Emetine resistance of Chinese hamster cells: structures of wild-type and mutant ribosomal protein S14 mRNAs

1985 ◽  
Vol 5 (7) ◽  
pp. 1655-1659
Author(s):  
D D Rhoads ◽  
D J Roufa
Keyword(s):  
Ribosomal Protein ◽  
Ribosomal Subunit ◽  
Chinese Hamster ◽  
Housekeeping Gene ◽  
Single Copy ◽  
Wild Type ◽  
Cho Cell ◽  
40S Ribosomal Subunit ◽  
And Function ◽  
Physical And Chemical

The Chinese hamster ovary (CHO) cell 40S ribosomal subunit protein S14 provides a unique opportunity to investigate an important mammalian housekeeping gene and its mRNA and protein products. The S14 gene appears to be single copy, and CHO cell S14 mutants have been isolated as emetine-resistant (emtB) clones in tissue culture. Thus, S14 is the only mammalian ribosomal protein whose gene structure and function are amenable to straightforward genetic and biochemical analysis. Recently, we isolated a wild-type Chinese hamster lung cell cDNA clone, pCS14-1, including an almost complete copy of the ribosomal protein S14 message (N. Nakamichi, D. D. Rhoads, and D. J. Roufa, J. Biol. Chem. 258: 13236-13242, 1983). Here we describe comparable cDNAs from wild-type and emtB CHO cells. We report both mRNA and polypeptide sequences of the wild-type and mutant ribosomal protein transcripts. As a consequence of the genetic methods used to obtain our emetine-resistant mutants, the emtB S14 cDNAs differ from wild-type cDNA by single-base changes. Physical and chemical features of polypeptides encoded by the cDNAs are consistent with well-characterized S14 protein polymorphisms. The three emtB mutations analyzed affect two adjacent arginine codons within the very basic S14 carboxyl region, indicating a significant role for this portion of the protein in the function and architecture of the mammalian 40S ribosomal subunit.

scholarly journals Emetine resistance of Chinese hamster cells: structures of wild-type and mutant ribosomal protein S14 mRNAs.

1985 ◽  
Vol 5 (7) ◽  
pp. 1655-1659 ◽  
Author(s):  
D D Rhoads ◽  
D J Roufa
Keyword(s):  
Ribosomal Protein ◽  
Ribosomal Subunit ◽  
Chinese Hamster ◽  
Housekeeping Gene ◽  
Single Copy ◽  
Wild Type ◽  
Cho Cell ◽  
40S Ribosomal Subunit ◽  
And Function ◽  
Physical And Chemical

The Chinese hamster ovary (CHO) cell 40S ribosomal subunit protein S14 provides a unique opportunity to investigate an important mammalian housekeeping gene and its mRNA and protein products. The S14 gene appears to be single copy, and CHO cell S14 mutants have been isolated as emetine-resistant (emtB) clones in tissue culture. Thus, S14 is the only mammalian ribosomal protein whose gene structure and function are amenable to straightforward genetic and biochemical analysis. Recently, we isolated a wild-type Chinese hamster lung cell cDNA clone, pCS14-1, including an almost complete copy of the ribosomal protein S14 message (N. Nakamichi, D. D. Rhoads, and D. J. Roufa, J. Biol. Chem. 258: 13236-13242, 1983). Here we describe comparable cDNAs from wild-type and emtB CHO cells. We report both mRNA and polypeptide sequences of the wild-type and mutant ribosomal protein transcripts. As a consequence of the genetic methods used to obtain our emetine-resistant mutants, the emtB S14 cDNAs differ from wild-type cDNA by single-base changes. Physical and chemical features of polypeptides encoded by the cDNAs are consistent with well-characterized S14 protein polymorphisms. The three emtB mutations analyzed affect two adjacent arginine codons within the very basic S14 carboxyl region, indicating a significant role for this portion of the protein in the function and architecture of the mammalian 40S ribosomal subunit.

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

1983 ◽  
Vol 3 (2) ◽  
pp. 190-197
Author(s):  
J J Madjar ◽  
M Frahm ◽  
S McGill ◽  
D J Roufa
Keyword(s):  
Ribosomal Protein ◽  
Ribosomal Proteins ◽  
Ribosomal Subunit ◽  
Chinese Hamster ◽  
Complementation Group ◽  
Resistance Mutations ◽  
Cho Cell ◽  
Ribosomal Subunits ◽  
40S Ribosomal Subunit ◽  
One Step

