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. 1994 Dec 11;22(24):5302–5309. doi: 10.1093/nar/22.24.5302

Evolutionarily conserved elements in the 5' untranslated region of beta globin mRNA mediate site-specific priming of a unique hairpin structure during cDNA synthesis.

V Z Volloch 1, B Schweitzer 1, S Rits 1
PMCID: PMC332075  PMID: 7816620

Abstract

Generation of double-stranded cDNA during reverse transcription of a variety of mRNA molecules is well known to involve the formation of covalently linked antisense and sense strands in a hairpin configuration. In the present study we have examined the sequence of molecular events which occurs during cDNA synthesis from mouse beta globin mRNA, in particular the self-priming event that initiates synthesis of sense-strand DNA. Upon completion of reverse transcription of globin mRNA and the removal of RNA template by RNase H activity associated with reverse transcriptase, the 3' end of cDNA snaps back to form a stable double-stranded structure, which is extended by reverse transcriptase to generate the sense DNA strand. Surprisingly, the fourteen 3' terminal nucleotides of the beta globin antisense DNA strand (cDNA) have strong complementarity with an internal segment of the same molecule corresponding to a portion of the 5'-untranslated region of the mRNA located just upstream of the translation start site. Efficient second strand cDNA synthesis appears to require the occurrence within the cDNA molecule of these two complementary elements, one of which must be 3'-terminal. A second surprising feature is that the strong complementarity between the terminal and the internal portions of the molecule exists in the antisense DNA and not in the sense mRNA strand. This is because A:C mismatches on the sense strand correspond to relatively stable T:G base pairs on the antisense strand. Such an extended region of complementarity within the segment of cDNA corresponding to the short 5' untranslated region of beta globin mRNA is unlikely to occur purely by chance, suggesting some underlying function. In this regard it is of interest that cDNAs of adult beta globin mRNAs from other mammalian species show a very similar arrangement of complementary elements, and that complementarity is heavily conserved, even when there are substitutions in nucleotide sequence.

