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Mol Cell Biol. 1992 April; 12(4): 1680–1686.
PMCID: PMC369611

Fine-structure map of the human ribosomal protein gene RPS14.

Abstract

We have used polymerase chain reaction-mediated chemical mutagenesis (J.-J. Diaz, D. D. Rhoads, and D. J. Roufa, BioTechniques 11:204-211, 1991) to analyze the genetic fine structure of a human ribosomal protein gene, RPS14. Eighty-three DNA clones containing 158 random single-base substitution mutations were isolated. Mutant RPS14 alleles were tested for biological activity by transfection into cultured Chinese hamster cells. The resulting data permitted us to construct a map of the S14-coding sequence that is comparable to available fine-structure genetic maps of many prokaryotic and lower eukaryotic gene loci. As predicted from the multiplicity of protein-protein and protein-RNA interactions required for ribosomal protein transport and assembly into functional ribosomal subunits, the distribution of null mutations indicated that S14 is composed of multiple, functionally distinct polypeptide domains. Two of the protein's internal domains, designated domains B and D, were essential for S14 biological activity. In contrast, mutations which altered or deleted S14's amino-terminal 20 amino acid residues (domain A) had no observable effect on the protein's assembly and function in mammalian ribosomes. Interestingly, S14 structural domains deduced by in vitro mutagenesis correlate well with the RPS14 gene's exon boundaries.

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Selected References

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  • Bataillé N, Helser T, Fried HM. Cytoplasmic transport of ribosomal subunits microinjected into the Xenopus laevis oocyte nucleus: a generalized, facilitated process. J Cell Biol. 1990 Oct;111(4):1571–1582. [PMC free article] [PubMed]
  • Boersma D, McGill S, Mollenkamp J, Roufa DJ. Emetine resistance in Chinese hamster cells. Analysis of ribosomal proteins prepared from mutant cells. J Biol Chem. 1979 Jan 25;254(2):559–567. [PubMed]
  • Brown SJ, Jewell A, Maki CG, Roufa DJ. A cDNA encoding human ribosomal protein S24. Gene. 1990 Jul 16;91(2):293–296. [PubMed]
  • Chen IT, Roufa DJ. The transcriptionally active human ribosomal protein S17 gene. Gene. 1988 Oct 15;70(1):107–116. [PubMed]
  • Diaz JJ, Rhoads DD, Roufa DJ. Genetic analysis of a vital mammalian housekeeping locus using CHO cells that express a transfected mutant allele. Somat Cell Mol Genet. 1990 Nov;16(6):517–528. [PubMed]
  • Diaz JJ, Rhoads DD, Roufa DJ. PCR-mediated chemical mutagenesis of cloned duplex DNAs. Biotechniques. 1991 Aug;11(2):204–211. [PubMed]
  • Dorit RL, Schoenbach L, Gilbert W. How big is the universe of exons? Science. 1990 Dec 7;250(4986):1377–1382. [PubMed]
  • Gilbert W. Why genes in pieces? Nature. 1978 Feb 9;271(5645):501–501. [PubMed]
  • Kozak M. Influences of mRNA secondary structure on initiation by eukaryotic ribosomes. Proc Natl Acad Sci U S A. 1986 May;83(9):2850–2854. [PubMed]
  • Lanford RE, Butel JS. Construction and characterization of an SV40 mutant defective in nuclear transport of T antigen. Cell. 1984 Jul;37(3):801–813. [PubMed]
  • Madjar JJ, Frahm M, McGill S, Roufa DJ. Ribosomal protein S14 is altered by two-step emetine resistance mutations in Chinese hamster cells. Mol Cell Biol. 1983 Feb;3(2):190–197. [PMC free article] [PubMed]
  • Madjar JJ, Nielsen-Smith K, Frahm M, Roufa DJ. Emetine resistance in chinese hamster ovary cells is associated with an altered ribosomal protein S14 mRNA. Proc Natl Acad Sci U S A. 1982 Feb;79(4):1003–1007. [PubMed]
  • Maki CG, Rhoads DD, Diaz JJ, Roufa DJ. A Drosophila ribosomal protein functions in mammalian cells. Mol Cell Biol. 1990 Sep;10(9):4524–4528. [PMC free article] [PubMed]
  • Marchionni M, Gilbert W. The triosephosphate isomerase gene from maize: introns antedate the plant-animal divergence. Cell. 1986 Jul 4;46(1):133–141. [PubMed]
  • Moreland RB, Nam HG, Hereford LM, Fried HM. Identification of a nuclear localization signal of a yeast ribosomal protein. Proc Natl Acad Sci U S A. 1985 Oct;82(19):6561–6565. [PubMed]
  • Nakamichi N, Rhoads DD, Roufa DJ. 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]
  • Perler F, Efstratiadis A, Lomedico P, Gilbert W, Kolodner R, Dodgson J. The evolution of genes: the chicken preproinsulin gene. Cell. 1980 Jun;20(2):555–566. [PubMed]
  • Rhoads DD, Dixit A, Roufa DJ. Primary structure of human ribosomal protein S14 and the gene that encodes it. Mol Cell Biol. 1986 Aug;6(8):2774–2783. [PMC free article] [PubMed]
  • Rhoads DD, Roufa DJ. Emetine resistance of Chinese hamster cells: structures of wild-type and mutant ribosomal protein S14 mRNAs. Mol Cell Biol. 1985 Jul;5(7):1655–1659. [PMC free article] [PubMed]
  • Rhoads DD, Roufa DJ. A cloned human ribosomal protein gene functions in rodent cells. Mol Cell Biol. 1987 Oct;7(10):3767–3774. [PMC free article] [PubMed]
  • Rhoads DD, Roufa DJ. Molecular evolution of the mammalian ribosomal protein gene, RPS14. Mol Biol Evol. 1991 Jul;8(4):503–514. [PubMed]
  • Sanger F, Coulson AR. A rapid method for determining sequences in DNA by primed synthesis with DNA polymerase. J Mol Biol. 1975 May 25;94(3):441–448. [PubMed]
  • Southern PJ, Berg P. Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J Mol Appl Genet. 1982;1(4):327–341. [PubMed]
  • Suzuki K, Olvera J, Wool IG. Primary structure of rat ribosomal protein S2. A ribosomal protein with arginine-glycine tandem repeats and RGGF motifs that are associated with nucleolar localization and binding to ribonucleic acids. J Biol Chem. 1991 Oct 25;266(30):20007–20010. [PubMed]
  • Underwood MR, Fried HM. Characterization of nuclear localizing sequences derived from yeast ribosomal protein L29. EMBO J. 1990 Jan;9(1):91–99. [PubMed]
  • 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. [PubMed]

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