Multiple regulatory elements ensure accurate transcription of a human ribosomal protein gene

1993 ◽  
Vol 19 (4) ◽  
pp. 347-362 ◽  
Author(s):  
Paul F. Overman ◽  
Douglas D. Rhoads ◽  
Elena S. Tasheva ◽  
Marla M. Pyle ◽  
Donald J. Roufa
Keyword(s):  
Ribosomal Protein ◽  
Protein Gene ◽  
Ribosomal Protein Gene ◽  
Regulatory Elements ◽  
Human Ribosomal Protein

Identification of highly specific localized sequence motifs in human ribosomal protein gene promoters

Gene ◽  
2006 ◽  
Vol 365 ◽  
pp. 48-56 ◽  
Author(s):  
Stefan Roepcke ◽  
Degui Zhi ◽  
Martin Vingron ◽  
Peter F. Arndt
Keyword(s):  
Ribosomal Protein ◽  
Protein Gene ◽  
Ribosomal Protein Gene ◽  
Gene Promoters ◽  
Sequence Motifs ◽  
Human Ribosomal Protein

Fine-structure map of the human ribosomal protein gene RPS14

1992 ◽  
Vol 12 (4) ◽  
pp. 1680-1686
Author(s):  
J J Diaz ◽  
D J Roufa
Keyword(s):  
Biological Activity ◽  
Fine Structure ◽  
Ribosomal Protein ◽  
Protein Gene ◽  
Ribosomal Protein Gene ◽  
Genetic Maps ◽  
Chemical Mutagenesis ◽  
In Vitro Mutagenesis ◽  
Amino Terminal ◽  
Human Ribosomal Protein

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.

Localization of transcriptional regulatory elements and nuclear factor binding sites in mouse ribosomal protein gene rpL32

1989 ◽  
Vol 9 (5) ◽  
pp. 2067-2074
Author(s):  
M L Atchison ◽  
O Meyuhas ◽  
R P Perry
Keyword(s):  
Ribosomal Protein ◽  
Dna Sequences ◽  
Binding Sites ◽  
Protein Gene ◽  
Transcriptional Start Site ◽  
Ribosomal Protein Gene ◽  
Regulatory Elements ◽  
Sequence Motif ◽  
Start Site ◽  
Nuclear Factors

The DNA sequences required for expression of the ribosomal protein gene rpL32 were identified by transient-expression assays of chimeric rpL32-chloramphenicol acetyltransferase genes. These studies showed that maximal rpL32 expression requires sequences in a 150- to 200-base-pair region spanning the transcriptional start site. Three discrete regions of importance were identified: one between positions -79 and -69 and two others located downstream of the transcriptional start site. Progressive 5' or 3' deletions caused stepwise decreases in expression, which suggested a complex interplay of redundant or compensatory elements. Gel mobility shift assays were used to identify trans-acting nuclear factors which bind to segments of the rpL32 promoter that are known to be important for transcription. Evidence for several distinct nuclear factors is presented. The binding sites for these factors were localized to the following regions: -79 to -69, -36 to -19, -19 to +11, +11 to +46 in exon I, and within the first 31 base pairs of intron 1. One of these factors may bind to multiple sites within the promoter region. Interestingly, the factor that binds to a sequence motif in the first exon also binds to similar motifs in a comparable region of the c-myc gene.

scholarly journals Localization of transcriptional regulatory elements and nuclear factor binding sites in mouse ribosomal protein gene rpL32.

1989 ◽  
Vol 9 (5) ◽  
pp. 2067-2074 ◽  
Author(s):  
M L Atchison ◽  
O Meyuhas ◽  
R P Perry
Keyword(s):  
Ribosomal Protein ◽  
Dna Sequences ◽  
Binding Sites ◽  
Protein Gene ◽  
Transcriptional Start Site ◽  
Ribosomal Protein Gene ◽  
Regulatory Elements ◽  
Sequence Motif ◽  
Start Site ◽  
Nuclear Factors

The DNA sequences required for expression of the ribosomal protein gene rpL32 were identified by transient-expression assays of chimeric rpL32-chloramphenicol acetyltransferase genes. These studies showed that maximal rpL32 expression requires sequences in a 150- to 200-base-pair region spanning the transcriptional start site. Three discrete regions of importance were identified: one between positions -79 and -69 and two others located downstream of the transcriptional start site. Progressive 5' or 3' deletions caused stepwise decreases in expression, which suggested a complex interplay of redundant or compensatory elements. Gel mobility shift assays were used to identify trans-acting nuclear factors which bind to segments of the rpL32 promoter that are known to be important for transcription. Evidence for several distinct nuclear factors is presented. The binding sites for these factors were localized to the following regions: -79 to -69, -36 to -19, -19 to +11, +11 to +46 in exon I, and within the first 31 base pairs of intron 1. One of these factors may bind to multiple sites within the promoter region. Interestingly, the factor that binds to a sequence motif in the first exon also binds to similar motifs in a comparable region of the c-myc gene.

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

1992 ◽  
Vol 12 (4) ◽  
pp. 1680-1686 ◽  
Author(s):  
J J Diaz ◽  
D J Roufa
Keyword(s):  
Biological Activity ◽  
Fine Structure ◽  
Ribosomal Protein ◽  
Protein Gene ◽  
Ribosomal Protein Gene ◽  
Genetic Maps ◽  
Chemical Mutagenesis ◽  
In Vitro Mutagenesis ◽  
Amino Terminal ◽  
Human Ribosomal Protein

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.

scholarly journals Cloning and charaterization of a human ribosomal protein gene with enhanced expression in fetal and neoplastic cells

1987 ◽  
Vol 15 (21) ◽  
pp. 8919-8934 ◽  
Author(s):  
Jing-hsiung Ou ◽  
T.S.Benedict Yen ◽  
Yan-Fei Wang ◽  
Wing K. Kam ◽  
William J. Rutter
Keyword(s):  
Ribosomal Protein ◽  
Protein Gene ◽  
Ribosomal Protein Gene ◽  
Neoplastic Cells ◽  
Enhanced Expression ◽  
Human Ribosomal Protein

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.

scholarly journals Cloning and characterization of a human ribosomal protein gene with enhanced expression in fetal and neoplastic cells

1988 ◽  
Vol 16 (9) ◽  
pp. 4196-4196 ◽  
Author(s):  
Jing-hsing Ou ◽  
T.S.Benedict Yen ◽  
Yan-Fei Wang ◽  
Wing K. Kam ◽  
William J. Rutter
Keyword(s):  
Ribosomal Protein ◽  
Protein Gene ◽  
Ribosomal Protein Gene ◽  
Neoplastic Cells ◽  
Enhanced Expression ◽  
Human Ribosomal Protein

scholarly journals 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.

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