Skip to main content
NCBI home page
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
. 1995 Apr 25;92(9):4016–4020. doi: 10.1073/pnas.92.9.4016

Rod photoreceptor-specific gene expression in human retinoblastoma cells.

A Di Polo 1, D B Farber 1
PMCID: PMC42093  PMID: 7732024

Abstract

Retinoblastoma cells in culture have previously been shown to express cone-specific genes but not their rod counterparts. We have detected the messages for the rod alpha, beta, and gamma subunits of cGMP phosphodiesterase (PDE), the rod alpha subunit of transducin, rod opsin, and the cone alpha' subunit of PDE in RNA of human Y-79 retinoblastoma cells by reverse transcription-PCR. Quantitative analysis of the mRNAs for the rod alpha and cone alpha' PDE subunits revealed that they were expressed at comparable levels; however, the transcript encoding the rod beta PDE subunit was 10 times more abundant in these cells. Northern hybridization analysis of Y-79 cell RNA confirmed the presence of the transcripts for rod and cone PDE catalytic subunits. To test whether the transcriptional machinery required for the expression of rod-specific genes was endogenous in Y-79 retinoblastoma cells, cultures were transfected with a construct containing the promoter region of the rod beta PDE subunit gene attached to the firefly luciferase reporter vector. Significant levels of reporter enzyme activity were observed in the cell lysates. Our results demonstrate that the Y-79 retinoblastoma cell line is a good model system for the study of transcriptional regulation of rod-specific genes.

Full text

PDF
4016

Images in this article

4018Fig. 1
on p.4018
4018Fig. 2
on p.4018
4018Fig. 3
on p.4018
4019Fig. 4
on p.4019

Selected References

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

  1. Amara F. M., Chen F. Y., Wright J. A. Phorbol ester modulation of a novel cytoplasmic protein binding activity at the 3'-untranslated region of mammalian ribonucleotide reductase R2 mRNA and role in message stability. J Biol Chem. 1994 Mar 4;269(9):6709–6715. [PubMed] [Google Scholar]
  2. Bogenmann E., Lochrie M. A., Simon M. I. Cone cell-specific genes expressed in retinoblastoma. Science. 1988 Apr 1;240(4848):76–78. doi: 10.1126/science.2451289. [DOI] [PubMed] [Google Scholar]
  3. Bowes C., Li T., Frankel W. N., Danciger M., Coffin J. M., Applebury M. L., Farber D. B. Localization of a retroviral element within the rd gene coding for the beta subunit of cGMP phosphodiesterase. Proc Natl Acad Sci U S A. 1993 Apr 1;90(7):2955–2959. doi: 10.1073/pnas.90.7.2955. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chelly J., Concordet J. P., Kaplan J. C., Kahn A. Illegitimate transcription: transcription of any gene in any cell type. Proc Natl Acad Sci U S A. 1989 Apr;86(8):2617–2621. doi: 10.1073/pnas.86.8.2617. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chelly J., Montarras D., Pinset C., Berwald-Netter Y., Kaplan J. C., Kahn A. Quantitative estimation of minor mRNAs by cDNA-polymerase chain reaction. Application to dystrophin mRNA in cultured myogenic and brain cells. Eur J Biochem. 1990 Feb 14;187(3):691–698. doi: 10.1111/j.1432-1033.1990.tb15355.x. [DOI] [PubMed] [Google Scholar]
  6. Chen C., Okayama H. High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol. 1987 Aug;7(8):2745–2752. doi: 10.1128/mcb.7.8.2745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
  8. Dryja T. P., Hahn L. B., Cowley G. S., McGee T. L., Berson E. L. Mutation spectrum of the rhodopsin gene among patients with autosomal dominant retinitis pigmentosa. Proc Natl Acad Sci U S A. 1991 Oct 15;88(20):9370–9374. doi: 10.1073/pnas.88.20.9370. [DOI] [PMC free article] [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. Fesenko E. E., Kolesnikov S. S., Lyubarsky A. L. Induction by cyclic GMP of cationic conductance in plasma membrane of retinal rod outer segment. Nature. 1985 Jan 24;313(6000):310–313. doi: 10.1038/313310a0. [DOI] [PubMed] [Google Scholar]
  11. Fong S. L. Characterization of the human rod transducin alpha-subunit gene. Nucleic Acids Res. 1992 Jun 11;20(11):2865–2870. doi: 10.1093/nar/20.11.2865. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fung B. K., Young J. H., Yamane H. K., Griswold-Prenner I. Subunit stoichiometry of retinal rod cGMP phosphodiesterase. Biochemistry. 1990 Mar 20;29(11):2657–2664. doi: 10.1021/bi00463a006. [DOI] [PubMed] [Google Scholar]
  13. Gal A., Orth U., Baehr W., Schwinger E., Rosenberg T. Heterozygous missense mutation in the rod cGMP phosphodiesterase beta-subunit gene in autosomal dominant stationary night blindness. Nat Genet. 1994 May;7(1):64–68. doi: 10.1038/ng0594-64. [DOI] [PubMed] [Google Scholar]
  14. Gillespie P. G., Beavo J. A. Characterization of a bovine cone photoreceptor phosphodiesterase purified by cyclic GMP-sepharose chromatography. J Biol Chem. 1988 Jun 15;263(17):8133–8141. [PubMed] [Google Scholar]
  15. Lantz V., Schedl P. Multiple cis-acting targeting sequences are required for orb mRNA localization during Drosophila oogenesis. Mol Cell Biol. 1994 Apr;14(4):2235–2242. doi: 10.1128/mcb.14.4.2235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lem J., Flannery J. G., Li T., Applebury M. L., Farber D. B., Simon M. I. Retinal degeneration is rescued in transgenic rd mice by expression of the cGMP phosphodiesterase beta subunit. Proc Natl Acad Sci U S A. 1992 May 15;89(10):4422–4426. doi: 10.1073/pnas.89.10.4422. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. McLaughlin M. E., Sandberg M. A., Berson E. L., Dryja T. P. Recessive mutations in the gene encoding the beta-subunit of rod phosphodiesterase in patients with retinitis pigmentosa. Nat Genet. 1993 Jun;4(2):130–134. doi: 10.1038/ng0693-130. [DOI] [PubMed] [Google Scholar]
  18. Nathans J., Hogness D. S. Isolation and nucleotide sequence of the gene encoding human rhodopsin. Proc Natl Acad Sci U S A. 1984 Aug;81(15):4851–4855. doi: 10.1073/pnas.81.15.4851. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Peterson G. L. A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal Biochem. 1977 Dec;83(2):346–356. doi: 10.1016/0003-2697(77)90043-4. [DOI] [PubMed] [Google Scholar]
  20. Piriev N. I., Purishko V. A., Khramtsov N. V., Lipkin V. M. Organizatsiia gena gamma-sub''edinitsy fotoretseptornoi fosfodiésterazy tsiklicheskogo GMF cheloveka. Dokl Akad Nauk SSSR. 1990;315(1):229–231. [PubMed] [Google Scholar]
  21. Pittler S. J., Baehr W. Identification of a nonsense mutation in the rod photoreceptor cGMP phosphodiesterase beta-subunit gene of the rd mouse. Proc Natl Acad Sci U S A. 1991 Oct 1;88(19):8322–8326. doi: 10.1073/pnas.88.19.8322. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Pittler S. J., Baehr W., Wasmuth J. J., McConnell D. G., Champagne M. S., vanTuinen P., Ledbetter D., Davis R. L. Molecular characterization of human and bovine rod photoreceptor cGMP phosphodiesterase alpha-subunit and chromosomal localization of the human gene. Genomics. 1990 Feb;6(2):272–283. doi: 10.1016/0888-7543(90)90567-e. [DOI] [PubMed] [Google Scholar]
  23. Rodrigues M. M., Wilson M. E., Wiggert B., Krishna G., Chader G. J. Retinoblastoma. A clinical, immunohistochemical, and electron microscopic case report. Ophthalmology. 1986 Aug;93(8):1010–1015. doi: 10.1016/s0161-6420(86)33630-3. [DOI] [PubMed] [Google Scholar]
  24. Rosenthal N. Identification of regulatory elements of cloned genes with functional assays. Methods Enzymol. 1987;152:704–720. doi: 10.1016/0076-6879(87)52075-4. [DOI] [PubMed] [Google Scholar]
  25. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Sarkar G., Sommer S. S. Access to a messenger RNA sequence or its protein product is not limited by tissue or species specificity. Science. 1989 Apr 21;244(4902):331–334. doi: 10.1126/science.2565599. [DOI] [PubMed] [Google Scholar]
  27. Stryer L. Visual excitation and recovery. J Biol Chem. 1991 Jun 15;266(17):10711–10714. [PubMed] [Google Scholar]
  28. Suber M. L., Pittler S. J., Qin N., Wright G. C., Holcombe V., Lee R. H., Craft C. M., Lolley R. N., Baehr W., Hurwitz R. L. Irish setter dogs affected with rod/cone dysplasia contain a nonsense mutation in the rod cGMP phosphodiesterase beta-subunit gene. Proc Natl Acad Sci U S A. 1993 May 1;90(9):3968–3972. doi: 10.1073/pnas.90.9.3968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Svitkin Y. V., Meerovitch K., Lee H. S., Dholakia J. N., Kenan D. J., Agol V. I., Sonenberg N. Internal translation initiation on poliovirus RNA: further characterization of La function in poliovirus translation in vitro. J Virol. 1994 Mar;68(3):1544–1550. doi: 10.1128/jvi.68.3.1544-1550.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Wang A. M., Doyle M. V., Mark D. F. Quantitation of mRNA by the polymerase chain reaction. Proc Natl Acad Sci U S A. 1989 Dec;86(24):9717–9721. doi: 10.1073/pnas.86.24.9717. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Weber B., Riess O., Hutchinson G., Collins C., Lin B. Y., Kowbel D., Andrew S., Schappert K., Hayden M. R. Genomic organization and complete sequence of the human gene encoding the beta-subunit of the cGMP phosphodiesterase and its localisation to 4p 16.3. Nucleic Acids Res. 1991 Nov 25;19(22):6263–6268. doi: 10.1093/nar/19.22.6263. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES