Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1984 Dec;4(12):2643–2652. doi: 10.1128/mcb.4.12.2643

Isolation and characterization of cloned DNA sequences containing ribosomal protein genes of Drosophila melanogaster.

D K Burns, B C Stark, M D Macklin, W Y Chooi
PMCID: PMC369273  PMID: 6084810

Abstract

Ribosomal (r) proteins encoded by polyadenylated RNA were specifically precipitated in vitro from polysomes by using antibodies raised against characterized Drosophila melanogaster r proteins. The immuno-purified mRNA in the polysome complex was used to prepare cDNA with which to probe a D. melanogaster genomic library. Selected recombinant phages were used to hybrid select mRNAs, which were analyzed by in vitro translation. Three clones containing the genes for r proteins 7/8, S18, and L12 were positively identified by electrophoresis of the translation products in one-dimensional and two-dimensional polyacrylamide gels. Sequences encoding r proteins S18 and L12 were found to be present in the genome in single copies. In contrast, the polynucleotide containing the region encoding 7/8 may be repeated or may contain or be flanked by short repeated sequences. The sizes of mRNAs that hybridized to the recombinant clone containing 7/8 were significantly larger than would be expected from the molecular weight of protein 7/8, implying that there were unusually long 5' and 3' noncoding sequences. The mRNAs for r proteins S18 and L12 were however, only about 10% larger. In situ hybridizations to salivary gland polytene chromosomes, using the recombinant phage, revealed that the recombinant clone containing the gene for r protein 7/8 hybridized to 5D on the X chromosome; the recombinant clone containing the gene for S18 hybridized to 15B on the same chromosome, and the recombinant phage containing the gene for L12 hybridized to 62E on chromosome 3L. It is of interest that the genomic locations of all three r protein clones were within the chromosomal intervals known to contain the Minute mutations [M(1)0, M(1)30, and M(3)LS2]. Although each clone contained sequences specifying two to four proteins, none had more than one identifiable r protein gene, suggesting that different D. melanogaster r protein genes may not be closely linked.

Full text

PDF
2643

Images in this article

2644Image
on p.2644
2645Image
on p.2645
2646Image
on p.2646
2648Image
on p.2648
2649Image
on p.2649
2649Image
on p.2649
2649Image
on p.2649
2650Image
on p.2650

Selected References

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

  1. Benton W. D., Davis R. W. Screening lambdagt recombinant clones by hybridization to single plaques in situ. Science. 1977 Apr 8;196(4286):180–182. doi: 10.1126/science.322279. [DOI] [PubMed] [Google Scholar]
  2. Bollen G. H., Cohen L. H., Mager W. H., Klaassen A. W., Planta R. J. Isolation of cloned ribosomal protein genes from the yeast Saccharomyces carlsbergensis. Gene. 1981 Sep;14(4):279–287. doi: 10.1016/0378-1119(81)90160-8. [DOI] [PubMed] [Google Scholar]
  3. Bozzoni I., Beccari E., Luo Z. X., Amaldi F. Xenopus laevis ribosomal protein genes: isolation of recombinant cDNA clones and study of the genomic organization. Nucleic Acids Res. 1981 Mar 11;9(5):1069–1086. doi: 10.1093/nar/9.5.1069. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bruskin A. M., Tyner A. L., Wells D. E., Showman R. M., Klein W. H. Accumulation in embryogenesis of five mRNAs enriched in the ectoderm of the sea urchin pluteus. Dev Biol. 1981 Oct 30;87(2):308–318. doi: 10.1016/0012-1606(81)90154-8. [DOI] [PubMed] [Google Scholar]
  5. Chooi W. Y., Leiby K. R. An electron microscopic method for localization of ribosomal proteins during transcription of ribosomal DNA: a method for studying protein assembly. Proc Natl Acad Sci U S A. 1981 Aug;78(8):4823–4827. doi: 10.1073/pnas.78.8.4823. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chooi W. Y., Macklin M. D., Leiby K. R., Hong T. H., Scofield S. R., Sabatini L. M., Burns D. K. Purification of Drosophila acidic ribosomal proteins. Eur J Biochem. 1982 Sep;127(1):199–205. doi: 10.1111/j.1432-1033.1982.tb06856.x. [DOI] [PubMed] [Google Scholar]
  7. Chooi W. Y., Otaka E. Immunological evidence for structural homology between Drosophila melanogaster (S14), rabbit liver (S12), Saccharomyces cerevisiae (S25), Bacillus subtilis (S6), and Escherichia coli (S6) ribosomal proteins. Mol Cell Biol. 1984 Nov;4(11):2535–2539. doi: 10.1128/mcb.4.11.2535. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chooi W. Y. Purification of Drosophila ribosomal proteins. Isolation of proteins S8, S13, S14, S16, S19, S20/L24, S22/L26, S24, S25/S27, S26, S29, L4, L10/L11, L12, L13, L16, L18, L19, L27, 1, 7/8, 9, and 11. Biochemistry. 1980 Jul 22;19(15):3469–3476. doi: 10.1021/bi00556a010. [DOI] [PubMed] [Google Scholar]
  9. Chooi W. Y., Sabatini L. M., Macklin M., Fraser D. Group fractionation and determination of the number of ribosomal subunit proteins from Drosophila melanogaster embryos. Biochemistry. 1980 Apr 1;19(7):1425–1433. doi: 10.1021/bi00548a025. [DOI] [PubMed] [Google Scholar]
  10. Chooi W. Y., Sabatini L. M., Macklin M. Structural homology between Drosophila melanogaster and Escherichia coli acidic ribosomal proteins. Biochem Genet. 1984 Aug;22(7-8):749–767. doi: 10.1007/BF00485858. [DOI] [PubMed] [Google Scholar]
  11. Collatz E., Lin A., Stöffler G., Tsurugi K., Wool I. G. Group fractionation of eukaryotic ribosomal proteins. J Biol Chem. 1976 Mar 25;251(6):1808–1816. [PubMed] [Google Scholar]
  12. D'Eustachio P., Meyuhas O., Ruddle F., Perry R. P. Chromosomal distribution of ribosomal protein genes in the mouse. Cell. 1981 May;24(2):307–312. doi: 10.1016/0092-8674(81)90320-2. [DOI] [PubMed] [Google Scholar]
  13. Fabijanski S., Pellegrini M. Isolation of a cloned DNA segment containing a ribosomal protein gene of Drosophila melanogaster. Gene. 1982 Jun;18(3):267–276. doi: 10.1016/0378-1119(82)90165-2. [DOI] [PubMed] [Google Scholar]
  14. Fried H. M., Pearson N. J., Kim C. H., Warner J. R. The genes for fifteen ribosomal proteins of Saccharomyces cerevisiae. J Biol Chem. 1981 Oct 10;256(19):10176–10183. [PubMed] [Google Scholar]
  15. Gough N. M., Adams J. M. Immunoprecipitation of specific polysomes using Staphylococcus aureus: purification of the immunoglobulin k chain messenger RNA from the mouse myeloma MPC11. Biochemistry. 1978 Dec 12;17(25):5560–5566. doi: 10.1021/bi00618a036. [DOI] [PubMed] [Google Scholar]
  16. Kaltschmidt E., Wittmann H. G. Ribosomal proteins. XII. Number of proteins in small and large ribosomal subunits of Escherichia coli as determined by two-dimensional gel electrophoresis. Proc Natl Acad Sci U S A. 1970 Nov;67(3):1276–1282. doi: 10.1073/pnas.67.3.1276. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kessler S. W. Rapid isolation of antigens from cells with a staphylococcal protein A-antibody adsorbent: parameters of the interaction of antibody-antigen complexes with protein A. J Immunol. 1975 Dec;115(6):1617–1624. [PubMed] [Google Scholar]
  18. 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]
  19. Lin A., Wittmann-Liebold B., McNally J., Wool I. G. The primary structure of the acidic phosphoprotein P2 from rat liver 60 S ribosomal subunits. Comparison with ribosomal 'A' proteins from other species. J Biol Chem. 1982 Aug 10;257(15):9189–9197. [PubMed] [Google Scholar]
  20. Lindsley D. L., Sandler L., Baker B. S., Carpenter A. T., Denell R. E., Hall J. C., Jacobs P. A., Miklos G. L., Davis B. K., Gethmann R. C. Segmental aneuploidy and the genetic gross structure of the Drosophila genome. Genetics. 1972 May;71(1):157–184. doi: 10.1093/genetics/71.1.157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Maniatis T., Hardison R. C., Lacy E., Lauer J., O'Connell C., Quon D., Sim G. K., Efstratiadis A. The isolation of structural genes from libraries of eucaryotic DNA. Cell. 1978 Oct;15(2):687–701. doi: 10.1016/0092-8674(78)90036-3. [DOI] [PubMed] [Google Scholar]
  22. McConkey E. H., Bielka H., Gordon J., Lastick S. M., Lin A., Ogata K., Reboud J. P., Traugh J. A., Traut R. R., Warner J. R. Proposed uniform nomenclature for mammalian ribosomal proteins. Mol Gen Genet. 1979 Jan 16;169(1):1–6. doi: 10.1007/BF00267538. [DOI] [PubMed] [Google Scholar]
  23. Meyuhas O., Perry R. P. Construction and identification of cDNA clones for mouse ribosomal proteins: application for the study of r-protein gene expression. Gene. 1980 Jul;10(2):113–129. doi: 10.1016/0378-1119(80)90129-8. [DOI] [PubMed] [Google Scholar]
  24. Nakamichi N., Rhoads D. D., Roufa D. J. The Chinese hamster cell emetine resistance gene. Analysis of cDNA and genomic sequences encoding ribosomal protein S14. J Biol Chem. 1983 Nov 10;258(21):13236–13242. [PubMed] [Google Scholar]
  25. Nomura M., Morgan E. A. Genetics of bacterial ribosomes. Annu Rev Genet. 1977;11:297–347. doi: 10.1146/annurev.ge.11.120177.001501. [DOI] [PubMed] [Google Scholar]
  26. Pardue M. L., Gall J. G. Nucleic acid hybridization to the DNA of cytological preparations. Methods Cell Biol. 1975;10:1–16. doi: 10.1016/s0091-679x(08)60727-x. [DOI] [PubMed] [Google Scholar]
  27. Ricciardi R. P., Miller J. S., Roberts B. E. Purification and mapping of specific mRNAs by hybridization-selection and cell-free translation. Proc Natl Acad Sci U S A. 1979 Oct;76(10):4927–4931. doi: 10.1073/pnas.76.10.4927. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Rigby P. W., Dieckmann M., Rhodes C., Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol. 1977 Jun 15;113(1):237–251. doi: 10.1016/0022-2836(77)90052-3. [DOI] [PubMed] [Google Scholar]
  29. Ritossa F. M., Atwood K. C., Spiegelman S. On the redundancy of DNA complementary to amino acid tranfer RNA and its absence from the nucleolar organizer region of Drosophila melanogaster. Genetics. 1966 Aug;54(2):663–676. doi: 10.1093/genetics/54.2.663. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Scott M. P., Weiner A. J., Hazelrigg T. I., Polisky B. A., Pirrotta V., Scalenghe F., Kaufman T. C. The molecular organization of the Antennapedia locus of Drosophila. Cell. 1983 Dec;35(3 Pt 2):763–776. doi: 10.1016/0092-8674(83)90109-5. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. Spradling A., Hui H., Penman S. Two very different components of messenger RNA in an insect cell line. Cell. 1975 Feb;4(2):131–137. doi: 10.1016/0092-8674(75)90119-1. [DOI] [PubMed] [Google Scholar]
  33. Thomas P. S. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5201–5205. doi: 10.1073/pnas.77.9.5201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. 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]
  35. Vaslet C. A., O'Connell P., Izquierdo M., Rosbash M. Isolation and mapping of a cloned ribosomal protein gene of Drosophila melanogaster. Nature. 1980 Jun 26;285(5767):674–676. doi: 10.1038/285674a0. [DOI] [PubMed] [Google Scholar]
  36. Vieira J., Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. doi: 10.1016/0378-1119(82)90015-4. [DOI] [PubMed] [Google Scholar]
  37. Warner J. R., Gorenstein C. The synthesis of eucaryotic ribosomal proteins in vitro. Cell. 1977 May;11(1):201–212. doi: 10.1016/0092-8674(77)90331-2. [DOI] [PubMed] [Google Scholar]
  38. Wool I. G. The structure and function of eukaryotic ribosomes. Annu Rev Biochem. 1979;48:719–754. doi: 10.1146/annurev.bi.48.070179.003443. [DOI] [PubMed] [Google Scholar]
  39. Woolford J. L., Jr, Hereford L. M., Rosbash M. Isolation of cloned DNA sequences containing ribosomal protein genes from Saccharomyces cerevisiae. Cell. 1979 Dec;18(4):1247–1259. doi: 10.1016/0092-8674(79)90236-8. [DOI] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES