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. 1992 Sep;12(9):4242–4248. doi: 10.1128/mcb.12.9.4242

Translation of the rat LINE bicistronic RNAs in vitro involves ribosomal reinitiation instead of frameshifting.

H Ilves 1, O Kahre 1, M Speek 1
PMCID: PMC360333  PMID: 1380649

Abstract

The genomic structure of the rat LINE (L1Rn) DNA element contains two overlapping open reading frames (ORFs) and apparently has a potential to code for a DNA/RNA-binding protein (in ORF1) and a reverse transcriptase (in ORF2). We have characterized a 1,630-bp L1Rn cDNA clone encompassing the overlapping ORFs and a 600-bp genomic fragment derived from a full-length L1Rn member and containing the beginning of ORF1. These DNAs were used to restore in part the ORF1-ORF2 organization of L1Rn after being cloned into the pSP65 vector under the control of SP6 polymerase promoter. To test whether L1Rn ORF1 and ORF2 are expressed as a fusion protein, a series of capped RNAs with progressive truncations containing one or both ORFs were prepared and translated in the rabbit reticulocyte lysate. Our analysis indicates that the expression of a putative reverse transcriptase-encoded L1Rn ORF2 in vitro is regulated by reinitiation or internal initiation of translation but not by ribosomal frameshifting.