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Selected References

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

  1. Aviv H., Voloch Z., Bastos R., Levy S. Biosynthesis and stability of globin mRNA in cultured erythroleukemic Friend cells. Cell. 1976 Aug;8(4):495–503. doi: 10.1016/0092-8674(76)90217-8. [DOI] [PubMed] [Google Scholar]
  2. Bastos R. N., Volloch Z., Aviv H. Messenger RNA population analysis during erythroid differentiation: a kinetical approach. J Mol Biol. 1977 Feb 25;110(2):191–203. doi: 10.1016/s0022-2836(77)80068-5. [DOI] [PubMed] [Google Scholar]
  3. Blum B., Bakalara N., Simpson L. A model for RNA editing in kinetoplastid mitochondria: "guide" RNA molecules transcribed from maxicircle DNA provide the edited information. Cell. 1990 Jan 26;60(2):189–198. doi: 10.1016/0092-8674(90)90735-w. [DOI] [PubMed] [Google Scholar]
  4. Cooper S. J., Hope R. M. Evolution and expression of a beta-like globin gene of the Australian marsupial Sminthopsis crassicaudata. Proc Natl Acad Sci U S A. 1993 Dec 15;90(24):11777–11781. doi: 10.1073/pnas.90.24.11777. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Efstratiadis A., Kafatos F. C., Maxam A. M., Maniatis T. Enzymatic in vitro synthesis of globin genes. Cell. 1976 Feb;7(2):279–288. doi: 10.1016/0092-8674(76)90027-1. [DOI] [PubMed] [Google Scholar]
  6. Efstratiadis A., Posakony J. W., Maniatis T., Lawn R. M., O'Connell C., Spritz R. A., DeRiel J. K., Forget B. G., Weissman S. M., Slightom J. L. The structure and evolution of the human beta-globin gene family. Cell. 1980 Oct;21(3):653–668. doi: 10.1016/0092-8674(80)90429-8. [DOI] [PubMed] [Google Scholar]
  7. Feagin J. E., Jasmer D. P., Stuart K. Developmentally regulated addition of nucleotides within apocytochrome b transcripts in Trypanosoma brucei. Cell. 1987 May 8;49(3):337–345. doi: 10.1016/0092-8674(87)90286-8. [DOI] [PubMed] [Google Scholar]
  8. Garner K. J., Lingrel J. B. A comparison of the beta A-and beta B-globin gene clusters of sheep. J Mol Evol. 1989 Mar;28(3):175–184. doi: 10.1007/BF02102474. [DOI] [PubMed] [Google Scholar]
  9. Hardison R. C., Butler E. T., 3rd, Lacy E., Maniatis T., Rosenthal N., Efstratiadis A. The structure and transcription of four linked rabbit beta-like globin genes. Cell. 1979 Dec;18(4):1285–1297. doi: 10.1016/0092-8674(79)90239-3. [DOI] [PubMed] [Google Scholar]
  10. Haynes J. R., Rosteck P., Jr, Lingrel J. B. Unusual sequence homology at the 5-ends of the developmentally regulated beta A-, beta C-, and gamma-globin genes of the goat. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7127–7131. doi: 10.1073/pnas.77.12.7127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Houts G. E., Miyagi M., Ellis C., Beard D., Beard J. W. Reverse transcriptase from avian myeloblastosis virus. J Virol. 1979 Feb;29(2):517–522. doi: 10.1128/jvi.29.2.517-522.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kang C., Wu C. W. Studies on SP6 promoter using a new plasmid vector that allows gene insertion at the transcription initiation site. Nucleic Acids Res. 1987 Mar 11;15(5):2279–2294. doi: 10.1093/nar/15.5.2279. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Konkel D. A., Maizel J. V., Jr, Leder P. The evolution and sequence comparison of two recently diverged mouse chromosomal beta--globin genes. Cell. 1979 Nov;18(3):865–873. doi: 10.1016/0092-8674(79)90138-7. [DOI] [PubMed] [Google Scholar]
  14. Kotewicz M. L., Sampson C. M., D'Alessio J. M., Gerard G. F. Isolation of cloned Moloney murine leukemia virus reverse transcriptase lacking ribonuclease H activity. Nucleic Acids Res. 1988 Jan 11;16(1):265–277. doi: 10.1093/nar/16.1.265. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kretschmer P. J., Coon H. C., Davis A., Harrison M., Nienhuis A. W. Hemoglobin switching in sheep. Isolation of the fetal gamma-globin gene and demonstration that the fetal gamma- and adult beta A-globin genes lie within eight kilobase segments of homologous DNA. J Biol Chem. 1981 Feb 25;256(4):1975–1982. [PubMed] [Google Scholar]
  16. Martin S. L., Zimmer E. A., Davidson W. S., Wilson A. C., Kan Y. W. The untranslated regions of beta-globin mRNA evolve at a functional rate in higher primates. Cell. 1981 Sep;25(3):737–741. doi: 10.1016/0092-8674(81)90181-1. [DOI] [PubMed] [Google Scholar]
  17. Myers J. C., Spiegelman S. Sodium pyrophosphate inhibition of RNA.DNA hybrid degradation by reverse transcriptase. Proc Natl Acad Sci U S A. 1978 Nov;75(11):5329–5333. doi: 10.1073/pnas.75.11.5329. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Perrin-Pecontal P., Gouy M., Nigon V. M., Trabuchet G. Evolution of the primate beta-globin gene region: nucleotide sequence of the delta-beta-globin intergenic region of gorilla and phylogenetic relationships between African apes and man. J Mol Evol. 1992 Jan;34(1):17–30. doi: 10.1007/BF00163849. [DOI] [PubMed] [Google Scholar]
  19. Schimenti J. C., Duncan C. H. Ruminant globin gene structures suggest an evolutionary role for Alu-type repeats. Nucleic Acids Res. 1984 Feb 10;12(3):1641–1655. doi: 10.1093/nar/12.3.1641. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Schwarz H., Whitton J. L. A rapid, inexpensive method for eluting DNA from agarose or acrylamide gel slices without using toxic or chaotropic materials. Biotechniques. 1992 Aug;13(2):205–206. [PubMed] [Google Scholar]
  21. Seeburg P. H., Shine J., Martial J. A., Baxter J. D., Goodman H. M. Nucleotide sequence and amplification in bacteria of structural gene for rat growth hormone. Nature. 1977 Dec 8;270(5637):486–494. doi: 10.1038/270486a0. [DOI] [PubMed] [Google Scholar]
  22. Volloch V. Cytoplasmic synthesis of globin RNA in differentiated murine erythroleukemia cells: possible involvement of RNA-dependent RNA polymerase. Proc Natl Acad Sci U S A. 1986 Mar;83(5):1208–1212. doi: 10.1073/pnas.83.5.1208. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Volloch V., Schweitzer B., Rits S. Synthesis of globin RNA in enucleated differentiating murine erythroleukemia cells. J Cell Biol. 1987 Jul;105(1):137–143. doi: 10.1083/jcb.105.1.137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Volloch V., Schweitzer B., Zhang X., Rits S. Identification of negative-strand complements to cytochrome oxidase subunit III RNA in Trypanosoma brucei. Proc Natl Acad Sci U S A. 1991 Dec 1;88(23):10671–10675. doi: 10.1073/pnas.88.23.10671. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Weiss M. A., Patel D. J., Sauer R. T., Karplus M. 1H-NMR study of the lambda operator site OL1: assignment of the imino and adenine H2 resonances. Nucleic Acids Res. 1984 May 11;12(9):4035–4047. doi: 10.1093/nar/12.9.4035. [DOI] [PMC free article] [PubMed] [Google Scholar]

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