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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1979 Jul;76(7):3299–3303. doi: 10.1073/pnas.76.7.3299

Molecular cloning and partial characterization of delta-crystallin cDNA sequences in a bacterial plasmid.

S P Bhat, J Piatigorsky
PMCID: PMC383812  PMID: 386337

Abstract

Double-stranded cDNA synthesized from delta-crystallin mRNA isolated from lens fiber cells of 15-day-old embryonic chicken was cloned in Escherichia coli chi 1776 in the Pst I site of the plasmid pBR322 by using the oligo(dC) . oligo(dG) joining procedure. Twelve Amps Tetr transformants contained sequences complementary to purified delta-crystallin [32P]cDNA. One of the recombinant clones (p delta Cr-2) had an insert of 1241 +/- 240 base pairs, as judged by R-looping analysis with purified delta-crystallin mRNA. The inserted cDNA represents at least 69% of the delta-crystallin coding sequences. p delta Cr-2 was further characterized by restriction analysis, protection of delta-crystallin [3H]cDNA from digestion by S1 nuclease, and hybrid-mediated arrest of delta-crystallin mRNA translation in vitro. p delta Cr-2 provides an invaluable probe for additional analysis of the primary structure, gene organization, and regulated synthesis of delta-crystallin, the principal protein synthesized during lens differentiation in the chicken embryo.