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Selected References

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

  1. Benton W. D., Davis R. W. Screening lambdagt recombinant clones by hybridization to single plaques in situ. Science. 1977 Apr 8;196(4286):180–182. doi: 10.1126/science.322279. [DOI] [PubMed] [Google Scholar]
  2. Boeke J. D., Corces V. G. Transcription and reverse transcription of retrotransposons. Annu Rev Microbiol. 1989;43:403–434. doi: 10.1146/annurev.mi.43.100189.002155. [DOI] [PubMed] [Google Scholar]
  3. D'Ambrosio E., Waitzkin S. D., Witney F. R., Salemme A., Furano A. V. Structure of the highly repeated, long interspersed DNA family (LINE or L1Rn) of the rat. Mol Cell Biol. 1986 Feb;6(2):411–424. doi: 10.1128/mcb.6.2.411. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Dasso M. C., Jackson R. J. On the fidelity of mRNA translation in the nuclease-treated rabbit reticulocyte lysate system. Nucleic Acids Res. 1989 Apr 25;17(8):3129–3144. doi: 10.1093/nar/17.8.3129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Deragon J. M., Sinnett D., Labuda D. Reverse transcriptase activity from human embryonal carcinoma cells NTera2D1. EMBO J. 1990 Oct;9(10):3363–3368. doi: 10.1002/j.1460-2075.1990.tb07537.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Di Nocera P. P., Sakaki Y. LINEs: a superfamily of retrotransposable ubiquitous DNA elements. Trends Genet. 1990 Feb;6(2):29–30. doi: 10.1016/0168-9525(90)90051-7. [DOI] [PubMed] [Google Scholar]
  7. Doolittle R. F., Feng D. F., Johnson M. S., McClure M. A. Origins and evolutionary relationships of retroviruses. Q Rev Biol. 1989 Mar;64(1):1–30. doi: 10.1086/416128. [DOI] [PubMed] [Google Scholar]
  8. Eckert K. A., Kunkel T. A. High fidelity DNA synthesis by the Thermus aquaticus DNA polymerase. Nucleic Acids Res. 1990 Jul 11;18(13):3739–3744. doi: 10.1093/nar/18.13.3739. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fanning T. G., Singer M. F. LINE-1: a mammalian transposable element. Biochim Biophys Acta. 1987 Dec 8;910(3):203–212. doi: 10.1016/0167-4781(87)90112-6. [DOI] [PubMed] [Google Scholar]
  10. Fanning T., Singer M. The LINE-1 DNA sequences in four mammalian orders predict proteins that conserve homologies to retrovirus proteins. Nucleic Acids Res. 1987 Mar 11;15(5):2251–2260. doi: 10.1093/nar/15.5.2251. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hattori M., Kuhara S., Takenaka O., Sakaki Y. L1 family of repetitive DNA sequences in primates may be derived from a sequence encoding a reverse transcriptase-related protein. Nature. 1986 Jun 5;321(6070):625–628. doi: 10.1038/321625a0. [DOI] [PubMed] [Google Scholar]
  12. Kozak M. An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 1987 Oct 26;15(20):8125–8148. doi: 10.1093/nar/15.20.8125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kozak M. Context effects and inefficient initiation at non-AUG codons in eucaryotic cell-free translation systems. Mol Cell Biol. 1989 Nov;9(11):5073–5080. doi: 10.1128/mcb.9.11.5073. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kozak M. Effects of intercistronic length on the efficiency of reinitiation by eucaryotic ribosomes. Mol Cell Biol. 1987 Oct;7(10):3438–3445. doi: 10.1128/mcb.7.10.3438. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kozak M. Evaluation of the fidelity of initiation of translation in reticulocyte lysates from commercial sources. Nucleic Acids Res. 1990 May 11;18(9):2828–2828. doi: 10.1093/nar/18.9.2828. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kozak M. The scanning model for translation: an update. J Cell Biol. 1989 Feb;108(2):229–241. doi: 10.1083/jcb.108.2.229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  18. Leibold D. M., Swergold G. D., Singer M. F., Thayer R. E., Dombroski B. A., Fanning T. G. Translation of LINE-1 DNA elements in vitro and in human cells. Proc Natl Acad Sci U S A. 1990 Sep;87(18):6990–6994. doi: 10.1073/pnas.87.18.6990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Loeb D. D., Padgett R. W., Hardies S. C., Shehee W. R., Comer M. B., Edgell M. H., Hutchison C. A., 3rd The sequence of a large L1Md element reveals a tandemly repeated 5' end and several features found in retrotransposons. Mol Cell Biol. 1986 Jan;6(1):168–182. doi: 10.1128/mcb.6.1.168. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Martin S. L. Ribonucleoprotein particles with LINE-1 RNA in mouse embryonal carcinoma cells. Mol Cell Biol. 1991 Sep;11(9):4804–4807. doi: 10.1128/mcb.11.9.4804. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Mathias S. L., Scott A. F., Kazazian H. H., Jr, Boeke J. D., Gabriel A. Reverse transcriptase encoded by a human transposable element. Science. 1991 Dec 20;254(5039):1808–1810. doi: 10.1126/science.1722352. [DOI] [PubMed] [Google Scholar]
  22. Melton D. A., Krieg P. A., Rebagliati M. R., Maniatis T., Zinn K., Green M. R. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 1984 Sep 25;12(18):7035–7056. doi: 10.1093/nar/12.18.7035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Mullis K. B., Faloona F. A. Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol. 1987;155:335–350. doi: 10.1016/0076-6879(87)55023-6. [DOI] [PubMed] [Google Scholar]
  24. Poch O., Sauvaget I., Delarue M., Tordo N. Identification of four conserved motifs among the RNA-dependent polymerase encoding elements. EMBO J. 1989 Dec 1;8(12):3867–3874. doi: 10.1002/j.1460-2075.1989.tb08565.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Saiki R. K., Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R., Horn G. T., Mullis K. B., Erlich H. A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. doi: 10.1126/science.2448875. [DOI] [PubMed] [Google Scholar]
  26. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Scott A. F., Schmeckpeper B. J., Abdelrazik M., Comey C. T., O'Hara B., Rossiter J. P., Cooley T., Heath P., Smith K. D., Margolet L. Origin of the human L1 elements: proposed progenitor genes deduced from a consensus DNA sequence. Genomics. 1987 Oct;1(2):113–125. doi: 10.1016/0888-7543(87)90003-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Shehee W. R., Chao S. F., Loeb D. D., Comer M. B., Hutchison C. A., 3rd, Edgell M. H. Determination of a functional ancestral sequence and definition of the 5' end of A-type mouse L1 elements. J Mol Biol. 1987 Aug 20;196(4):757–767. doi: 10.1016/0022-2836(87)90402-5. [DOI] [PubMed] [Google Scholar]
  29. Skowronski J., Fanning T. G., Singer M. F. Unit-length line-1 transcripts in human teratocarcinoma cells. Mol Cell Biol. 1988 Apr;8(4):1385–1397. doi: 10.1128/mcb.8.4.1385. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Skowronski J., Singer M. F. Expression of a cytoplasmic LINE-1 transcript is regulated in a human teratocarcinoma cell line. Proc Natl Acad Sci U S A. 1985 Sep;82(18):6050–6054. doi: 10.1073/pnas.82.18.6050. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Speek M., Ilves H., Lind A. Consecutive DNA terminator sequencing by using enzymatically generated primers. Anal Biochem. 1986 Nov 1;158(2):242–249. doi: 10.1016/0003-2697(86)90544-0. [DOI] [PubMed] [Google Scholar]
  32. Swergold G. D. Identification, characterization, and cell specificity of a human LINE-1 promoter. Mol Cell Biol. 1990 Dec;10(12):6718–6729. doi: 10.1128/mcb.10.12.6718. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Varmus H. Retroviruses. Science. 1988 Jun 10;240(4858):1427–1435. doi: 10.1126/science.3287617. [DOI] [PubMed] [Google Scholar]
  34. Xiong Y., Eickbush T. H. Similarity of reverse transcriptase-like sequences of viruses, transposable elements, and mitochondrial introns. Mol Biol Evol. 1988 Nov;5(6):675–690. doi: 10.1093/oxfordjournals.molbev.a040521. [DOI] [PubMed] [Google Scholar]
  35. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  36. Zabarovsky E. R., Allikmets R. L. An improved technique for the efficient construction of gene libraries by partial filling-in of cohesive ends. Gene. 1986;42(1):119–123. doi: 10.1016/0378-1119(86)90158-7. [DOI] [PubMed] [Google Scholar]

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