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. 1984 Oct;52(1):255–265. doi: 10.1128/jvi.52.1.255-265.1984

Transcription of overlapping sets of RNAs from the genome of Autographa californica nuclear polyhedrosis virus: a novel method for mapping RNAs.

H Lübbert, W Doerfler
PMCID: PMC254513  PMID: 6090702

Abstract

The insect baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) contains a double-stranded, supercoiled circular genome of 126 to 129 kilobase pairs in length. In cultured Spodoptera frugiperda insect cells the virus replications, and early and late phases of viral genome expression are discernible. We previously mapped 5 early and at least 32 late different viral polypeptides on the viral genome (H. Esche, H. Lübbert, B. Siegmann, and W. Doerfler, EMBO J. 1:1629-1633, 1982). However, at the same time we located 11 early and more than 90 late different size-classes of viral RNA on the AcNPV genome. Evidence for extensive RNA splicing in this virus system has not been adduced, although tiny splices cannot yet be ruled out (H. Lübbert and W. Doerfler, J. Virol. 50:497-506, 1984). The large number of AcNPV transcripts and the apparent lack of splicing have raised tantalizing questions about the mechanisms involved in the expression of AcNPV DNA and its regulation. It is also unknown how the widely differing numbers of RNAs and polypeptides can be correlated. For this reason, we have started to analyze in detail the map locations of some of the RNA size-classes in three different segments of the viral genome. For this purpose a novel method has been devised which will prove useful for the analyses of transcriptional patterns in complex viral genomes. The EcoRI fragments J, O through F, and Q, comprising viral DNA segments between 81.8 and 86.4, 32.6 and 41.0, and 88.2 and 89.7 map units, respectively, were investigated. Surprisingly, overlapping sets of viral RNAs of various lengths and with apparently common 3' termini in EcoRI fragments J (seven size-classes) and O through F (four size-classes) or with common 5' termini in EcoRI fragment Q (two size-classes) have been detected. At present, the functional significance of this mode of transcription is unknown. EcoRI fragment Q of AcNPV DNA encodes a 10,000-molecular-weight polypeptide which is expressed abundantly late after infection. The function of this protein has not yet been elucidated. The promoter and 5' part of the gene for the 10,000-molecular-weight polypeptide have been sequenced, and we have shown that at least two RNAs of different lengths are transcribed in this region and initiated at one site of three nucleotides. Studies on the expression of the AcNPV genome have revealed interesting properties not commonly found in other eucaryotic systems.

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Selected References

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

  1. Adang M. J., Miller L. K. Molecular cloning of DNA complementary to mRNA of the baculovirus Autographa californica nuclear polyhedrosis virus: location and gene products of RNA transcripts found late in infection. J Virol. 1982 Dec;44(3):782–793. doi: 10.1128/jvi.44.3.782-793.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Anderson K. P., Frink R. J., Devi G. B., Gaylord B. H., Costa R. H., Wagner E. K. Detailed characterization of the mRNA mapping in the HindIII fragment K region of the herpes simplex virus type 1 genome. J Virol. 1981 Mar;37(3):1011–1027. doi: 10.1128/jvi.37.3.1011-1027.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Arif B. M., Doerfler W. Identification and localization of reiterated sequences in the Choristoneura fumiferana MNPV genome. EMBO J. 1984 Mar;3(3):525–529. doi: 10.1002/j.1460-2075.1984.tb01841.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Aviv H., Leder P. Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose. Proc Natl Acad Sci U S A. 1972 Jun;69(6):1408–1412. doi: 10.1073/pnas.69.6.1408. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bajszár G., Wittek R., Weir J. P., Moss B. Vaccinia virus thymidine kinase and neighboring genes: mRNAs and polypeptides of wild-type virus and putative nonsense mutants. J Virol. 1983 Jan;45(1):62–72. doi: 10.1128/jvi.45.1.62-72.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Benyajati C., Spoerel N., Haymerle H., Ashburner M. The messenger RNA for alcohol dehydrogenase in Drosophila melanogaster differs in its 5' end in different developmental stages. Cell. 1983 May;33(1):125–133. doi: 10.1016/0092-8674(83)90341-0. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Breathnach R., Chambon P. Organization and expression of eucaryotic split genes coding for proteins. Annu Rev Biochem. 1981;50:349–383. doi: 10.1146/annurev.bi.50.070181.002025. [DOI] [PubMed] [Google Scholar]
  9. Carstens E. B., Tjia S. T., Doerfler W. Infection of Spodoptera frugiperda cells with Autographa californica nuclear polyhedrosis virus I. Synthesis of intracellular proteins after virus infection. Virology. 1979 Dec;99(2):386–398. doi: 10.1016/0042-6822(79)90017-5. [DOI] [PubMed] [Google Scholar]
  10. Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
  11. Cochran M. A., Carstens E. B., Eaton B. T., Faulkner P. Molecular Cloning and Physical Mapping of Restriction Endonuclease Fragments of Autographa californica Nuclear Polyhedrosis Virus DNA. J Virol. 1982 Mar;41(3):940–946. doi: 10.1128/jvi.41.3.940-946.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Cochran M. A., Faulkner P. Location of Homologous DNA Sequences Interspersed at Five Regions in the Baculovirus AcMNPV Genome. J Virol. 1983 Mar;45(3):961–970. doi: 10.1128/jvi.45.3.961-970.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Dierks P., van Ooyen A., Mantei N., Weissmann C. DNA sequences preceding the rabbit beta-globin gene are required for formation in mouse L cells of beta-globin RNA with the correct 5' terminus. Proc Natl Acad Sci U S A. 1981 Mar;78(3):1411–1415. doi: 10.1073/pnas.78.3.1411. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Doerfler W., Stabel S., Ibelgaufts H., Sutter D., Neumann R., Groneberg J., Scheidtmann K. H., Deuring R., Winterhoff U. Selectivity in integration sites of adenoviral DNA. Cold Spring Harb Symp Quant Biol. 1980;44(Pt 1):551–564. doi: 10.1101/sqb.1980.044.01.057. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Eick D., Fritz H. J., Doerfler W. Quantitative determination of 5-methylcytosine in DNA by reverse-phase high-performance liquid chromatography. Anal Biochem. 1983 Nov;135(1):165–171. doi: 10.1016/0003-2697(83)90746-7. [DOI] [PubMed] [Google Scholar]
  17. Esche H., Lübbert H., Siegmann B., Doerfler W. The translational map of the Autographa californica nuclear polyhedrosis virus (AcNPV) genome. EMBO J. 1982;1(12):1629–1633. doi: 10.1002/j.1460-2075.1982.tb01365.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Favaloro J., Treisman R., Kamen R. Transcription maps of polyoma virus-specific RNA: analysis by two-dimensional nuclease S1 gel mapping. Methods Enzymol. 1980;65(1):718–749. doi: 10.1016/s0076-6879(80)65070-8. [DOI] [PubMed] [Google Scholar]
  19. Klenow H., Overgaard-Hansen K., Patkar S. A. Proteolytic cleavage fo native DNA polymerase into two different catalytic fragments. Influence of assay condtions on the change of exonuclease activity and polymerase activity accompanying cleavage. Eur J Biochem. 1971 Oct 14;22(3):371–381. doi: 10.1111/j.1432-1033.1971.tb01554.x. [DOI] [PubMed] [Google Scholar]
  20. Kozlov E. A., Levitina T. L., Sidorova N. M., Radavski Y. L., Serebryani S. B. Comparative chemical studies of the polyherdral proteins of the nuclear polyhedrosis viruses of Bombyx mori and Galleria mellonella. J Invertebr Pathol. 1975 Jan;25(1):103–107. doi: 10.1016/0022-2011(75)90289-x. [DOI] [PubMed] [Google Scholar]
  21. LEHMAN I. R., BESSMAN M. J., SIMMS E. S., KORNBERG A. Enzymatic synthesis of deoxyribonucleic acid. I. Preparation of substrates and partial purification of an enzyme from Escherichia coli. J Biol Chem. 1958 Jul;233(1):163–170. [PubMed] [Google Scholar]
  22. Lübbert H., Kruczek I., Tjia S., Doerfler W. The cloned EcoRI fragments of Autographa californica nuclear polyhedrosis virus DNA. Gene. 1981 Dec;16(1-3):343–345. doi: 10.1016/0378-1119(81)90092-5. [DOI] [PubMed] [Google Scholar]
  23. Maxam A. M., Gilbert W. A new method for sequencing DNA. Proc Natl Acad Sci U S A. 1977 Feb;74(2):560–564. doi: 10.1073/pnas.74.2.560. [DOI] [PMC free article] [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. Miller D. W., Miller L. K. A virus mutant with an insertion of a copia-like transposable element. Nature. 1982 Oct 7;299(5883):562–564. doi: 10.1038/299562a0. [DOI] [PubMed] [Google Scholar]
  26. Miller L. K., Dawes K. P. Physical Map of the DNA Genome of Autographa californica Nuclear Polyhedrosis Virus. J Virol. 1979 Mar;29(3):1044–1055. doi: 10.1128/jvi.29.3.1044-1055.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Reiser J., Renart J., Stark G. R. Transfer of small DNA fragments from polyacrylamide gels to diazobenzyloxymethyl-paper and detection by hybridization with DNA probes. Biochem Biophys Res Commun. 1978 Dec 14;85(3):1104–1112. doi: 10.1016/0006-291x(78)90656-3. [DOI] [PubMed] [Google Scholar]
  28. Richardson C. C. Phosphorylation of nucleic acid by an enzyme from T4 bacteriophage-infected Escherichia coli. Proc Natl Acad Sci U S A. 1965 Jul;54(1):158–165. doi: 10.1073/pnas.54.1.158. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Rigby P. W., Dieckmann M., Rhodes C., Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol. 1977 Jun 15;113(1):237–251. doi: 10.1016/0022-2836(77)90052-3. [DOI] [PubMed] [Google Scholar]
  30. Rohel D. Z., Faulkner P. Time Course Analysis and Mapping of Autographa californica Nuclear Polyhedrosis Virus Transcripts. J Virol. 1984 Jun;50(3):739–747. doi: 10.1128/jvi.50.3.739-747.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Smith G. E., Summers M. D. Restriction Maps of Five Autographa californica MNPV Variants, Trichoplusia ni MNPV, and Galleria mellonella MNPV DNAs with Endonucleases SmaI, KpnI, BamHI, SacI, XhoI, and EcoRI. J Virol. 1979 Jun;30(3):828–838. doi: 10.1128/jvi.30.3.828-838.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Smith G. E., Vlak J. M., Summers M. D. In Vitro Translation of Autographa californica Nuclear Polyhedrosis Virus Early and Late mRNAs. J Virol. 1982 Oct;44(1):199–208. doi: 10.1128/jvi.44.1.199-208.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Smith G. E., Vlak J. M., Summers M. D. Physical Analysis of Autographa californica Nuclear Polyhedrosis Virus Transcripts for Polyhedrin and 10,000-Molecular-Weight Protein. J Virol. 1983 Jan;45(1):215–225. doi: 10.1128/jvi.45.1.215-225.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  35. Tjia S. T., Carstens E. B., Doerfler W. Infection of Spodoptera frugiperda cells with Autographa californica nuclear polyhedrosis virus II. The viral DNA and the kinetics of its replication. Virology. 1979 Dec;99(2):399–409. doi: 10.1016/0042-6822(79)90018-7. [DOI] [PubMed] [Google Scholar]
  36. Villarreal L. P., White R. T., Berg P. Mutational alterations within the simian virus 40 leader segment generate altered 16S and 19S mRNA's. J Virol. 1979 Jan;29(1):209–219. doi: 10.1128/jvi.29.1.209-219.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Vlak J. M., Smith G. E. Orientation of the Genome of Autographa californica Nuclear Polyhedrosis Virus: a Proposal. J Virol. 1982 Mar;41(3):1118–1121. doi: 10.1128/jvi.41.3.1118-1121.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Vlak J. M., Smith G. E., Summers M. D. Hybridization Selection and In Vitro Translation of Autographa californica Nuclear Polyhedrosis Virus mRNA. J Virol. 1981 Dec;40(3):762–771. doi: 10.1128/jvi.40.3.762-771.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Vlak J. M., van der Krol S. Transcription of the Autographa californica nuclear polyhedrosis virus genome: location of late cytoplasmic mRNA. Virology. 1982 Nov;123(1):222–228. doi: 10.1016/0042-6822(82)90309-9. [DOI] [PubMed] [Google Scholar]
  40. WYATT G. R., COHEN S. S. The bases of the nucleic acids of some bacterial and animal viruses: the occurrence of 5-hydroxymethylcytosine. Biochem J. 1953 Dec;55(5):774–782. doi: 10.1042/bj0550774. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Wahl G. M., Stern M., Stark G. R. Efficient transfer of large DNA fragments from agarose gels to diazobenzyloxymethyl-paper and rapid hybridization by using dextran sulfate. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3683–3687. doi: 10.1073/pnas.76.8.3683. [DOI] [PMC free article] [PubMed] [Google Scholar]

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