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. 1981 Oct;78(10):6091–6095. doi: 10.1073/pnas.78.10.6091

Synthesis of stable unspliced mRNA from an intronless simian virus 40--rat preproinsulin gene recombinant.

P Gruss, A Efstratiadis, S Karathanasis, M König, G Khoury
PMCID: PMC348983  PMID: 6171810

Abstract

We constructed a recombinant DNA molecule carrying the coding region and the 3' flanking sequences of the rat preproinsulin I gene, replacing the late region of a simian virus 40 (SV40) viral vector. The sequences making up the single intron of the preproinsulin I gene, located within the nontranslated leader region, as well as all of the 5' flanking sequences, have been deleted. The sequences remaining within the late transcriptional unit of SV40 do not retain a functional intron. This "intronless" mutant nevertheless directs the synthesis of substantial quantities of unspliced stable hybrid transcripts that are translated into polypeptides with insulin immunoreactivity. Two novel 5' termini, not used in wild-type SV40 transcripts, have been detected in the majority of the hybrid RNA molecules. These are located at analogous positions in both of the late SV40 72-base-pair repeats.

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Selected References

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  1. Aleström P., Akusjärvi G., Perricaudet M., Mathews M. B., Klessig D. F., Pettersson U. The gene for polypeptide IX of adenovirus type 2 and its unspliced messenger RNA. Cell. 1980 Mar;19(3):671–681. doi: 10.1016/s0092-8674(80)80044-4. [DOI] [PubMed] [Google Scholar]
  2. Benoist C., Chambon P. In vivo sequence requirements of the SV40 early promotor region. Nature. 1981 Mar 26;290(5804):304–310. doi: 10.1038/290304a0. [DOI] [PubMed] [Google Scholar]
  3. Berk A. J., Sharp P. A. Sizing and mapping of early adenovirus mRNAs by gel electrophoresis of S1 endonuclease-digested hybrids. Cell. 1977 Nov;12(3):721–732. doi: 10.1016/0092-8674(77)90272-0. [DOI] [PubMed] [Google Scholar]
  4. Berk A. J., Sharp P. A. Spliced early mRNAs of simian virus 40. Proc Natl Acad Sci U S A. 1978 Mar;75(3):1274–1278. doi: 10.1073/pnas.75.3.1274. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brockman W. W., Nathans D. The isolation of simian virus 40 variants with specifically altered genomes. Proc Natl Acad Sci U S A. 1974 Mar;71(3):942–946. doi: 10.1073/pnas.71.3.942. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Casey J., Davidson N. Rates of formation and thermal stabilities of RNA:DNA and DNA:DNA duplexes at high concentrations of formamide. Nucleic Acids Res. 1977;4(5):1539–1552. doi: 10.1093/nar/4.5.1539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Ghosh P. K., Lebowitz P., Frisque R. J., Gluzman Y. Identification of a promoter component involved in positioning the 5' termini of simian virus 40 early mRNAs. Proc Natl Acad Sci U S A. 1981 Jan;78(1):100–104. doi: 10.1073/pnas.78.1.100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Ghosh P. K., Reddy V. B., Swinscoe J., Choudary P. V., Lebowitz P., Weissman S. M. The 5'-terminal leader sequence of late 16 S mRNA from cells infected with simian virus 40. J Biol Chem. 1978 May 25;253(10):3643–3647. [PubMed] [Google Scholar]
  9. Ghosh P. K., Reddy V. B., Swinscoe J., Lebowitz P., Weissman S. M. Heterogeneity and 5'-terminal structures of the late RNAs of simian virus 40. J Mol Biol. 1978 Dec 25;126(4):813–846. doi: 10.1016/0022-2836(78)90022-0. [DOI] [PubMed] [Google Scholar]
  10. Ghosh P. K., Roy P., Barkan A., Mertz J. E., Weissman S. M., Lebowitz P. Unspliced functional late 19S mRNAs containing intervening sequences are produced by a late leader mutant of simian virus 40. Proc Natl Acad Sci U S A. 1981 Mar;78(3):1386–1390. doi: 10.1073/pnas.78.3.1386. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Goff S. P., Berg P. Construction of hybrid viruses containing SV40 and lambda phage DNA segments and their propagation in cultured monkey cells. Cell. 1976 Dec;9(4 Pt 2):695–705. doi: 10.1016/0092-8674(76)90133-1. [DOI] [PubMed] [Google Scholar]
  12. Goldberg D. A. Isolation and partial characterization of the Drosophila alcohol dehydrogenase gene. Proc Natl Acad Sci U S A. 1980 Oct;77(10):5794–5798. doi: 10.1073/pnas.77.10.5794. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gruss P., Dhar R., Khoury G. Simian virus 40 tandem repeated sequences as an element of the early promoter. Proc Natl Acad Sci U S A. 1981 Feb;78(2):943–947. doi: 10.1073/pnas.78.2.943. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gruss P., Khoury G. Expression of simian virus 40-rat preproinsulin recombinants in monkey kidney cells: use of preproinsulin RNA processing signals. Proc Natl Acad Sci U S A. 1981 Jan;78(1):133–137. doi: 10.1073/pnas.78.1.133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gruss P., Khoury G. Rescue of a splicing defective mutant by insertion of an heterologous intron. Nature. 1980 Aug 7;286(5773):634–637. doi: 10.1038/286634a0. [DOI] [PubMed] [Google Scholar]
  16. Gruss P., Lai C. J., Dhar R., Khoury G. Splicing as a requirement for biogenesis of functional 16S mRNA of simian virus 40. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4317–4321. doi: 10.1073/pnas.76.9.4317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hamer D. H., Leder P. Splicing and the formation of stable RNA. Cell. 1979 Dec;18(4):1299–1302. doi: 10.1016/0092-8674(79)90240-x. [DOI] [PubMed] [Google Scholar]
  18. Hamer D. H., Smith K. D., Boyer S. H., Leder P. SV40 recombinants carrying rabbit beta-globin gene coding sequences. Cell. 1979 Jul;17(3):725–735. doi: 10.1016/0092-8674(79)90279-4. [DOI] [PubMed] [Google Scholar]
  19. Jay G., Nomura S., Anderson C. W., Khoury G. Identification of the SV40 agnogene product: a DNA binding protein. Nature. 1981 May 28;291(5813):346–349. doi: 10.1038/291346a0. [DOI] [PubMed] [Google Scholar]
  20. Lai C. J., Dhar R., Khoury G. Mapping the spliced and unspliced late lytic SV40 RNAs. Cell. 1978 Aug;14(4):971–982. doi: 10.1016/0092-8674(78)90351-3. [DOI] [PubMed] [Google Scholar]
  21. Lai C. J., Khoury G. Deletion mutants of simian virus 40 defective in biosynthesis of late viral mRNA. Proc Natl Acad Sci U S A. 1979 Jan;76(1):71–75. doi: 10.1073/pnas.76.1.71. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lawn R. M., Adelman J., Franke A. E., Houck C. M., Gross M., Najarian R., Goeddel D. V. Human fibroblast interferon gene lacks introns. Nucleic Acids Res. 1981 Mar 11;9(5):1045–1052. doi: 10.1093/nar/9.5.1045. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lomedico P., Rosenthal N., Efstratidadis A., Gilbert W., Kolodner R., Tizard R. The structure and evolution of the two nonallelic rat preproinsulin genes. Cell. 1979 Oct;18(2):545–558. doi: 10.1016/0092-8674(79)90071-0. [DOI] [PubMed] [Google Scholar]
  24. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  25. McKnight S. L. The nucleotide sequence and transcript map of the herpes simplex virus thymidine kinase gene. Nucleic Acids Res. 1980 Dec 20;8(24):5949–5964. doi: 10.1093/nar/8.24.5949. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Nagata S., Mantei N., Weissmann C. The structure of one of the eight or more distinct chromosomal genes for human interferon-alpha. Nature. 1980 Oct 2;287(5781):401–408. doi: 10.1038/287401a0. [DOI] [PubMed] [Google Scholar]
  27. Penman S. RNA metabolism in the HeLa cell nucleus. J Mol Biol. 1966 May;17(1):117–130. doi: 10.1016/s0022-2836(66)80098-0. [DOI] [PubMed] [Google Scholar]
  28. Schaffner W., Gross K., Telford J., Birnstiel M. Molecular analysis of the histone gene cluster of psammechinus miliaris: II. The arrangement of the five histone-coding and spacer sequences. Cell. 1976 Aug;8(4):471–478. doi: 10.1016/0092-8674(76)90214-2. [DOI] [PubMed] [Google Scholar]
  29. Tegtmeyer P., Ozer H. L. Temperature-sensitive mutants of simian virus 40: infection of permissive cells. J Virol. 1971 Oct;8(4):516–524. doi: 10.1128/jvi.8.4.516-524.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Villa-Komaroff L., Efstratiadis A., Broome S., Lomedico P., Tizard R., Naber S. P., Chick W. L., Gilbert W. A bacterial clone synthesizing proinsulin. Proc Natl Acad Sci U S A. 1978 Aug;75(8):3727–3731. doi: 10.1073/pnas.75.8.3727. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Wagner M. J., Sharp J. A., Summers W. C. Nucleotide sequence of the thymidine kinase gene of herpes simplex virus type 1. Proc Natl Acad Sci U S A. 1981 Mar;78(3):1441–1445. doi: 10.1073/pnas.78.3.1441. [DOI] [PMC free article] [PubMed] [Google Scholar]

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