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.

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.

Construction and characterization of Chinese hamster cell EmtA EmtB double mutants

1983 ◽  
Vol 3 (5) ◽  
pp. 761-772
Author(s):  
S Chang ◽  
J J Wasmuth
Keyword(s):  
Ribosomal Protein ◽  
Cell Lines ◽  
Ribosomal Proteins ◽  
Hybrid Cell ◽  
Polyacrylamide Gel Electrophoresis ◽  
Chinese Hamster ◽  
Chinese Hamster Cell ◽  
Altered Form ◽  
Hybrid Cell Lines

Starting with hybrid cell lines between a Chinese hamster cell EmtA mutant and a Chinese hamster cell EmtB mutant, we have constructed cell lines that are homozygous for mutant alleles at both the emtA locus and the emtB locus, by using a two-step segregation protocol. The EmtA EmtB double mutants are approximately 10-fold more resistant to emetine inhibition than either of the parental mutants. Having both the EmtA mutation and the EmtB mutation expressed in the same cell also results in a level of resistance to cryptopleurine that is significantly higher than a simple additive effect of the two mutations alone. Analysis of ribosomal proteins by two-dimensional polyacrylamide gel electrophoresis demonstrated that a parental hybrid and a first-step segregant, which has lost the wild-type emtA allele, synthesize both a normal and an altered form of ribosomal protein S14, whereas an EmtA EmtB double mutant synthesizes only the altered form of this ribosomal protein. This result confirms that the emtB locus is the structural gene for ribosomal protein S14. Our results also suggest that the products of the emtA and emtB loci interact directly, indicating that the emtA locus, like the emtB locus, encodes a component of the ribosome.

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.

Centromeric DNA cloned from functional kinetochore fragments in mitotic cells with unreplicated genomes

1993 ◽  
Vol 105 (2) ◽  
pp. 359-367 ◽  
Author(s):  
I.I. Ouspenski ◽  
B.R. Brinkley
Keyword(s):  
Topoisomerase Ii ◽  
Chinese Hamster ◽  
Single Copy ◽  
Chromosome 1 ◽  
Sequence Motifs ◽  
Mitotic Chromosomes ◽  
Distinctive Features ◽  
Repetitive Structure ◽  
Species Specific

Treatment of cells arrested in the cell cycle at the G1/S-phase boundary with 5 mM caffeine induces premature mitosis, resulting in chromosomal fragmentation and detachment of centromere-kinetochore fragments, which are subsequently attached to the mitotic spindle and segregated in anaphase. Taking advantage of this in vivo separation of the centromere, we have developed a procedure for isolation of a centromere-enriched fraction of mitotic chromatin. Using this method, we have isolated and cloned DNA from the centromere-enriched material of Chinese hamster cells. One of the clones thus obtained was characterized in detail. It contains 6 kb of centromere-associated sequence that exhibits no recognizable homology with other mammalian centromeric sequences and is devoid of any extensive repetitive structure. This sequence is present in a single copy on chromosome 1 and is species-specific. Distinctive features of the clone include the presence of several A+T-rich regions and clusters of multiple topoisomerase II consensus cleavage sites and other sequence motifs characteristic of nuclear matrix-associated regions. We hypothesize that these features might be related to the more compact packaging of centromeric chromatin in interphase nuclei and mitotic chromosomes.

The ABF1 factor is the transcriptional activator of the L2 ribosomal protein genes in Saccharomyces cerevisiae

1990 ◽  
Vol 10 (5) ◽  
pp. 2437-2441
Author(s):  
F Della Seta ◽  
S A Ciafré ◽  
C Marck ◽  
B Santoro ◽  
C Presutti ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ribosomal Protein ◽  
Binding Sites ◽  
Housekeeping Genes ◽  
Ribosomal Protein Genes ◽  
General Role ◽  
Gene Copies ◽  
Vitro Binding

The same factor, ABF1, binds to the promoters of the two gene copies (L2A and L2B) coding for the ribosomal protein L2 in Saccharomyces cerevisiae. In vitro binding experiments and in vivo functional analysis showed that the different affinities of the L2A and L2B promoters for the ABF1 factor are responsible for the differential transcriptional activities of the two gene copies. The presence of ABF1-binding sites in front of many housekeeping genes suggests a general role for ABF1 in the regulation of gene activity.

scholarly journals Construction and characterization of Chinese hamster cell EmtA EmtB double mutants.

1983 ◽  
Vol 3 (5) ◽  
pp. 761-772 ◽  
Author(s):  
S Chang ◽  
J J Wasmuth
Keyword(s):  
Ribosomal Protein ◽  
Cell Lines ◽  
Ribosomal Proteins ◽  
Hybrid Cell ◽  
Polyacrylamide Gel Electrophoresis ◽  
Chinese Hamster ◽  
Chinese Hamster Cell ◽  
Altered Form ◽  
Hybrid Cell Lines

Starting with hybrid cell lines between a Chinese hamster cell EmtA mutant and a Chinese hamster cell EmtB mutant, we have constructed cell lines that are homozygous for mutant alleles at both the emtA locus and the emtB locus, by using a two-step segregation protocol. The EmtA EmtB double mutants are approximately 10-fold more resistant to emetine inhibition than either of the parental mutants. Having both the EmtA mutation and the EmtB mutation expressed in the same cell also results in a level of resistance to cryptopleurine that is significantly higher than a simple additive effect of the two mutations alone. Analysis of ribosomal proteins by two-dimensional polyacrylamide gel electrophoresis demonstrated that a parental hybrid and a first-step segregant, which has lost the wild-type emtA allele, synthesize both a normal and an altered form of ribosomal protein S14, whereas an EmtA EmtB double mutant synthesizes only the altered form of this ribosomal protein. This result confirms that the emtB locus is the structural gene for ribosomal protein S14. Our results also suggest that the products of the emtA and emtB loci interact directly, indicating that the emtA locus, like the emtB locus, encodes a component of the ribosome.

Localization of three chloroplast ribosomal protein genes at the left junction of the large single copy region and the inverted repeat of Spirodela oligorhiza chloroplast DNA

1985 ◽  
Vol 9 (3) ◽  
pp. 211-219 ◽  
Author(s):  
Mark Posno ◽  
Dick J. Torenvliet ◽  
Henk Lustig ◽  
Marjolein van Noort ◽  
Gert S. P. Groot
Keyword(s):  
Chloroplast Dna ◽  
Ribosomal Protein ◽  
Inverted Repeat ◽  
Single Copy ◽  
Ribosomal Protein Genes ◽  
Large Single Copy ◽  
Spirodela Oligorhiza ◽  
Large Single Copy Region

A cloned human ribosomal protein gene functions in rodent cells

1987 ◽  
Vol 7 (10) ◽  
pp. 3767-3774
Author(s):  
D D Rhoads ◽  
D J Roufa
Keyword(s):  
Ribosomal Protein ◽  
Cho Cells ◽  
Protein Gene ◽  
Chinese Hamster ◽  
Ribosomal Protein Gene ◽  
Mrna Levels ◽  
Flanking Sequence ◽  
Base Pairs ◽  
Exon 1 ◽  
Human Ribosomal Protein

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.

Tripartite upstream promoter element essential for expression of Saccharomyces cerevisiae ribosomal protein genes

1986 ◽  
Vol 6 (2) ◽  
pp. 674-687
Author(s):  
M O Rotenberg ◽  
J L Woolford
Keyword(s):  
Ribosomal Protein ◽  
Protein Gene ◽  
Ribosomal Protein Gene ◽  
Yeast Cells ◽  
Lacz Gene ◽  
Sequence Motifs ◽  
Ribosomal Protein Genes ◽  
Rich Region ◽  
Flanking Region ◽  
Beta Galactosidase

To initiate a genetic analysis of yeast ribosomal protein gene promoters, we have constructed a gene fusion between the yeast ribosomal protein gene RP39A and the Escherichia coli lacZ gene. This gene fusion contains approximately 1,030 nucleotides of the 5' flanking region and the first 49 1/3 codons of RP39A fused in frame to a large 3' end fragment of lacZ. Whether it is introduced into yeast cells on a moderately high-copy-number plasmid, or integrated into the yeast genome at the RP39A locus, this RP39A-lacZ gene directs the synthesis of a hybrid transcript which encodes beta-galactosidase activity. Deletions in the 5' flanking region of RP39A-lacZ were constructed by linker insertion and BAL 31 mutagenesis. The expression of the mutant genes in yeast cells was assayed by measuring RP39A-lacZ mRNA and beta-galactosidase levels. By these means we have shown that the sequences between nucleotides -256 and -170 upstream of RP39A are essential for expression of this gene. Three sequence motifs, HOMOL1, RPG, and a T-rich region, which were found in that order 5'----3' upstream of most yeast ribosomal protein genes, were present within this interval. We found that substitution of the CYC1-lacZ upstream activation site with the fragment from nucleotides -298 to -172 upstream of RP39A, containing the HOMOL1-RPG-T-rich motif in that 5'----3' orientation, fully restored expression of the CYC1-lacZ gene. The essentially of HOMOL1, the RPG sequence, and the T-rich region for wild-type levels of expression of RP39A, the conserved location and order of these sequence motifs in yeast ribosomal protein genes, and the ability of a DNA fragment carrying these three sequence elements to substitute for the upstream activation site regions of CYC1 indicate that these three oligonucleotides may be essential to the transcription of yeast ribosomal protein genes.

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