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. 1990 May 1;267(3):631–637. doi: 10.1042/bj2670631

A new recombinant DNA strategy for the molecular cloning of rare membrane proteins.

B Brake 1, P Braghetta 1, G Banting 1, G Bressan 1, J P Luzio 1, K K Stanley 1
PMCID: PMC1131344  PMID: 2339979

Abstract

We have constructed a cDNA library in the plasmid expression vector pUEX enriched in sequences encoding membrane proteins. The procedure involved positive selection of sequences common to two different rat tissues (thus excluding tissue-specific mRNA) followed by positive selection between this material and RNA extracted from membrane bound polysomes (thus excluding cytoplasmic proteins). The resultant library prepared from rat kidney cDNA hybridized with rat liver poly(A)+ RNA, contained 30,000 clones and was shown to be enriched in cDNAs encoding membrane proteins. Seventeen clones selected because they encode large fusion proteins were shown to be single copy in the library, and not present in nucleotide data banks. Thus the strategy is particularly suitable for cloning low abundance cDNAs encoding membrane proteins.

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Selected References

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

  1. Alt F. W., Kellems R. E., Bertino J. R., Schimke R. T. Selective multiplication of dihydrofolate reductase genes in methotrexate-resistant variants of cultured murine cells. J Biol Chem. 1978 Mar 10;253(5):1357–1370. [PubMed] [Google Scholar]
  2. Banting G., Brake B., Braghetta P., Luzio J. P., Stanley K. K. Intracellular targetting signals of polymeric immunoglobulin receptors are highly conserved between species. FEBS Lett. 1989 Aug 28;254(1-2):177–183. doi: 10.1016/0014-5793(89)81034-8. [DOI] [PubMed] [Google Scholar]
  3. Beisiegel U., Weber W., Ihrke G., Herz J., Stanley K. K. The LDL-receptor-related protein, LRP, is an apolipoprotein E-binding protein. Nature. 1989 Sep 14;341(6238):162–164. doi: 10.1038/341162a0. [DOI] [PubMed] [Google Scholar]
  4. Bishop J. O., Morton J. G., Rosbash M., Richardson M. Three abundance classes in HeLa cell messenger RNA. Nature. 1974 Jul 19;250(463):199–204. doi: 10.1038/250199a0. [DOI] [PubMed] [Google Scholar]
  5. Bordier C. Phase separation of integral membrane proteins in Triton X-114 solution. J Biol Chem. 1981 Feb 25;256(4):1604–1607. [PubMed] [Google Scholar]
  6. Bressan G. M., Stanley K. K. pUEX, a bacterial expression vector related to pEX with universal host specificity. Nucleic Acids Res. 1987 Dec 10;15(23):10056–10056. doi: 10.1093/nar/15.23.10056. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Burnette W. N. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
  8. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  9. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  10. Gubler U., Hoffman B. J. A simple and very efficient method for generating cDNA libraries. Gene. 1983 Nov;25(2-3):263–269. doi: 10.1016/0378-1119(83)90230-5. [DOI] [PubMed] [Google Scholar]
  11. Haymerle H., Herz J., Bressan G. M., Frank R., Stanley K. K. Efficient construction of cDNA libraries in plasmid expression vectors using an adaptor strategy. Nucleic Acids Res. 1986 Nov 11;14(21):8615–8624. doi: 10.1093/nar/14.21.8615. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hedrick S. M., Cohen D. I., Nielsen E. A., Davis M. M. Isolation of cDNA clones encoding T cell-specific membrane-associated proteins. Nature. 1984 Mar 8;308(5955):149–153. doi: 10.1038/308149a0. [DOI] [PubMed] [Google Scholar]
  13. Herz J., Hamann U., Rogne S., Myklebost O., Gausepohl H., Stanley K. K. Surface location and high affinity for calcium of a 500-kd liver membrane protein closely related to the LDL-receptor suggest a physiological role as lipoprotein receptor. EMBO J. 1988 Dec 20;7(13):4119–4127. doi: 10.1002/j.1460-2075.1988.tb03306.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Lacal J. C., Santos E., Notario V., Barbacid M., Yamazaki S., Kung H., Seamans C., McAndrew S., Crowl R. Expression of normal and transforming H-ras genes in Escherichia coli and purification of their encoded p21 proteins. Proc Natl Acad Sci U S A. 1984 Sep;81(17):5305–5309. doi: 10.1073/pnas.81.17.5305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Louvard D., Reggio H., Warren G. Antibodies to the Golgi complex and the rough endoplasmic reticulum. J Cell Biol. 1982 Jan;92(1):92–107. doi: 10.1083/jcb.92.1.92. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Mechler B., Rabbitts T. H. Membrane-bound ribosomes of myeloma cells. IV. mRNA complexity of free and membrane-bound polysomes. J Cell Biol. 1981 Jan;88(1):29–36. doi: 10.1083/jcb.88.1.29. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Morrison D. C., Ryan J. L. Bacterial endotoxins and host immune responses. Adv Immunol. 1979;28:293–450. doi: 10.1016/s0065-2776(08)60802-0. [DOI] [PubMed] [Google Scholar]
  18. Omlin F. X., Webster H. D., Palkovits C. G., Cohen S. R. Immunocytochemical localization of basic protein in major dense line regions of central and peripheral myelin. J Cell Biol. 1982 Oct;95(1):242–248. doi: 10.1083/jcb.95.1.242. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Senapathy P. Origin of eukaryotic introns: a hypothesis, based on codon distribution statistics in genes, and its implications. Proc Natl Acad Sci U S A. 1986 Apr;83(7):2133–2137. doi: 10.1073/pnas.83.7.2133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Seth A., Vande Woude G. F. Nucleotide sequence and biochemical activities of the Moloney murine sarcoma virus strain HT-1 mos gene. J Virol. 1985 Oct;56(1):144–152. doi: 10.1128/jvi.56.1.144-152.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Smith Z. D., D'Eugenio-Gumkowski F., Yanagisawa K., Jamieson J. D. Endogenous and monoclonal antibodies to the rat pancreatic acinar cell Golgi complex. J Cell Biol. 1984 Jun;98(6):2035–2046. doi: 10.1083/jcb.98.6.2035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Soutar A. K. Lipoprotein-remnant receptors: second receptor verified? Nature. 1989 Sep 14;341(6238):106–107. doi: 10.1038/341106a0. [DOI] [PubMed] [Google Scholar]
  23. Tooze S. A., Stanley K. K. Identification of two epitopes in the carboxyterminal 15 amino acids of the E1 glycoprotein of mouse hepatitis virus A59 by using hybrid proteins. J Virol. 1986 Dec;60(3):928–934. doi: 10.1128/jvi.60.3.928-934.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]

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