Four two-dimensional polyacrylamide gel electrophoresis systems were used to identify 78 Chinese hamster cell ribosomal proteins by the uniform nomenclature based on rat liver ribosomal proteins. The 40S ribosomal subunit protein affected by Chinese hamster ovary (CHO) cell one-step emetine resistance mutations is designated S14 in the standard nomenclature. To seek unambiguous genetic evidence for a cause and effect relationship between CHO cell emetine resistance and mutations in the S14 gene, we mutagenized a one-step CHO cell mutant and isolated second-step mutant clones resistant to 10-fold-higher concentrations of emetine. All of the highly resistant, two-step CHO cell mutants obtained displayed additional alterations in ribosomal protein S14. Hybridization complementation tests revealed that the two-step CHO cell emetine resistance mutants were members of the same complementation group defined by one-step CHO cell mutants, EmtB. Two-step mutants obtained from a Chinese hamster lung cell emetine-resistant clone belong to the EmtA complementation group. The two-step and EmtB mutants elaborated 40S ribosomal subunits, which dissociated to 32S and 40S core particles in buffers containing 0.5 M KCl at 4 degrees C. In contrast, 40S ribosomal subunits purified from all EmtA, one-step EmtB EmtC mutants, and wild-type CHO and lung cells were stable at this temperature in buffers containing substantially higher concentrations of salt. Thus, two-step emtB mutations affect the structure of S14 protein directly and the stability of the 40S ribosomal subunit indirectly.

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

1983 ◽  
Vol 3 (2) ◽  
pp. 190-197 ◽  
Author(s):  
J J Madjar ◽  
M Frahm ◽  
S McGill ◽  
D J Roufa
Keyword(s):  
Ribosomal Protein ◽  
Ribosomal Proteins ◽  
Ribosomal Subunit ◽  
Chinese Hamster ◽  
Complementation Group ◽  
Resistance Mutations ◽  
Cho Cell ◽  
Ribosomal Subunits ◽  
40S Ribosomal Subunit ◽  
One Step

Four two-dimensional polyacrylamide gel electrophoresis systems were used to identify 78 Chinese hamster cell ribosomal proteins by the uniform nomenclature based on rat liver ribosomal proteins. The 40S ribosomal subunit protein affected by Chinese hamster ovary (CHO) cell one-step emetine resistance mutations is designated S14 in the standard nomenclature. To seek unambiguous genetic evidence for a cause and effect relationship between CHO cell emetine resistance and mutations in the S14 gene, we mutagenized a one-step CHO cell mutant and isolated second-step mutant clones resistant to 10-fold-higher concentrations of emetine. All of the highly resistant, two-step CHO cell mutants obtained displayed additional alterations in ribosomal protein S14. Hybridization complementation tests revealed that the two-step CHO cell emetine resistance mutants were members of the same complementation group defined by one-step CHO cell mutants, EmtB. Two-step mutants obtained from a Chinese hamster lung cell emetine-resistant clone belong to the EmtA complementation group. The two-step and EmtB mutants elaborated 40S ribosomal subunits, which dissociated to 32S and 40S core particles in buffers containing 0.5 M KCl at 4 degrees C. In contrast, 40S ribosomal subunits purified from all EmtA, one-step EmtB EmtC mutants, and wild-type CHO and lung cells were stable at this temperature in buffers containing substantially higher concentrations of salt. Thus, two-step emtB mutations affect the structure of S14 protein directly and the stability of the 40S ribosomal subunit indirectly.

Yeast ribosomal protein L1 is required for the stability of newly synthesized 5S rRNA and the assembly of 60S ribosomal subunits

1993 ◽  
Vol 13 (5) ◽  
pp. 2835-2845
Author(s):  
M Deshmukh ◽  
Y F Tsay ◽  
A G Paulovich ◽  
J L Woolford
Keyword(s):  
Ribosomal Protein ◽  
Ribosomal Subunit ◽  
Single Copy ◽  
5S Rrna ◽  
Gene Encoding ◽  
Ribosomal Subunits ◽  
Ribosomal Protein L1 ◽  
The Stability ◽  
And Function

Ribosomal protein L1 from Saccharomyces cerevisiae binds 5S rRNA and can be released from intact 60S ribosomal subunits as an L1-5S ribonucleoprotein (RNP) particle. To understand the nature of the interaction between L1 and 5S rRNA and to assess the role of L1 in ribosome assembly and function, we cloned the RPL1 gene encoding L1. We have shown that RPL1 is an essential single-copy gene. A conditional null mutant in which the only copy of RPL1 is under control of the repressible GAL1 promoter was constructed. Depletion of L1 causes instability of newly synthesized 5S rRNA in vivo. Cells depleted of L1 no longer assemble 60S ribosomal subunits, indicating that L1 is required for assembly of stable 60S ribosomal subunits but not 40S ribosomal subunits. An L1-5S RNP particle not associated with ribosomal particles was detected by coimmunoprecipitation of L1 and 5S rRNA. This pool of L1-5S RNP remained stable even upon cessation of 60S ribosomal subunit assembly by depletion of another ribosomal protein, L16. Preliminary results suggest that transcription of RPL1 is not autogenously regulated by L1.

scholarly journals Yeast ribosomal protein L1 is required for the stability of newly synthesized 5S rRNA and the assembly of 60S ribosomal subunits.

1993 ◽  
Vol 13 (5) ◽  
pp. 2835-2845 ◽  
Author(s):  
M Deshmukh ◽  
Y F Tsay ◽  
A G Paulovich ◽  
J L Woolford
Keyword(s):  
Ribosomal Protein ◽  
Ribosomal Subunit ◽  
Single Copy ◽  
5S Rrna ◽  
Gene Encoding ◽  
Ribosomal Subunits ◽  
Ribosomal Protein L1 ◽  
The Stability ◽  
And Function

Ribosomal protein L1 from Saccharomyces cerevisiae binds 5S rRNA and can be released from intact 60S ribosomal subunits as an L1-5S ribonucleoprotein (RNP) particle. To understand the nature of the interaction between L1 and 5S rRNA and to assess the role of L1 in ribosome assembly and function, we cloned the RPL1 gene encoding L1. We have shown that RPL1 is an essential single-copy gene. A conditional null mutant in which the only copy of RPL1 is under control of the repressible GAL1 promoter was constructed. Depletion of L1 causes instability of newly synthesized 5S rRNA in vivo. Cells depleted of L1 no longer assemble 60S ribosomal subunits, indicating that L1 is required for assembly of stable 60S ribosomal subunits but not 40S ribosomal subunits. An L1-5S RNP particle not associated with ribosomal particles was detected by coimmunoprecipitation of L1 and 5S rRNA. This pool of L1-5S RNP remained stable even upon cessation of 60S ribosomal subunit assembly by depletion of another ribosomal protein, L16. Preliminary results suggest that transcription of RPL1 is not autogenously regulated by L1.

scholarly journals Cloning and characterization of small circular DNA from Chinese hamster ovary cells.

1984 ◽  
Vol 4 (1) ◽  
pp. 173-180 ◽  
Author(s):  
S W Stanfield ◽  
D R Helinski
Keyword(s):  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Single Copy ◽  
Chromosomal Dna ◽  
Cho Cell ◽  
Ovary Cells ◽  
Chromosomal Sequences

Small polydisperse circular (spc) DNA was isolated and cloned, using BglII from Chinese hamster ovary (CHO) cells. The properties of 47 clones containing at least 43 different BglII fragments are reported. The majority of the clones probably contain entire sequences from individual spcDNA molecules. Most of the clones were homologous to sequences in CHO cell chromosomal DNA, and many were also homologous to mouse LMTK- cell chromosomal sequences. The majority of homologous CHO cell chromosomal sequences were repetitive, although a few may be single copy. Only a small fraction of cloned spcDNA molecules were present in every cell; most occurred less frequently than once in 15 cells. Localization studies indicated that at least a portion of spcDNA is associated with the nucleus in CHO cells.

A mammalian origin of bidirectional DNA replication within the Chinese hamster RPS14 locus

1994 ◽  
Vol 14 (9) ◽  
pp. 5628-5635
Author(s):  
E S Tasheva ◽  
D J Roufa
Keyword(s):  
Dna Replication ◽  
Chinese Hamster ◽  
Housekeeping Gene ◽  
Single Copy ◽  
The Other ◽  
Replication Origins ◽  
Chromosomal Replication ◽  
Dna Binding Sites ◽  
Sequence Encoding ◽  
Copy Dna

Two complementary experimental approaches have been used to identify a chromosomal origin of bidirectional DNA replication within or immediately downstream of the Chinese hamster ribosomal protein S14 gene (RPS14). The replication origin, designated oriS14, maps within a 1.6- to 2.0-kbp region of RPS14 that includes the gene's third and fourth introns, exons IV plus V, and approximately 500 bp of proximal downstream flanking DNA. The nucleic acid sequence encoding oriS14 closely resembles the other mammalian chromosomal replication origins whose primary structures are known. It contains DNA binding sites for a large number of transcription factors, replication proteins, and mammalian oncogenes as well as several dinucleotide repeat motifs, an AT-rich region, and a sequence that is likely to bend the DNA. In contrast to the other well-characterized mammalian replication origins, which are autosomal and therefore carried as two copies per somatic cell, oriS14 is encoded by single-copy DNA within a hemizygous segment of chromosome 2q in CHO-K1 cells. Also, other known mammalian replication origins are situated in nontranscribed, intergenic DNA, whereas the DNA sequence encoding oriS14 substantially overlaps the transcribed portion of a constitutively expressed housekeeping gene.

scholarly journals The Carboxyl-Terminal Region Is a Determinant for the Intracellular Behavior of the Chorionic Gonadotropin β Subunit: Effects on the Processing of the Asn-Linked Oligosaccharides

1998 ◽  
Vol 12 (5) ◽  
pp. 766-772
Author(s):  
Mesut Muyan ◽  
Irving Boime
Keyword(s):  
Intramolecular Interaction ◽  
Chinese Hamster ◽  
Glycoprotein Hormones ◽  
Wild Type ◽  
Cho Cell ◽  
Α Subunit ◽  
Glycosylation Sites ◽  
Carboxyl Terminal ◽  
Complex Forms ◽  
Placental Hormone

Abstract The placental hormone human CG (hCG) consists of two noncovalently linked α- and β-subunits similar to the other glycoprotein hormones LH, FSH, and TSH. These heterodimers share a common α subunit but differ in their structurally distinct β subunits. The CGβ subunit is distinguished among the β subunits by the presence of a C-terminal extension with four serine-linked oligosaccharides (carboxyl terminal peptide or CTP). In previous studies we observed that deleting this sequence decreased assembly of the truncated CGβ subunit (CGβ114) with the α-subunit and increased the heterogeneity of the secreted forms of the uncombined subunit synthesized in transfected Chinese hamster ovary (CHO) cells. The latter result was attributed to alterations in the processing of the two N-linked oligosaccharides. To examine at what step this heterogeneity occurs, the CGβ and CGβ114 genes were transfected into wild-type and mutant CHO cell lines that are defective in the late steps of the N-linked carbohydrate-processing pathway. We show here that removal of the CTP alters the processing of the core mannosyl unit of the subunit to complex forms at both glycosylation sites and that the oligosaccharides contain polylactosamine. Although it has been presumed that there is little intramolecular interaction between the CTP and the proximal domains of the subunit, our data suggest that the CTP sequence participates in the folding of the newly synthesized subunit, which is manifest by the posttranslational changes observed here.

Identification of a protein altered in mutants resistant to microtubule inhibitors as a member of the major heat shock protein (hsp70) family

1990 ◽  
Vol 10 (10) ◽  
pp. 5160-5165
Author(s):  
S Ahmad ◽  
R Ahuja ◽  
T J Venner ◽  
R S Gupta
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Cho Cells ◽  
Chinese Hamster ◽  
Immunofluorescent Staining ◽  
Mutant Form ◽  
Two Dimensional ◽  
Wild Type ◽  
Cho Cell ◽  
Microtubule Inhibitors

A major cellular protein (P2; approximately 70 kilodaltons) which is altered in Chinese hamster ovary (CHO) cell mutants resistant to the microtubule inhibitors colchicine and podophyllotoxin has been shown to correspond to the constitutive form of the 70-kilodalton heat shock protein (hsc70). The inference that P2 and hsc70 are the same protein is based on the following observations: (i) migration of P2 in two-dimensional polyacrylamide gels in the same position as that reported for hsc70; (ii) cross-reactivity of a monoclonal antibody which reacts with both the constitutive and induced forms of hsp70 with the P2 spot from wild-type CHO cells and with both P2 and a mutant form of P2 in a CHO cell mutant; (iii) specific reactivity of a polyclonal antibody to P2 with both the constitutive and heat-induced forms of hsp70 in human cells; (iv) identical immunofluorescent staining of dot/patchlike structures with both P2 and hsp70 antibodies in human and CHO cells; and (v) a cDNA clone for hsc70 has been isolated and sequenced from wild-type CHO cells. The in vitro transcription and translation product of this cDNA has been shown to comigrate with the P2 protein spot in two-dimensional gels, indicating their identity. The fact that there is an alteration in hsc70 in mutants resistant to antimitotic drugs suggests a role for this protein in the in vivo assembly and function of microtubules.

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

1986 ◽  
Vol 6 (8) ◽  
pp. 2774-2783 ◽  
Author(s):  
D D Rhoads ◽  
A Dixit ◽  
D J Roufa
Keyword(s):  
Ribosomal Protein ◽  
Hybrid Cell ◽  
Chinese Hamster ◽  
Housekeeping Genes ◽  
Single Copy ◽  
Sequence Motifs ◽  
Ribosomal Protein Genes ◽  
Alu Sequence ◽  
Exon 1 ◽  
Human Genomic

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 cytoplasma 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.

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