar;10(3):453–462. doi: 10.1016/0092-8674(77)90032-0. [DOI] [PubMed] [Google Scholar]
  9. Higuchi R., Paddock G. V., Wall R., Salser W. A general method for cloning eukaryotic structural gene sequences. Proc Natl Acad Sci U S A. 1976 Sep;73(9):3146–3150. doi: 10.1073/pnas.73.9.3146. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Imaizumi T., Diggelmann H., Scherrer K. Demonstration of globin messenger sequences in giant nuclear precursors of messenger RNA of avian erythroblasts. Proc Natl Acad Sci U S A. 1973 Apr;70(4):1122–1126. doi: 10.1073/pnas.70.4.1122. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Leis J. P. RNA-dependent DNA polymerase activity of RNA tumor virus. VI. Processive mode of action of avian myeloblastosis virus polymerase. J Virol. 1976 Sep;19(3):932–939. doi: 10.1128/jvi.19.3.932-939.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Maniatis T., Jeffrey A., van deSande H. Chain length determination of small double- and single-stranded DNA molecules by polyacrylamide gel electrophoresis. Biochemistry. 1975 Aug 26;14(17):3787–3794. doi: 10.1021/bi00688a010. [DOI] [PubMed] [Google Scholar]
  13. Maniatis T., Kee S. G., Efstratiadis A., Kafatos F. C. Amplification and characterization of a beta-globin gene synthesized in vitro. Cell. 1976 Jun;8(2):163–182. doi: 10.1016/0092-8674(76)90001-5. [DOI] [PubMed] [Google Scholar]
  14. Marotta C. A., Forget B. G., Weissman S. M., Verma I. M., McCaffrey R. P., Baltimore D. Nucleotide sequences of human globin messenger RNA. Proc Natl Acad Sci U S A. 1974 Jun;71(6):2300–2304. doi: 10.1073/pnas.71.6.2300. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. McClure W. R., Jovin T. M. The steady state kinetic parameters and non-processivity of Escherichia coli deoxyribonucleic acid polymerase I. J Biol Chem. 1975 Jun 10;250(11):4073–4080. [PubMed] [Google Scholar]
  16. Monahan J. J., Harris S. E., Woo S. L., Robberson D. L., O'Malley B. W. The synthesis and properties of the complete complementary DNA transcript of ovalbumin mRNA. Biochemistry. 1976 Jan 13;15(1):223–233. doi: 10.1021/bi00646a034. [DOI] [PubMed] [Google Scholar]
  17. Mölling K., Bolognesi D. P., Bauer H., Büsen W., Plassmann H. W., Hausen P. Association of viral reverse transcriptase with an enzyme degrading the RNA moiety of RNA-DNA hybrids. Nat New Biol. 1971 Dec 22;234(51):240–243. doi: 10.1038/newbio234240a0. [DOI] [PubMed] [Google Scholar]
  18. Poon R., Paddock G. V., Heindell H., Whitcome P., Salser W., Kacian D., Bank A., Gambino R., Ramirez F. Nucleotide sequence analysis of RNA synthesized from rabbit globin complementary DNA. Proc Natl Acad Sci U S A. 1974 Sep;71(9):3502–3506. doi: 10.1073/pnas.71.9.3502. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Proudfoot N. J., Brownlee G. G. Sequence at the 3' end of globin mRNA shows homology with immunoglobulin light chain mRNA. Nature. 1974 Nov 29;252(5482):359–362. doi: 10.1038/252359a0. [DOI] [PubMed] [Google Scholar]
  20. Rabbitts T. H. Bacterial cloning of plasmids carrying copies of rabbit globin messenger RNA. Nature. 1976 Mar 18;260(5548):221–225. doi: 10.1038/260221a0. [DOI] [PubMed] [Google Scholar]
  21. Ross J., Aviv H., Scolnick E., Leder P. In vitro synthesis of DNA complementary to purified rabbit globin mRNA (RNA-dependent DNA polymerase-reticulocyte-hemoglobin-density gradient centrifugation-oligo(dT) primer). Proc Natl Acad Sci U S A. 1972 Jan;69(1):264–268. doi: 10.1073/pnas.69.1.264. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Rougeon F., Kourilsky P., Mach B. Insertion of a rabbit beta-globin gene sequence into an E. coli plasmid. Nucleic Acids Res. 1975 Dec;2(12):2365–2378. doi: 10.1093/nar/2.12.2365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Verma I. M., Temple G. F., Fan H., Baltimore D. In vitro synthesis of DNA complementary to rabbit reticulocyte 10S RNA. Nat New Biol. 1972 Feb 9;235(58):163–167. doi: 10.1038/newbio235163a0. [DOI] [PubMed] [Google Scholar]
  24. Weiss G. B., Wilson G. N., Steggles A. W., Anderson W. F. Importance of full size complementary DNA in nucleic acid hybridization. J Biol Chem. 1976 Jun 10;251(11):3425–3431. [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES