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. 1984 Aug;81(15):4965–4969. doi: 10.1073/pnas.81.15.4965

Cloning and physical mapping of a gene fragment coding for a 64-kilodalton major late antigen of human cytomegalovirus.

H Pande, S W Baak, A D Riggs, B R Clark, J E Shively, J A Zaia
PMCID: PMC391613  PMID: 6087357

Abstract

We have isolated a clone containing a gene fragment coding for a 64-kilodalton glycoprotein that is the major late antigen of human cytomegalovirus (HCMV). Based upon the amino acid sequence of a tryptic peptide of this glycoprotein (HCMVgp64), two sets of mixed-sequence probes, one consisting of a mixture of 16 heptadecadeoxyribonucleotides and the other a mixture of 32 icosadeoxyribonucleotides, were synthesized. A subgenomic library of HCMV (Towne strain) DNA was constructed in plasmid pBR327 and transformants were screened with 32P-labeled aliquots of these synthetic oligodeoxyribonucleotide probes. Two clones among 15,000 gave strong positive signals. Plasmid DNA was isolated from the positive clones and characterized by restriction mapping and Southern blot analysis using both probes. The plasmid DNA contained a 2.3-kilobase insert, which yielded an 800-base-pair and a 1500-base-pair fragment after Sau3A digestion. Only the 800-base-pair fragment hybridized to the mixed probes, and DNA sequence analysis revealed that it contains nucleotide sequences compatible with amino acid sequences of tryptic peptides of HCMVgp64. Restriction mapping studies of HCMV DNA using this 32P-labeled 800-base-pair cloned DNA have allowed us to locate this gene fragment in the long unique region of HCMV (Towne strain) genome at approximately equal to 0.5-0.51 map unit.

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Selected References

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

  1. Boldogh I., Beth E., Huang E. S., Kyalwazi S. K., Giraldo G. Kaposi's sarcoma. IV. Detection of CMV DNA, CMV RNA and CMNA in tumor biopsies. Int J Cancer. 1981 Oct 15;28(4):469–474. doi: 10.1002/ijc.2910280412. [DOI] [PubMed] [Google Scholar]
  2. Clark B. R., Zaia J. A., Balce-Directo L., Ting Y. P. Isolation and partial chemical characterization of a 64,000-dalton glycoprotein of human cytomegalovirus. J Virol. 1984 Jan;49(1):279–282. doi: 10.1128/jvi.49.1.279-282.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. DeMarchi J. M., Blankenship M. L., Brown G. D., Kaplan A. S. Size and complexity of human cytomegalovirus DNA. Virology. 1978 Sep;89(2):643–646. doi: 10.1016/0042-6822(78)90209-x. [DOI] [PubMed] [Google Scholar]
  4. Del Valle U., Shively J. E. Two-column system for determination of glucosamine, galactosamine, and amino acids on a Beckman 121MB amino acid analyzer: separation of the anomers of glucosamine and galactosamine. Anal Biochem. 1979 Jul 1;96(1):77–83. doi: 10.1016/0003-2697(79)90556-6. [DOI] [PubMed] [Google Scholar]
  5. Demarchi J. M. Human cytomegalovirus DNA: restriction enzyme cleavage maps and map locations for immediate-early, early, and late RNAs. Virology. 1981 Oct 15;114(1):23–38. doi: 10.1016/0042-6822(81)90249-x. [DOI] [PubMed] [Google Scholar]
  6. Ebeling A., Keil G., Nowak B., Fleckenstein B., Berthelot N., Sheldrick P. Genome structure and virion polypeptides of the primate herpesviruses Herpesvirus aotus types 1 and 3: comparison with human cytomegalovirus. J Virol. 1983 Feb;45(2):715–726. doi: 10.1128/jvi.45.2.715-726.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Fiala M., Honess R. W., Heiner D. C., Heine J. W., Jr, Murnane J., Wallace R., Guze L. B. Cytomegalovirus proteins. I. Polypeptides of virions and dense bodies. J Virol. 1976 Jul;19(1):243–254. doi: 10.1128/jvi.19.1.243-254.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Fleckenstein B., Müller I., Collins J. Cloning of the complete human cytomegalovirus genome in cosmids. Gene. 1982 Apr;18(1):39–46. doi: 10.1016/0378-1119(82)90054-3. [DOI] [PubMed] [Google Scholar]
  9. Gergen J. P., Stern R. H., Wensink P. C. Filter replicas and permanent collections of recombinant DNA plasmids. Nucleic Acids Res. 1979 Dec 20;7(8):2115–2136. doi: 10.1093/nar/7.8.2115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gibson W. Protein counterparts of human and simian cytomegaloviruses. Virology. 1983 Jul 30;128(2):391–406. doi: 10.1016/0042-6822(83)90265-9. [DOI] [PubMed] [Google Scholar]
  11. Gibson W. Structural and nonstructural proteins of strain Colburn cytomegalovirus. Virology. 1981 Jun;111(2):516–537. doi: 10.1016/0042-6822(81)90354-8. [DOI] [PubMed] [Google Scholar]
  12. Giraldo G., Beth E., Huang E. S. Kaposi's sarcoma and its relationship to cytomegalovirus (CMNV). III. CMV DNA and CMV early antigens in Kaposi's sarcoma. Int J Cancer. 1980 Jul 15;26(1):23–29. doi: 10.1002/ijc.2910260105. [DOI] [PubMed] [Google Scholar]
  13. Greenaway P. J., Oram J. D., Downing R. G., Patel K. Human cytomegalovirus DNA: BamHI, EcoRI and PstI restriction endonuclease cleavage maps. Gene. 1982 Jun;18(3):355–360. doi: 10.1016/0378-1119(82)90174-3. [DOI] [PubMed] [Google Scholar]
  14. Ish-Horowicz D., Burke J. F. Rapid and efficient cosmid cloning. Nucleic Acids Res. 1981 Jul 10;9(13):2989–2998. doi: 10.1093/nar/9.13.2989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kim K. S., Sapienza V. J., Carp R. I., Moon H. M. Analysis of structural polypeptides of purified human cytomegalovirus. J Virol. 1976 Dec;20(3):604–611. doi: 10.1128/jvi.20.3.604-611.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kornblihtt A. R., Vibe-Pedersen K., Baralle F. E. Isolation and characterization of cDNA clones for human and bovine fibronectins. Proc Natl Acad Sci U S A. 1983 Jun;80(11):3218–3222. doi: 10.1073/pnas.80.11.3218. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Marsden H. S., Stow N. D., Preston V. G., Timbury M. C., Wilkie N. M. Physical mapping of herpes simplex virus-induced polypeptides. J Virol. 1978 Nov;28(2):624–642. doi: 10.1128/jvi.28.2.624-642.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Messing J., Vieira J. A new pair of M13 vectors for selecting either DNA strand of double-digest restriction fragments. Gene. 1982 Oct;19(3):269–276. doi: 10.1016/0378-1119(82)90016-6. [DOI] [PubMed] [Google Scholar]
  19. Morse L. S., Pereira L., Roizman B., Schaffer P. A. Anatomy of herpes simplex virus (HSV) DNA. X. Mapping of viral genes by analysis of polypeptides and functions specified by HSV-1 X HSV-2 recombinants. J Virol. 1978 May;26(2):389–410. doi: 10.1128/jvi.26.2.389-410.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Nelson J. A., Fleckenstein B., Galloway D. A., McDougall J. K. Transformation of NIH 3T3 cells with cloned fragments of human cytomegalovirus strain AD169. J Virol. 1982 Jul;43(1):83–91. doi: 10.1128/jvi.43.1.83-91.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Oram J. D., Downing R. G., Akrigg A., Dollery A. A., Duggleby C. J., Wilkinson G. W., Greenaway P. J. Use of recombinant plasmids to investigate the structure of the human cytomegalovirus genome. J Gen Virol. 1982 Mar;59(Pt 1):111–129. doi: 10.1099/0022-1317-59-1-111. [DOI] [PubMed] [Google Scholar]
  22. Pereira L., Hoffman M., Cremer N. Electrophoretic analysis of polypeptides immune precipitated from cytomegalovirus-infected cell extracts by human sera. Infect Immun. 1982 Jun;36(3):933–942. doi: 10.1128/iai.36.3.933-942.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Pereira L., Hoffman M., Gallo D., Cremer N. Monoclonal antibodies to human cytomegalovirus: three surface membrane proteins with unique immunological and electrophoretic properties specify cross-reactive determinants. Infect Immun. 1982 Jun;36(3):924–932. doi: 10.1128/iai.36.3.924-932.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Pereira L., Stagno S., Hoffman M., Volanakis J. E. Cytomegalovirus-infected cell polypeptides immune-precipitated by sera from children with congenital and perinatal infections. Infect Immun. 1983 Jan;39(1):100–108. doi: 10.1128/iai.39.1.100-108.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Rodriguez R. L., West R. W., Heyneker H. L., Bolivar F., Boyer H. W. Characterizing wild-type and mutant promoters of the tetracycline resistance gene in pBR313. Nucleic Acids Res. 1979 Jul 25;6(10):3267–3287. doi: 10.1093/nar/6.10.3267. [DOI] [PMC free article] [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. Sarov I., Abady I. The morphogenesis of human cytomegalovirus. Isolation and polypeptide characterization of cytomegalovirions and dense bodies. Virology. 1975 Aug;66(2):464–473. doi: 10.1016/0042-6822(75)90218-4. [DOI] [PubMed] [Google Scholar]
  28. Shively J. E. Sequence determinations of proteins and peptides at the nanomole and subnanomole level with a modified spinning cup sequenator. Methods Enzymol. 1981;79(Pt B):31–48. doi: 10.1016/s0076-6879(81)79011-6. [DOI] [PubMed] [Google Scholar]
  29. Singer-Sam J., Simmer R. L., Keith D. H., Shively L., Teplitz M., Itakura K., Gartler S. M., Riggs A. D. Isolation of a cDNA clone for human X-linked 3-phosphoglycerate kinase by use of a mixture of synthetic oligodeoxyribonucleotides as a detection probe. Proc Natl Acad Sci U S A. 1983 Feb;80(3):802–806. doi: 10.1073/pnas.80.3.802. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Soberon X., Covarrubias L., Bolivar F. Construction and characterization of new cloning vehicles. IV. Deletion derivatives of pBR322 and pBR325. Gene. 1980 May;9(3-4):287–305. doi: 10.1016/0378-1119(90)90328-o. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. Stinski M. F. Human cytomegalovirus: glycoproteins associated with virions and dense bodies. J Virol. 1976 Aug;19(2):594–609. doi: 10.1128/jvi.19.2.594-609.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Stinski M. F., Mocarski E. S., Thomsen D. R. DNA of human cytomegalovirus: size heterogeneity and defectiveness resulting from serial undiluted passage. J Virol. 1979 Jul;31(1):231–239. doi: 10.1128/jvi.31.1.231-239.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Stinski M. F. Sequence of protein synthesis in cells infected by human cytomegalovirus: early and late virus-induced polypeptides. J Virol. 1978 Jun;26(3):686–701. doi: 10.1128/jvi.26.3.686-701.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Stinski M. F. Synthesis of proteins and glycoproteins in cells infected with human cytomegalovirus. J Virol. 1977 Sep;23(3):751–767. doi: 10.1128/jvi.23.3.751-767.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Stinski M. F., Thomsen D. R., Stenberg R. M., Goldstein L. C. Organization and expression of the immediate early genes of human cytomegalovirus. J Virol. 1983 Apr;46(1):1–14. doi: 10.1128/jvi.46.1.1-14.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Tamashiro J. C., Hock L. J., Spector D. H. Construction of a cloned library of the EcoRI fragments from the human cytomegalovirus genome (strain AD169). J Virol. 1982 May;42(2):547–557. doi: 10.1128/jvi.42.2.547-557.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Tan Z. K., Ikuta S., Huang T., Dugaiczyk A., Itakura K. Solid-phase synthesis of polynucleotides. VIII: A simplified synthesis of oligodeoxyribonucleotides. Cold Spring Harb Symp Quant Biol. 1983;47(Pt 1):383–391. doi: 10.1101/sqb.1983.047.01.045. [DOI] [PubMed] [Google Scholar]
  39. Thomsen D. R., Stinski M. F. Cloning of the human cytomegalovirus genome as endonuclease XbaI fragments. Gene. 1981 Dec;16(1-3):207–216. doi: 10.1016/0378-1119(81)90077-9. [DOI] [PubMed] [Google Scholar]
  40. 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]
  41. Wallace R. B., Johnson M. J., Hirose T., Miyake T., Kawashima E. H., Itakura K. The use of synthetic oligonucleotides as hybridization probes. II. Hybridization of oligonucleotides of mixed sequence to rabbit beta-globin DNA. Nucleic Acids Res. 1981 Feb 25;9(4):879–894. doi: 10.1093/nar/9.4.879. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Wallace R. B., Schold M., Johnson M. J., Dembek P., Itakura K. Oligonucleotide directed mutagenesis of the human beta-globin gene: a general method for producing specific point mutations in cloned DNA. Nucleic Acids Res. 1981 Aug 11;9(15):3647–3656. doi: 10.1093/nar/9.15.3647. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Wathen M. W., Stinski M. F. Temporal patterns of human cytomegalovirus transcription: mapping the viral RNAs synthesized at immediate early, early, and late times after infection. J Virol. 1982 Feb;41(2):462–477. doi: 10.1128/jvi.41.2.462-477.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Weiner D., Gibson W. Phosphorylation, maturational processing, and relatedness of strain Colburn matrix proteins. Virology. 1983 Aug;129(1):155–169. doi: 10.1016/0042-6822(83)90403-8. [DOI] [PubMed] [Google Scholar]
  45. Weller T. H. The cytomegaloviruses: ubiquitous agents with protean clinical manifestations. I. N Engl J Med. 1971 Jul 22;285(4):203–214. doi: 10.1056/NEJM197107222850406. [DOI] [PubMed] [Google Scholar]
  46. Yuan P. M., Pande H., Clark B. R., Shively J. E. Microsequence analysis of peptides and proteins. I. Preparation of samples by reverse-phase liquid chromatography. Anal Biochem. 1982 Mar 1;120(2):289–301. doi: 10.1016/0003-2697(82)90350-5. [DOI] [PubMed] [Google Scholar]

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