Abstract
We placed UAA, UAG and UGA nonsense mutations at two leucine codons, Leu(205) and Leu(309), in Drosophila's major rhodopsin gene, ninaE, by site-directed mutagenesis, and then created the corresponding mutants by P element-mediated transformation of a ninaE deficiency strain. In the absence of a genetic suppressor, flies harboring any of the nonsense mutations at the 309 site, but not the 205 site, show increased rhodopsin activity. Additionally, all flies with nonsense mutations at either site have better rhabdomere structure than does the ninaE deficiency strain. Construction and analysis of a 3'-deletion mutant of ninaE indicates that translational readthrough accounts for the extra photoreceptor activity of the ninaE(309) alleles and that truncated opsins are responsible for the improved rhabdomere structure. The presence of leucine-inserting tRNA nonsense suppressors DtL(a) Su(+) and DtL(b) Su(+) in the mutant strains produced a small increase (less than 0.04%) in functional rhodopsin. The opal (UGA) suppressor derived from the DtL(a) tRNA gene is more efficient than the amber (UAG) or opal suppressor derived from the DtL(b) gene, and both DtL(a) and DtL(b) derived suppressors are more efficient at site 205 than 309.
Full Text
The Full Text of this article is available as a PDF (7.1 MB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Bossi L. Context effects: translation of UAG codon by suppressor tRNA is affected by the sequence following UAG in the message. J Mol Biol. 1983 Feb 15;164(1):73–87. doi: 10.1016/0022-2836(83)90088-8. [DOI] [PubMed] [Google Scholar]
- Cathala G., Savouret J. F., Mendez B., West B. L., Karin M., Martial J. A., Baxter J. D. A method for isolation of intact, translationally active ribonucleic acid. DNA. 1983;2(4):329–335. doi: 10.1089/dna.1983.2.329. [DOI] [PubMed] [Google Scholar]
- Church G. M., Gilbert W. Genomic sequencing. Proc Natl Acad Sci U S A. 1984 Apr;81(7):1991–1995. doi: 10.1073/pnas.81.7.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Doerig R. E., Suter B., Gray M., Kubli E. Identification of an amber nonsense mutation in the rosy516 gene by germline transformation of an amber suppressor tRNA gene. EMBO J. 1988 Aug;7(8):2579–2584. doi: 10.1002/j.1460-2075.1988.tb03107.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Feng Y. X., Levin J. G., Hatfield D. L., Schaefer T. S., Gorelick R. J., Rein A. Suppression of UAA and UGA termination codons in mutant murine leukemia viruses. J Virol. 1989 Jun;63(6):2870–2873. doi: 10.1128/jvi.63.6.2870-2873.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Franceschini N., Kirschfeld K. Etude optique in vivo des éléments photorécepteurs dans l'oeil composé de Drosophila. Kybernetik. 1971 Jan;8(1):1–13. doi: 10.1007/BF00270828. [DOI] [PubMed] [Google Scholar]
- Garza D., Medhora M. M., Hartl D. L. Drosophila nonsense suppressors: functional analysis in Saccharomyces cerevisiae, Drosophila tissue culture cells and Drosophila melanogaster. Genetics. 1990 Nov;126(3):625–637. doi: 10.1093/genetics/126.3.625. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Geller A. I., Rich A. A UGA termination suppression tRNATrp active in rabbit reticulocytes. Nature. 1980 Jan 3;283(5742):41–46. doi: 10.1038/283041a0. [DOI] [PubMed] [Google Scholar]
- Johnson E. C., Pak W. L. Electrophysiological study of Drosophila rhodopsin mutants. J Gen Physiol. 1986 Nov;88(5):651–673. doi: 10.1085/jgp.88.5.651. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Karnik S. S., Sakmar T. P., Chen H. B., Khorana H. G. Cysteine residues 110 and 187 are essential for the formation of correct structure in bovine rhodopsin. Proc Natl Acad Sci U S A. 1988 Nov;85(22):8459–8463. doi: 10.1073/pnas.85.22.8459. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kondo K., Makovec B., Waterston R. H., Hodgkin J. Genetic and molecular analysis of eight tRNA(Trp) amber suppressors in Caenorhabditis elegans. J Mol Biol. 1990 Sep 5;215(1):7–19. doi: 10.1016/S0022-2836(05)80090-7. [DOI] [PubMed] [Google Scholar]
- Kubli E. The genetics of transfer RNA in Drosophila. Adv Genet. 1982;21:123–172. doi: 10.1016/s0065-2660(08)60298-9. [DOI] [PubMed] [Google Scholar]
- Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
- Larrivee D. C., Conrad S. K., Stephenson R. S., Pak W. L. Mutation that selectively affects rhodopsin concentration in the peripheral photoreceptors of Drosophila melanogaster. J Gen Physiol. 1981 Nov;78(5):521–545. doi: 10.1085/jgp.78.5.521. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Laski F. A., Ganguly S., Sharp P. A., RajBhandary U. L., Rubin G. M. Construction, stable transformation, and function of an amber suppressor tRNA gene in Drosophila melanogaster. Proc Natl Acad Sci U S A. 1989 Sep;86(17):6696–6698. doi: 10.1073/pnas.86.17.6696. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lee Y. J., Dobbs M. B., Verardi M. L., Hyde D. R. dgq: a drosophila gene encoding a visual system-specific G alpha molecule. Neuron. 1990 Dec;5(6):889–898. doi: 10.1016/0896-6273(90)90349-k. [DOI] [PubMed] [Google Scholar]
- Li G. P., Rice C. M. Mutagenesis of the in-frame opal termination codon preceding nsP4 of Sindbis virus: studies of translational readthrough and its effect on virus replication. J Virol. 1989 Mar;63(3):1326–1337. doi: 10.1128/jvi.63.3.1326-1337.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Montell C., Jones K., Zuker C., Rubin G. A second opsin gene expressed in the ultraviolet-sensitive R7 photoreceptor cells of Drosophila melanogaster. J Neurosci. 1987 May;7(5):1558–1566. doi: 10.1523/JNEUROSCI.07-05-01558.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
- O'Connell P. O., Rosbash M. Sequence, structure, and codon preference of the Drosophila ribosomal protein 49 gene. Nucleic Acids Res. 1984 Jul 11;12(13):5495–5513. doi: 10.1093/nar/12.13.5495. [DOI] [PMC free article] [PubMed] [Google Scholar]
- O'Tousa J. E., Baehr W., Martin R. L., Hirsh J., Pak W. L., Applebury M. L. The Drosophila ninaE gene encodes an opsin. Cell. 1985 Apr;40(4):839–850. doi: 10.1016/0092-8674(85)90343-5. [DOI] [PubMed] [Google Scholar]
- O'Tousa J. E., Leonard D. S., Pak W. L. Morphological defects in oraJK84 photoreceptors caused by mutation in R1-6 opsin gene of Drosophila. J Neurogenet. 1989 Sep;6(1):41–52. doi: 10.3109/01677068909107099. [DOI] [PubMed] [Google Scholar]
- Robertson H. M., Preston C. R., Phillis R. W., Johnson-Schlitz D. M., Benz W. K., Engels W. R. A stable genomic source of P element transposase in Drosophila melanogaster. Genetics. 1988 Mar;118(3):461–470. doi: 10.1093/genetics/118.3.461. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Robinson R. R., Davidson N. Analysis of a drosophila tRNA gene cluster: two tRNALeu genes contain intervening sequences. Cell. 1981 Jan;23(1):251–259. doi: 10.1016/0092-8674(81)90289-0. [DOI] [PubMed] [Google Scholar]
- Smith D., Yarus M. tRNA-tRNA interactions within cellular ribosomes. Proc Natl Acad Sci U S A. 1989 Jun;86(12):4397–4401. doi: 10.1073/pnas.86.12.4397. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sprague K. U., Hagenbüchle O., Zuniga M. C. The nucleotide sequence of two silk gland alanine tRNAs: implications for fibroin synthesis and for initiator tRNA structure. Cell. 1977 Jul;11(3):561–570. doi: 10.1016/0092-8674(77)90074-5. [DOI] [PubMed] [Google Scholar]
- Stark W. S., Carlson S. D. Ultrastructure of the compound eye and first optic neuropile of the photoreceptor mutant oraJK84 of Drosophila. Cell Tissue Res. 1983;233(2):305–317. doi: 10.1007/BF00238298. [DOI] [PubMed] [Google Scholar]
- Valle R. P., Morch M. D. Stop making sense: or Regulation at the level of termination in eukaryotic protein synthesis. FEBS Lett. 1988 Aug 1;235(1-2):1–15. doi: 10.1016/0014-5793(88)81225-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Vieira J., Messing J. Production of single-stranded plasmid DNA. Methods Enzymol. 1987;153:3–11. doi: 10.1016/0076-6879(87)53044-0. [DOI] [PubMed] [Google Scholar]
- Washburn T., O'Tousa J. E. Molecular defects in Drosophila rhodopsin mutants. J Biol Chem. 1989 Sep 15;264(26):15464–15466. [PubMed] [Google Scholar]
- Wills N., Gesteland R. F., Karn J., Barnett L., Bolten S., Waterston R. H. The genes sup-7 X and sup-5 III of C. elegans suppress amber nonsense mutations via altered transfer RNA. Cell. 1983 Jun;33(2):575–583. doi: 10.1016/0092-8674(83)90438-5. [DOI] [PubMed] [Google Scholar]
- Zuker C. S., Montell C., Jones K., Laverty T., Rubin G. M. A rhodopsin gene expressed in photoreceptor cell R7 of the Drosophila eye: homologies with other signal-transducing molecules. J Neurosci. 1987 May;7(5):1550–1557. doi: 10.1523/JNEUROSCI.07-05-01550.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]