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. 1987 Aug;7(8):2671–2679. doi: 10.1128/mcb.7.8.2671

Gamma-crystallins of the human eye lens: expression analysis of five members of the gene family.

S O Meakin 1, R P Du 1, L C Tsui 1, M L Breitman 1
PMCID: PMC367883  PMID: 3670288

Abstract

While only two gamma-crystallins have been identified in the human eye lens, molecular studies indicate that the human gamma-crystallins are encoded in a multigene family comprising at least seven closely related members. Sequence analysis of five of these genes has suggested that three (gamma 1-2, G3, and G4) are potentially active, while two (G1 psi and G2 psi) correspond to closely related pseudogenes. Here we report on the detailed structure of a sixth gamma-crystallin gene, G5, and our results obtained with transient expression assays to characterize both the promoter activity and translation products of five members of the gene family. We show that 5'-flanking sequences of G1 psi and G2 psi lacked detectable promoter activity, while the corresponding sequences of G3, G4, and G5 were able to direct high levels of expression of the bacterial chloramphenicol acetyltransferase gene in primary lens epithelia, but not in cultures of nonlens origin. Detailed sequence comparisons indicated that active genes contained several conserved sequence tracts 5' of the TATA box which may constitute functional elements of a lens-specific gamma-crystallin promoter. Expression of the gamma-crystallin coding sequences from the human metallothionein IIA promoter in nonlens cells facilitated characterization of the polypeptides encoded by individual gamma-genes and, in future studies, should permit comparison of these proteins with distinct gamma-crystallins in the human lens.

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Selected References

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  1. Blundell T., Lindley P., Miller L., Moss D., Slingsby C., Tickle I., Turnell B., Wistow G. The molecular structure and stability of the eye lens: x-ray analysis of gamma-crystallin II. Nature. 1981 Feb 26;289(5800):771–777. doi: 10.1038/289771a0. [DOI] [PubMed] [Google Scholar]
  2. Breitman M. L., Lok S., Wistow G., Piatigorsky J., Tréton J. A., Gold R. J., Tsui L. C. Gamma-crystallin family of the mouse lens: structural and evolutionary relationships. Proc Natl Acad Sci U S A. 1984 Dec;81(24):7762–7766. doi: 10.1073/pnas.81.24.7762. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chepelinsky A. B., King C. R., Zelenka P. S., Piatigorsky J. Lens-specific expression of the chloramphenicol acetyltransferase gene promoted by 5' flanking sequences of the murine alpha A-crystallin gene in explanted chicken lens epithelia. Proc Natl Acad Sci U S A. 1985 Apr;82(8):2334–2338. doi: 10.1073/pnas.82.8.2334. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Delaye M., Tardieu A. Short-range order of crystallin proteins accounts for eye lens transparency. 1983 Mar 31-Apr 6Nature. 302(5907):415–417. doi: 10.1038/302415a0. [DOI] [PubMed] [Google Scholar]
  5. Garber A. T., Gold R. J. Comparative two-dimensional electrophoretic analysis of water soluble proteins from bovine and murine lenses. Exp Eye Res. 1982 Dec;35(6):585–596. doi: 10.1016/s0014-4835(82)80072-9. [DOI] [PubMed] [Google Scholar]
  6. Gluzman Y. SV40-transformed simian cells support the replication of early SV40 mutants. Cell. 1981 Jan;23(1):175–182. doi: 10.1016/0092-8674(81)90282-8. [DOI] [PubMed] [Google Scholar]
  7. Goring D. R., Rossant J., Clapoff S., Breitman M. L., Tsui L. C. In situ detection of beta-galactosidase in lenses of transgenic mice with a gamma-crystallin/lacZ gene. Science. 1987 Jan 23;235(4787):456–458. doi: 10.1126/science.3099390. [DOI] [PubMed] [Google Scholar]
  8. Gorman C. M., Moffat L. F., Howard B. H. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol. 1982 Sep;2(9):1044–1051. doi: 10.1128/mcb.2.9.1044. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Graham F. L., van der Eb A. J. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973 Apr;52(2):456–467. doi: 10.1016/0042-6822(73)90341-3. [DOI] [PubMed] [Google Scholar]
  10. Hoenders H. J., Bloemendal H. Lens proteins and aging. J Gerontol. 1983 May;38(3):278–286. doi: 10.1093/geronj/38.3.278. [DOI] [PubMed] [Google Scholar]
  11. Karin M., Richards R. I. Human metallothionein genes--primary structure of the metallothionein-II gene and a related processed gene. Nature. 1982 Oct 28;299(5886):797–802. doi: 10.1038/299797a0. [DOI] [PubMed] [Google Scholar]
  12. Kondoh H., Yasuda K., Okada T. S. Tissue-specific expression of a cloned chick delta-crystallin gene in mouse cells. Nature. 1983 Feb 3;301(5899):440–442. doi: 10.1038/301440a0. [DOI] [PubMed] [Google Scholar]
  13. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  14. Lerman S. An experimental and clinical evaluation of lens transparency and aging. J Gerontol. 1983 May;38(3):293–301. doi: 10.1093/geronj/38.3.293. [DOI] [PubMed] [Google Scholar]
  15. Lok S., Breitman M. L., Chepelinsky A. B., Piatigorsky J., Gold R. J., Tsui L. C. Lens-specific promoter activity of a mouse gamma-crystallin gene. Mol Cell Biol. 1985 Sep;5(9):2221–2230. doi: 10.1128/mcb.5.9.2221. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lok S., Tsui L. C., Shinohara T., Piatigorsky J., Gold R., Breitman M. Analysis of the mouse gamma-crystallin gene family: assignment of multiple cDNAs to discrete genomic sequences and characterization of a representative gene. Nucleic Acids Res. 1984 Jun 11;12(11):4517–4529. doi: 10.1093/nar/12.11.4517. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lubsen N. H., Renwick J. H., Tsui L. C., Breitman M. L., Schoenmakers J. G. A locus for a human hereditary cataract is closely linked to the gamma-crystallin gene family. Proc Natl Acad Sci U S A. 1987 Jan;84(2):489–492. doi: 10.1073/pnas.84.2.489. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. McAvoy J. W. Cell division, cell elongation and distribution of alpha-, beta- and gamma-crystallins in the rat lens. J Embryol Exp Morphol. 1978 Apr;44:149–165. [PubMed] [Google Scholar]
  20. McAvoy J. W. Cell division, cell elongation and the co-ordination of crystallin gene expression during lens morphogenesis in the rat. J Embryol Exp Morphol. 1978 Jun;45:271–281. [PubMed] [Google Scholar]
  21. Meakin S. O., Breitman M. L., Tsui L. C. Structural and evolutionary relationships among five members of the human gamma-crystallin gene family. Mol Cell Biol. 1985 Jun;5(6):1408–1414. doi: 10.1128/mcb.5.6.1408. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Moormann R. J., den Dunnen J. T., Heuyerjans J., Jongbloed R. J., van Leen R. W., Lubsen N. H., Schoenmakers J. G. Characterization of the rat gamma-crystallin gene family and its expression in the eye lens. J Mol Biol. 1985 Apr 5;182(3):419–430. doi: 10.1016/0022-2836(85)90201-3. [DOI] [PubMed] [Google Scholar]
  23. Mulligan R. C., Berg P. Selection for animal cells that express the Escherichia coli gene coding for xanthine-guanine phosphoribosyltransferase. Proc Natl Acad Sci U S A. 1981 Apr;78(4):2072–2076. doi: 10.1073/pnas.78.4.2072. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Murer-Orlando M., Paterson R. C., Lok S., Tsui L. C., Breitman M. L. Differential regulation of gamma-crystallin genes during mouse lens development. Dev Biol. 1987 Jan;119(1):260–267. doi: 10.1016/0012-1606(87)90227-2. [DOI] [PubMed] [Google Scholar]
  25. Okazaki K., Yasuda K., Kondoh H., Okada T. S. DNA sequences responsible for tissue-specific expression of a chicken alpha-crystallin gene in mouse lens cells. EMBO J. 1985 Oct;4(10):2589–2595. doi: 10.1002/j.1460-2075.1985.tb03975.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Parker B. A., Stark G. R. Regulation of simian virus 40 transcription: sensitive analysis of the RNA species present early in infections by virus or viral DNA. J Virol. 1979 Aug;31(2):360–369. doi: 10.1128/jvi.31.2.360-369.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Ramaekers F., Dodemont H., Vorstenbosch P., Bloemendal H. Classification of rat lens crystallins and identification of proteins encoded by rat lens mRNA. Eur J Biochem. 1982 Nov 15;128(2-3):503–508. doi: 10.1111/j.1432-1033.1982.tb06993.x. [DOI] [PubMed] [Google Scholar]
  28. Ringens P. J., Hoenders H. J., Bloemendal H. Effect of aging on the water-soluble and water-insoluble protein pattern in normal human lens. Exp Eye Res. 1982 Feb;34(2):201–207. doi: 10.1016/0014-4835(82)90054-9. [DOI] [PubMed] [Google Scholar]
  29. Ringens P. J., Hoenders H. J., Bloemendal H. Protein distribution and characterization in the prenatal and postnatal human lens. Exp Eye Res. 1982 May;34(5):815–823. doi: 10.1016/s0014-4835(82)80041-9. [DOI] [PubMed] [Google Scholar]
  30. Shiloh Y., Donlon T., Bruns G., Breitman M. L., Tsui L. C. Assignment of the human gamma-crystallin gene cluster (CRYG) to the long arm of chromosome 2, region q33-36. Hum Genet. 1986 May;73(1):17–19. doi: 10.1007/BF00292656. [DOI] [PubMed] [Google Scholar]
  31. Summers L., Slingsby C., White H., Narebor M., Moss D., Miller L., Mahadevan D., Lindley P., Driessen H., Blundell T. The molecular structures and interactions of bovine and human gamma-crystallins. Ciba Found Symp. 1984;106:219–236. doi: 10.1002/9780470720875.ch13. [DOI] [PubMed] [Google Scholar]
  32. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Tripathi R. C., Tripathi B. J. Lens morphology, aging, and cataract. J Gerontol. 1983 May;38(3):258–270. doi: 10.1093/geronj/38.3.258. [DOI] [PubMed] [Google Scholar]
  34. Willard H. F., Meakin S. O., Tsui L. C., Breitman M. L. Assignment of human gamma crystallin multigene family to chromosome 2. Somat Cell Mol Genet. 1985 Sep;11(5):511–516. doi: 10.1007/BF01534846. [DOI] [PubMed] [Google Scholar]
  35. Wistow G., Turnell B., Summers L., Slingsby C., Moss D., Miller L., Lindley P., Blundell T. X-ray analysis of the eye lens protein gamma-II crystallin at 1.9 A resolution. J Mol Biol. 1983 Oct 15;170(1):175–202. doi: 10.1016/s0022-2836(83)80232-0. [DOI] [PubMed] [Google Scholar]
  36. Zigler J. S., Jr, Russell P., Horwitz J., Reddy V. N., Kinoshita J. H. Further studies on low molecular weight crystallins: relationship between the bovine beta s, the human 24kD protein and the gamma-crystallins. Curr Eye Res. 1986 May;5(5):395–401. doi: 10.3109/02713688609025179. [DOI] [PubMed] [Google Scholar]
  37. Zigler J. S., Jr, Russell P., Takemoto L. J., Schwab S. J., Hansen J. S., Horwitz J., Kinoshita J. H. Partial characterization of three distinct populations of human gamma-crystallins. Invest Ophthalmol Vis Sci. 1985 Apr;26(4):525–531. [PubMed] [Google Scholar]
  38. den Dunnen J. T., Jongbloed R. J., Geurts van Kessel A. H., Schoenmakers J. G. Human lens gamma-crystallin sequences are located in the p12-qter region of chromosome 2. Hum Genet. 1985;70(3):217–221. doi: 10.1007/BF00273445. [DOI] [PubMed] [Google Scholar]
  39. den Dunnen J. T., Moormann R. J., Cremers F. P., Schoenmakers J. G. Two human gamma-crystallin genes are linked and riddled with Alu-repeats. Gene. 1985;38(1-3):197–204. doi: 10.1016/0378-1119(85)90218-5. [DOI] [PubMed] [Google Scholar]
  40. den Dunnen J. T., Moormann R. J., Lubsen N. H., Schoenmakers J. G. Concerted and divergent evolution within the rat gamma-crystallin gene family. J Mol Biol. 1986 May 5;189(1):37–46. doi: 10.1016/0022-2836(86)90379-7. [DOI] [PubMed] [Google Scholar]
  41. van Leen R. W., van Roozendaal K. E., Lubsen N. H., Schoenmakers J. G. Differential expression of crystallin genes during development of the rat eye lens. Dev Biol. 1987 Apr;120(2):457–464. doi: 10.1016/0012-1606(87)90249-1. [DOI] [PubMed] [Google Scholar]

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