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Selected References

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  1. Bolivar F., Rodriguez R. L., Greene P. J., Betlach M. C., Heyneker H. L., Boyer H. W., Crosa J. H., Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [PubMed] [Google Scholar]
  2. Breathnach R., Mandel J. L., Chambon P. Ovalbumin gene is split in chicken DNA. Nature. 1977 Nov 24;270(5635):314–319. doi: 10.1038/270314a0. [DOI] [PubMed] [Google Scholar]
  3. Clayton R. M., Truman D. E. Molecular structure and antigenicity of lens proteins. Nature. 1967 Jun 17;214(5094):1201–1204. doi: 10.1038/2141201a0. [DOI] [PubMed] [Google Scholar]
  4. Denhardt D. T. A membrane-filter technique for the detection of complementary DNA. Biochem Biophys Res Commun. 1966 Jun 13;23(5):641–646. doi: 10.1016/0006-291x(66)90447-5. [DOI] [PubMed] [Google Scholar]
  5. Eguchi G., Okada T. S. Differentiation of lens tissue from the progeny of chick retinal pigment cells cultured in vitro: a demonstration of a switch of cell types in clonal cell culture. Proc Natl Acad Sci U S A. 1973 May;70(5):1495–1499. doi: 10.1073/pnas.70.5.1495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Enea V., Vovis G. F., Zinder N. D. Genetic studies with heteroduplex DNA of bacteriophage fl. Asymmetric segregation, base correction and implications for the mechanism of genetic recombination. J Mol Biol. 1975 Aug 15;96(3):495–509. doi: 10.1016/0022-2836(75)90175-8. [DOI] [PubMed] [Google Scholar]
  7. Genis-Galves J. M., Maisel H., Castro J. Changes in chick lens proteins with aging. Exp Eye Res. 1968 Oct;7(4):593–602. doi: 10.1016/s0014-4835(68)80014-4. [DOI] [PubMed] [Google Scholar]
  8. Glover D. M., Hogness D. S. A novel arrangement of the 18S and 28S sequences in a repeating unit of Drosophila melanogaster rDNA. Cell. 1977 Feb;10(2):167–176. doi: 10.1016/0092-8674(77)90212-4. [DOI] [PubMed] [Google Scholar]
  9. Grunstein M., Hogness D. S. Colony hybridization: a method for the isolation of cloned DNAs that contain a specific gene. Proc Natl Acad Sci U S A. 1975 Oct;72(10):3961–3965. doi: 10.1073/pnas.72.10.3961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Guerry P., LeBlanc D. J., Falkow S. General method for the isolation of plasmid deoxyribonucleic acid. J Bacteriol. 1973 Nov;116(2):1064–1066. doi: 10.1128/jb.116.2.1064-1066.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Harding J. J., Dilley K. J. Structural proteins of the mammalian lens: a review with emphasis on changes in development, aging and cataract. Exp Eye Res. 1976 Jan;22(1):1–73. doi: 10.1016/0014-4835(76)90033-6. [DOI] [PubMed] [Google Scholar]
  12. Humphries P., Old R., Coggins L. W., McShane T., Watson C., Paul J. Recombinant plasmids containing Xenopus laevis globin structural genes derived from complementary DNA. Nucleic Acids Res. 1978 Mar;5(3):905–924. doi: 10.1093/nar/5.3.905. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Meagher R. B., Tait R. C., Betlach M., Boyer H. W. Protein expression in E. coli minicells by recombinant plasmids. Cell. 1977 Mar;10(3):521–536. doi: 10.1016/0092-8674(77)90039-3. [DOI] [PubMed] [Google Scholar]
  14. Meyer J., Neuwald P. D., Lai S. P., Maizel J. V., Jr, Westphal H. Electron microscopy of late adenovirus type 2 mRNA hybridized to double-stranded viral DNA. J Virol. 1977 Mar;21(3):1010–1018. doi: 10.1128/jvi.21.3.1010-1018.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Milstone L. M., Zelenka P., Piatigorsky J. Delta-crystallin mRNA in chick lens cells: mRNA accumulates during differential stimulation of delta-crystallin synthesis in cultured cells. Dev Biol. 1976 Feb;48(2):197–204. doi: 10.1016/0012-1606(76)90084-1. [DOI] [PubMed] [Google Scholar]
  16. Nguyen-Huu M. C., Stratmann M., Groner B., Wurtz T., Land H., Giesecke K., Sippel A. E., Schütz G. Chicken lysozyme gene contains several intervening sequences. Proc Natl Acad Sci U S A. 1979 Jan;76(1):76–80. doi: 10.1073/pnas.76.1.76. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Okada T. S., Eguchi G., Takeichi M. The retention of differentiated properties by lens epithelial cells in clonal cell culture. Dev Biol. 1973 Oct;34(2):321–333. doi: 10.1016/0012-1606(73)90361-8. [DOI] [PubMed] [Google Scholar]
  18. PHILPOTT G. W., COULOMBRE A. J. LENS DEVELOPMENT. II. THE DIFFERENTIATION OF EMBRYONIC CHICK LENS EPITHELIAL CELLS IN VITRO AND IN VIVO. Exp Cell Res. 1965 Jun;38:635–644. doi: 10.1016/0014-4827(65)90387-3. [DOI] [PubMed] [Google Scholar]
  19. Paterson B. M., Roberts B. E., Kuff E. L. Structural gene identification and mapping by DNA-mRNA hybrid-arrested cell-free translation. Proc Natl Acad Sci U S A. 1977 Oct;74(10):4370–4374. doi: 10.1073/pnas.74.10.4370. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Piatigorsky J. Subunit composition of delta-crystallin from embryonic chick lens. Analysis of methionine-containing tryptic peptides and cyanogen bromide peptides. J Biol Chem. 1976 Jul 25;251(14):4416–4420. [PubMed] [Google Scholar]
  21. Piatigorsky J., Webster H. D., Craig S. P. Protein synthesis and ultrastructure during the formation of embryonic chick lens fibers in vivo and in vitro. Dev Biol. 1972 Feb;27(2):176–189. doi: 10.1016/0012-1606(72)90096-6. [DOI] [PubMed] [Google Scholar]
  22. RABAEY M. Electrophoretic and immunoelectrophoretic studies on the soluble proteins in the developing lens of birds. Exp Eye Res. 1962 Jun;1:310–316. doi: 10.1016/s0014-4835(62)80017-7. [DOI] [PubMed] [Google Scholar]
  23. Reszelbach R., Shinohara T., Piatigorsky J. Resolution of two distinct embryonic chick delta-crystallin bands by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and urea. Exp Eye Res. 1977 Dec;25(6):583–593. doi: 10.1016/0014-4835(77)90137-3. [DOI] [PubMed] [Google Scholar]
  24. Shinohara T., Piatigorsky J. Quantitation of delta-crystallin messenger RNA during lens induction in chick embryos. Proc Natl Acad Sci U S A. 1976 Aug;73(8):2808–2812. doi: 10.1073/pnas.73.8.2808. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. Sutcliffe J. G. pBR322 restriction map derived from the DNA sequence: accurate DNA size markers up to 4361 nucleotide pairs long. Nucleic Acids Res. 1978 Aug;5(8):2721–2728. doi: 10.1093/nar/5.8.2721. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Thomas M., White R. L., Davis R. W. Hybridization of RNA to double-stranded DNA: formation of R-loops. Proc Natl Acad Sci U S A. 1976 Jul;73(7):2294–2298. doi: 10.1073/pnas.73.7.2294. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Thomson I., Wilkinson C. E., Jackson J. F., de Pomerai D. I., Clayton R. M., Truman D. E., Williamson R. Isolation and cell-free translation of chick lens crystallin mRNA during normal development and transdifferentiation of neural retina. Dev Biol. 1978 Aug;65(2):372–382. doi: 10.1016/0012-1606(78)90033-7. [DOI] [PubMed] [Google Scholar]
  29. Tilghman S. M., Tiemeier D. C., Seidman J. G., Peterlin B. M., Sullivan M., Maizel J. V., Leder P. Intervening sequence of DNA identified in the structural portion of a mouse beta-globin gene. Proc Natl Acad Sci U S A. 1978 Feb;75(2):725–729. doi: 10.1073/pnas.75.2.725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Truman D. E., Brown A. G., Rao K. V. Estimation of the molecular weights of chick -and -crystallins and their subunits by gel filtration. Exp Eye Res. 1971 Nov;12(3):304–310. doi: 10.1016/0014-4835(71)90154-0. [DOI] [PubMed] [Google Scholar]
  31. Yoshida K., Katoh A. Crystallin synthesis by chick lens. II. Changes in synthetic activities of epithelial and fiber cells during embryonic development. Exp Eye Res. 1971 Mar;11(2):184–194. doi: 10.1016/s0014-4835(71)80022-2. [DOI] [PubMed] [Google Scholar]
  32. Zelenka P., Piatigorsky J. Isolation and in vitro translation of delta-crystallin mRNA from embryonic chick lens fibers. Proc Natl Acad Sci U S A. 1974 May;71(5):1896–1900. doi: 10.1073/pnas.71.5.1896. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Zelenka P., Piatigorsky J. Molecular weight and sequence complexity of delta-crystallin mRNA. Exp Eye Res. 1976 Feb;22(2):115–124. doi: 10.1016/0014-4835(76)90038-5. [DOI] [PubMed] [Google Scholar]
  34. Zwaan J., Ikeda A. Macromolecular events during differentiation of the chicken lens. Exp Eye Res. 1968 Apr;7(2):301–311. doi: 10.1016/s0014-4835(68)80081-8. [DOI] [PubMed] [Google Scholar]

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