Abstract
Here we describe a reliable method for isolating genes that are differentially expressed in two cell populations. The method is a combination of subtractive hybridization and PCR. Among many improvements to previously described methods is the incorporation of a new technology into the procedure which sterilizes(inactivates) PCR amplicons, and thereby overcomes the limitation of similar procedures. To test this improved method, we conducted a search for estrogen-responsive genes. Estrogen-regulated genes dominated the subtracted libraries after four rounds of subtractive hybridizations. Four estrogen-regulated genes were identified from the initial screening.
Full Text
The Full Text of this article is available as a PDF (116.0 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Brown A. M., Jeltsch J. M., Roberts M., Chambon P. Activation of pS2 gene transcription is a primary response to estrogen in the human breast cancer cell line MCF-7. Proc Natl Acad Sci U S A. 1984 Oct;81(20):6344–6348. doi: 10.1073/pnas.81.20.6344. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cimino G. D., Metchette K. C., Tessman J. W., Hearst J. E., Isaacs S. T. Post-PCR sterilization: a method to control carryover contamination for the polymerase chain reaction. Nucleic Acids Res. 1991 Jan 11;19(1):99–107. doi: 10.1093/nar/19.1.99. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Clauss I. M., Gravallese E. M., Darling J. M., Shapiro F., Glimcher M. J., Glimcher L. H. In situ hybridization studies suggest a role for the basic region-leucine zipper protein hXBP-1 in exocrine gland and skeletal development during mouse embryogenesis. Dev Dyn. 1993 Jun;197(2):146–156. doi: 10.1002/aja.1001970207. [DOI] [PubMed] [Google Scholar]
- Davis R. L., Weintraub H., Lassar A. B. Expression of a single transfected cDNA converts fibroblasts to myoblasts. Cell. 1987 Dec 24;51(6):987–1000. doi: 10.1016/0092-8674(87)90585-x. [DOI] [PubMed] [Google Scholar]
- Duguid J. R., Dinauer M. C. Library subtraction of in vitro cDNA libraries to identify differentially expressed genes in scrapie infection. Nucleic Acids Res. 1990 May 11;18(9):2789–2792. doi: 10.1093/nar/18.9.2789. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Duguid J. R., Rohwer R. G., Seed B. Isolation of cDNAs of scrapie-modulated RNAs by subtractive hybridization of a cDNA library. Proc Natl Acad Sci U S A. 1988 Aug;85(15):5738–5742. doi: 10.1073/pnas.85.15.5738. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Isaacs S. T., Tessman J. W., Metchette K. C., Hearst J. E., Cimino G. D. Post-PCR sterilization: development and application to an HIV-1 diagnostic assay. Nucleic Acids Res. 1991 Jan 11;19(1):109–116. doi: 10.1093/nar/19.1.109. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Liou H. C., Boothby M. R., Finn P. W., Davidon R., Nabavi N., Zeleznik-Le N. J., Ting J. P., Glimcher L. H. A new member of the leucine zipper class of proteins that binds to the HLA DR alpha promoter. Science. 1990 Mar 30;247(4950):1581–1584. doi: 10.1126/science.2321018. [DOI] [PubMed] [Google Scholar]
- Nardulli A. M., Greene G. L., O'Malley B. W., Katzenellenbogen B. S. Regulation of progesterone receptor messenger ribonucleic acid and protein levels in MCF-7 cells by estradiol: analysis of estrogen's effect on progesterone receptor synthesis and degradation. Endocrinology. 1988 Mar;122(3):935–944. doi: 10.1210/endo-122-3-935. [DOI] [PubMed] [Google Scholar]
- Ono S. J., Liou H. C., Davidon R., Strominger J. L., Glimcher L. H. Human X-box-binding protein 1 is required for the transcription of a subset of human class II major histocompatibility genes and forms a heterodimer with c-fos. Proc Natl Acad Sci U S A. 1991 May 15;88(10):4309–4312. doi: 10.1073/pnas.88.10.4309. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wang Z., Brown D. D. A gene expression screen. Proc Natl Acad Sci U S A. 1991 Dec 15;88(24):11505–11509. doi: 10.1073/pnas.88.24.11505. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wang Z., Brown D. D. Thyroid hormone-induced gene expression program for amphibian tail resorption. J Biol Chem. 1993 Aug 5;268(22):16270–16278. [PubMed] [Google Scholar]
- Weisz A., Bresciani F. Estrogen regulation of proto-oncogenes coding for nuclear proteins. Crit Rev Oncog. 1993;4(4):361–388. [PubMed] [Google Scholar]
- Wieland I., Bolger G., Asouline G., Wigler M. A method for difference cloning: gene amplification following subtractive hybridization. Proc Natl Acad Sci U S A. 1990 Apr;87(7):2720–2724. doi: 10.1073/pnas.87.7.2720. [DOI] [PMC free article] [PubMed] [Google Scholar]