Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1984 Dec 21;12(24):9309–9321. doi: 10.1093/nar/12.24.9309

Generation and functional analyses for base-substitution mutants of the adenovirus 2 major late promoter.

Y T Yu, J L Manley
PMCID: PMC320463  PMID: 6514578

Abstract

The function of guanosine residues surrounding the TATA box of the adenovirus 2 major late promoter (MLP) in promoting efficient transcription initiation and in selecting a specific transcription start site in vitro has been examined. Multiple and single base substitutions (G----A) were generated in this region (from -63 to +25 relative to the cap site, +1) of the MLP. The promoter activities of the wild type and 21 mutants were assayed in an in vitro transcription system using whole cell extract (WCE) prepared from HeLa cells. The results suggest that the strings of G residues immediately adjacent to the TATA box are not required for full promoter activity in vitro. These G residues also appear not to be involved in the selection of a specific transcription start site by RNA polymerase II in vitro, since the identical cap site was used by wild-type and mutated MLP's. However, two G residues (-55 and -57) were identified as part of an upstream promoter element: a G----A transition at either -55 or -57 resulted in a 2.6 fold reduction in promoter activity. However, neither single nor double G----A transitions at -62 and -63 had an effect on promoter activity.

Full text

PDF
9309

Images in this article

9314Image
on p.9314
9316Image
on p.9316
9318Image
on p.9318

Selected References

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

  1. 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]
  2. Bensimhon M., Gabarro-Arpa J., Ehrlich R., Reiss C. Physical characteristics in eucaryotic promoters. Nucleic Acids Res. 1983 Jul 11;11(13):4521–4540. doi: 10.1093/nar/11.13.4521. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. Concino M., Goldman R. A., Caruthers M. H., Weinmann R. Point mutations of the adenovirus major late promoter with different transcriptional efficiencies in vitro. J Biol Chem. 1983 Jul 10;258(13):8493–8496. [PubMed] [Google Scholar]
  5. Corden J., Wasylyk B., Buchwalder A., Sassone-Corsi P., Kedinger C., Chambon P. Promoter sequences of eukaryotic protein-coding genes. Science. 1980 Sep 19;209(4463):1406–1414. doi: 10.1126/science.6251548. [DOI] [PubMed] [Google Scholar]
  6. Dierks P., van Ooyen A., Cochran M. D., Dobkin C., Reiser J., Weissmann C. Three regions upstream from the cap site are required for efficient and accurate transcription of the rabbit beta-globin gene in mouse 3T6 cells. Cell. 1983 Mar;32(3):695–706. doi: 10.1016/0092-8674(83)90055-7. [DOI] [PubMed] [Google Scholar]
  7. Everett R. D., Chambon P. A rapid and efficient method for region- and strand-specific mutagenesis of cloned DNA. EMBO J. 1982;1(4):433–437. doi: 10.1002/j.1460-2075.1982.tb01187.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Fire A., Baker C. C., Manley J. L., Ziff E. B., Sharp P. A. In vitro transcription of adenovirus. J Virol. 1981 Dec;40(3):703–719. doi: 10.1128/jvi.40.3.703-719.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Grosschedl R., Birnstiel M. L. Identification of regulatory sequences in the prelude sequences of an H2A histone gene by the study of specific deletion mutants in vivo. Proc Natl Acad Sci U S A. 1980 Mar;77(3):1432–1436. doi: 10.1073/pnas.77.3.1432. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Grosschedl R., Wasylyk B., Chambon P., Birnstiel M. L. Point mutation in the TATA box curtails expression of sea urchin H2A histone gene in vivo. Nature. 1981 Nov 12;294(5837):178–180. doi: 10.1038/294178a0. [DOI] [PubMed] [Google Scholar]
  11. Grosveld G. C., Rosenthal A., Flavell R. A. Sequence requirements for the transcription of the rabbit beta-globin gene in vivo: the -80 region. Nucleic Acids Res. 1982 Aug 25;10(16):4951–4971. doi: 10.1093/nar/10.16.4951. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Grosveld G. C., Shewmaker C. K., Jat P., Flavell R. A. Localization of DNA sequences necessary for transcription of the rabbit beta-globin gene in vitro. Cell. 1981 Jul;25(1):215–226. doi: 10.1016/0092-8674(81)90246-4. [DOI] [PubMed] [Google Scholar]
  13. Hen R., Sassone-Corsi P., Corden J., Gaub M. P., Chambon P. Sequences upstream from the T-A-T-A box are required in vivo and in vitro for efficient transcription from the adenovirus serotype 2 major late promoter. Proc Natl Acad Sci U S A. 1982 Dec;79(23):7132–7136. doi: 10.1073/pnas.79.23.7132. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hirose S., Takeuchi K., Suzuki Y. In vitro characterization of the fibroin gene promoter by the use of single-base substitution mutants. Proc Natl Acad Sci U S A. 1982 Dec;79(23):7258–7262. doi: 10.1073/pnas.79.23.7258. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Jove R., Manley J. L. In vitro transcription from the adenovirus 2 major late promoter utilizing templates truncated at promoter-proximal sites. J Biol Chem. 1984 Jul 10;259(13):8513–8521. [PubMed] [Google Scholar]
  16. Kalderon D., Oostra B. A., Ely B. K., Smith A. E. Deletion loop mutagenesis: a novel method for the construction of point mutations using deletion mutants. Nucleic Acids Res. 1982 Sep 11;10(17):5161–5171. doi: 10.1093/nar/10.17.5161. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lee D. C., Roeder R. G., Wold W. S. DNA sequences affecting specific initiation of transcription in vitro from the EIII promoter of adenovirus 2. Proc Natl Acad Sci U S A. 1982 Jan;79(1):41–45. doi: 10.1073/pnas.79.1.41. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Maniatis T., Jeffrey A., van deSande H. Chain length determination of small double- and single-stranded DNA molecules by polyacrylamide gel electrophoresis. Biochemistry. 1975 Aug 26;14(17):3787–3794. doi: 10.1021/bi00688a010. [DOI] [PubMed] [Google Scholar]
  19. Manley J. L. Analysis of the expression of genes encoding animal mRNA by in vitro techniques. Prog Nucleic Acid Res Mol Biol. 1983;30:195–244. doi: 10.1016/s0079-6603(08)60687-x. [DOI] [PubMed] [Google Scholar]
  20. Manley J. L., Fire A., Cano A., Sharp P. A., Gefter M. L. DNA-dependent transcription of adenovirus genes in a soluble whole-cell extract. Proc Natl Acad Sci U S A. 1980 Jul;77(7):3855–3859. doi: 10.1073/pnas.77.7.3855. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Manley J. L., Fire A., Samuels M., Sharp P. A. In vitro transcription: whole-cell extract. Methods Enzymol. 1983;101:568–582. doi: 10.1016/0076-6879(83)01038-1. [DOI] [PubMed] [Google Scholar]
  22. Mathis D. J., Chambon P. The SV40 early region TATA box is required for accurate in vitro initiation of transcription. Nature. 1981 Mar 26;290(5804):310–315. doi: 10.1038/290310a0. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. McKnight S. L., Kingsbury R. Transcriptional control signals of a eukaryotic protein-coding gene. Science. 1982 Jul 23;217(4557):316–324. doi: 10.1126/science.6283634. [DOI] [PubMed] [Google Scholar]
  25. McMaster G. K., Carmichael G. G. Analysis of single- and double-stranded nucleic acids on polyacrylamide and agarose gels by using glyoxal and acridine orange. Proc Natl Acad Sci U S A. 1977 Nov;74(11):4835–4838. doi: 10.1073/pnas.74.11.4835. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Messing J. New M13 vectors for cloning. Methods Enzymol. 1983;101:20–78. doi: 10.1016/0076-6879(83)01005-8. [DOI] [PubMed] [Google Scholar]
  27. Sassone-Corsi P., Corden J., Kédinger C., Chambon P. Promotion of specific in vitro transcription by excised "TATA" box sequences inserted in a foreign nucleotide environment. Nucleic Acids Res. 1981 Aug 25;9(16):3941–3958. doi: 10.1093/nar/9.16.3941. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Shortle D., Nathans D. Local mutagenesis: a method for generating viral mutants with base substitutions in preselected regions of the viral genome. Proc Natl Acad Sci U S A. 1978 May;75(5):2170–2174. doi: 10.1073/pnas.75.5.2170. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Smith H. O. Recovery of DNA from gels. Methods Enzymol. 1980;65(1):371–380. doi: 10.1016/s0076-6879(80)65048-4. [DOI] [PubMed] [Google Scholar]
  30. Tsuda M., Suzuki Y. Transcription modulation in vitro of the fibroin gene exerted by a 200-base-pair region upstream from the "TATA" box. Proc Natl Acad Sci U S A. 1983 Dec;80(24):7442–7446. doi: 10.1073/pnas.80.24.7442. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Tsujimoto Y., Hirose S., Tsuda M., Suzuki Y. Promoter sequence of fibroin gene assigned by in vitro transcription system. Proc Natl Acad Sci U S A. 1981 Aug;78(8):4838–4842. doi: 10.1073/pnas.78.8.4838. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Wasylyk B., Kédinger C., Corden J., Brison O., Chambon P. Specific in vitro initiation of transcription on conalbumin and ovalbumin genes and comparison with adenovirus-2 early and late genes. Nature. 1980 Jun 5;285(5764):367–373. doi: 10.1038/285367a0. [DOI] [PubMed] [Google Scholar]
  33. Ziff E. B., Evans R. M. Coincidence of the promoter and capped 5' terminus of RNA from the adenovirus 2 major late transcription unit. Cell. 1978 Dec;15(4):1463–1475. doi: 10.1016/0092-8674(78)90070-3. [DOI] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES