Ribosomal protein S14 is altered by two-step emetine resistance mutations in Chinese hamster cells.

Madjar, Jean‐Jacques; Frahm, Michael; McGill, S; Roufa, D J

doi:10.1128/mcb.3.2.190

Cited by 35 publications

(36 citation statements)

References 23 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…S14 cDNA probes detect multiple bands in restriction digests of Chinese hamster genomic DNA (34,35). However, because recessive emtB (S14) CHO cell clones are obtainable by single-step mutagenesis, it appears that they express only a single S14 gene (5,27,35). Others mapped the emtB locus to a region of chromosome 2 that is hemizygous in CHO cells (64,65).…”

mentioning

confidence: 99%

“…This gene is the target of emetine resistance mutations, as emtB Chinese hamster ovary (CHO) cell mutants elaborate altered S14 mRNAs and polypeptides (4,5,27,28,35,37,45). We have isolated Chinese hamster S14 and other r-protein cDNAs (35) and have demonstrated that emtB S14 alleles differ from wild type by single base changes in two adjacent arginine codons at the 3' ends of their coding sequences (45).…”

mentioning

confidence: 99%

“…ing the cells in monolayer tissue culture also have been described previously (4,5,27,28 6,1986 on Restriction endonuclease map and DNA sequencing strategy for human genomic S14 clones. (A) Restriction endonuclease map of two overlapping human placental S14 genomic clones isolated from a Charon 28 DNA library.…”

mentioning

confidence: 99%

See 2 more Smart Citations

Primary structure of human ribosomal protein S14 and the gene that encodes it.

Rhoads¹,

Dixit²,

Roufa³

1986

Mol. Cell. Biol.

Self Cite

137

105

View full text Add to dashboard Cite

Chinese hamster ribosomal protein S14 cDNA was used to recognize homologous human cDNA and genomic clones. Human and Chinese hamster S14 protein sequences deduced from the cDNAs are identical. Two overlapping human genomic S14 DNA clones were isolated from a Charon 28 placental DNA library. A fragment of single-copy DNA derived from an intron region of one clone was mapped to the functional RPS14 locus on human chromosome 5q by using a panel of human x Chinese hamster hybrid cell DNAs. The human S14 gene consists of five exons and four introns spanning 5.9 kilobase pairs of DNA. Polyadenylated S14 transcripts purified from HeLa cell cytoplasm display heterogeneous 5' ends that map within noncoding RPS14 exon 1. This precludes assignment of a unique 5' boundary of RPS14 transcripts with respect to the cloned human genomic DNA. Apparently HeLa cells either initiate transcription at multiple sites within RPS14 exon 1, or capped 5' oligonucleotides are removed from most S14 mRNAs posttranscription. In contrast to the few murine ribosomal protein and several other mammalian housekeeping genes whose structures are known, human RPS14 contains a TATA sequence (TATACTT) upstream from exon 1. Three related short sequence motifs, also observed in murine and yeast ribosomal protein genes, occur in this region of the RPS14 gene. RPS14 introns 3 and 4 both contain Alu sequences. Interestingly, the Alu sequence in intron 3 is located slightly downstream from a chromosome 5 deletion breakpoint in one human x hamster hybrid clone analyzed.Ribosomal proteins (r-proteins) are encoded by complex multigene families in eucaryotes and procaryotes. Several bacterial and yeast r-protein genes have been investigated by both biochemical and genetic approaches. Substantial evidence indicates that bacterial and yeast r-protein genes are stringently coregulated by transcriptional, translational, and posttranslational mechanisms (16, 18, 22, 31, 38-40, 42, 47, 59, 60, 66-68). A few r-protein genes have been isolated from the DNAs of higher eucaryotes-Drosophila melanogaster (8,14,41,57), Xenopus laevis (1, 6), mice (13, 43, 58, 63), rats (25, 36), and Chinese hamsters (34,35,45). For lack of appropriate genetic methods and markers, progress in elucidating mechanisms that regulate r-protein genes in these organisms has been slow. Despite this, recombinant DNA techniques permit us to distinguish active, intron-containing animal r-protein genes from processed pseudogenes (13,23,43,58,63) and indicate that they are regulated coordinately under a variety of experimental conditions (15,17,21,24,30,33,44,51,62).The gene encoding Chinese hamster 40S ribosomal subunit protein S14 is the only mammalian r-protein gene currently amenable to somatic genetic analysis in tissue culture. This gene is the target of emetine resistance mutations, as emtB Chinese hamster ovary (CHO) cell mutants elaborate altered S14 mRNAs and polypeptides (4,5,27,28,35,37,45). We have isolated Chinese hamster S14 and other r-protein cDNAs (35) and have demonstrated that emtB ...

show abstract

mentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

Primary structure of human ribosomal protein S14 and the gene that encodes it.

Rhoads¹,

Dixit²,

Roufa³

1986

Mol. Cell. Biol.

Self Cite

137

105

View full text Add to dashboard Cite

show abstract

“…The human RPS14 locus encodes ribosomal protein S14 and is especially well suited for these studies. It is the only mammalian r-protein locus in which a drug selection system facilitates somatic genetics (Boersma et al 1979;Madjar et al 1982Madjar et al , 1983Diaz et al 1990;Diaz and Roufa 1992;Tasheva and Roufa 1993) and for which both genomic and eDNA clones carrying numerous wild-type and mutant alleles are available (Rhoads et al 1986;Rhoads and Roufa 1987;Diaz et al 1990;Overman et al 1993).…”

mentioning

confidence: 99%

Regulation of human RPS14 transcription by intronic antisense RNAs and ribosomal protein S14.

Tasheva

Roufa²

1995

Genes Dev.

Self Cite

View full text Add to dashboard Cite

RNase protection studies reveal two stable RNAs (250 and 280 nucleotides) transcribed from the antisense strand of the human ribosomal protein gene RPS14's first intron. These transcripts, designated a-250 and a-280, map to overlapping segments of the intron's 5' sequence. Neither RNA encodes a polypeptide sequence, and both are expressed in all human cells and tissues examined. Although a-280 is detected among both the cells' nuclear and cytoplasmic RNAs, the great majority of o~-250 is found in the cytoplasmic subcellular compartment. As judged by its resistance to high concentrations of c~-amanitin, cell-free transcription of oL-250 and 0~-280 appears to involve RNA polymerase I. Tissue culture transfection and cell-free transcription experiments demonstrate that oL-250 and ot-280 stimulate S14 mRNA transcription, whereas free ribosomal protein S14 inhibits it. Electrophoretic mobility shift experiments indicate specific binary molecular interactions between r-protein S14, its message and the antisense RNAs. In light of these data, we propose a model for fine regulation of human RPS14 transcription that involves RPS14 intron 1 antisense RNAs as positive effectors and S14 protein as a negative effector.

show abstract

“…Recessive mutations (EmtB) affecting the CHO cell r-protein S14 gene (RPS14) result in resistance to emetine, an alkaloid inhibitor of protein biosynthesis (2,3,16,17,24). EmtB S14 protein is slightly more acidic than wild-type S14 (2), and ribosomes containing mutant S14 are relatively unstable (23,45). RPS14 genomic and cDNA sequences have been cloned from Chinese hamster and human cells, and their complete nucleic acid sequences are known (32,37,38).…”

mentioning

confidence: 99%

A cloned human ribosomal protein gene functions in rodent cells.

Rhoads¹,

Roufa²

1987

Mol. Cell. Biol.

Self Cite

View full text Add to dashboard Cite

Cloned fragments of human ribosomal protein S14 DNA (RPS14) were transfected into cultured Chinese hamster (CHO) cells. Transient expression assays indicated that DNA with as little as 31 base pairs of upstream flanking sequence was transcribed into a polyadenylated, 650-base mRNA that is largely bound to the polyribosomes. In these respects the exogenous human S14 message appeared to function normally in CHO cells. Interestingly, transcription of human RPS14 did not require the TATA sequence located 26 base pairs upstream of exon 1. Stably transformed clones were selected from cultures of emetine-resistant CHO cells (Emr-2) after transfection with pSV2Neo-human RPS14 constructs. Human RPS14 complemented the mutationally based drug resistance of the Chinese hamster cells, demonstrating that the cloned human ribosomal protein gene is functional in rodent cells. Analysis of transformed cells with different amounts of integrated RPS14 indicated that human S14 mRNA levels are not tightly regulated by CHO cells. In contrast, the steady-state S14 level fluctuated only slightly, if at all, in transformed clones whose S14 message contents differed by more than 30-fold. These data support the conclusion that expression of human RPS14 is regulated, at least partially, posttranscriptionally.Ribosomal components are encoded by a complex family of genes in eucaryotic and procaryotic organisms. Because approximately equal amounts of ribosomal proteins (rproteins) and rRNAs are synthesized under various growth conditions (11,13,19,21,28,30,36,40,45), ribosomal genes appear to be controlled by stringent biochemical and genetic mechanisms. Expression of r-protein genes is governed by a combination of transcriptional, translational, and posttranslational mechanisms in bacteria and lower eucaryotes (12,15,20,29,33,35,39,43,44,(47)(48)(49). Comparable information about eucaryotic, especially mammalian, r-protein genes is sparse owing to a lack of high-resolution genetic systems.Only one of the mammalian r-protein genes so far isolated is amenable to genetic analysis in tissue culture. Recessive mutations (EmtB) affecting the CHO cell r-protein S14 gene (RPS14) result in resistance to emetine, an alkaloid inhibitor of protein biosynthesis (2,3,16,17,24). EmtB S14 protein is slightly more acidic than wild-type S14 (2), and ribosomes containing mutant S14 are relatively unstable (23, 45). RPS14 genomic and cDNA sequences have been cloned from Chinese hamster and human cells, and their complete nucleic acid sequences are known (32,37,38). Human RPS14 consists of five exons and four introns spanning 6 kilobase pairs (kbp) of chromosome Sq DNA (37). It contains a TATA box 28 bp upstream of exon 1. In this regard RPS14 differs from the few other well-characterized mammalian (mouse) r-protein genes, which all lack TATA sequences (9,42,46 protein. Our data demonstrated that the cloned DNAs encode a complete and functional r-protein gene. Disrupting the TATA sequence immediately 5' of exon 1 (37) did not preclude transcription of S14 mRNA, w...

show abstract

Ribosomal protein S14 is altered by two-step emetine resistance mutations in Chinese hamster cells.

Cited by 35 publications

References 23 publications

Primary structure of human ribosomal protein S14 and the gene that encodes it.

Primary structure of human ribosomal protein S14 and the gene that encodes it.

Regulation of human RPS14 transcription by intronic antisense RNAs and ribosomal protein S14.

A cloned human ribosomal protein gene functions in rodent cells.

Contact Info

Product

Resources

About