scholarly journals DNase I footprinting shows three protected regions in the promoter of the rRNA genes of Xenopus laevis.

1985 ◽  
Vol 5 (2) ◽  
pp. 313-319 ◽  
Author(s):  
M Dunaway ◽  
R H Reeder
Keyword(s):  
Xenopus Laevis ◽  
Base Pair ◽  
Transcription Initiation ◽  
Protein S ◽  
Dnase I ◽  
Rrna Genes ◽  
Xenopus Laevis Oocytes ◽  
Dnase I Footprinting ◽  
Region I ◽  
Region Ii

Extracts prepared from Xenopus laevis oocytes contain a protein(s) which specifically protects three discrete regions of the RNA polymerase I promoter from digestion by DNase I. Protected region I, from nucleotide +15 to nucleotide -10, spans the site of transcription initiation. Protected region II extends from nucleotide -70 to nucleotide -100 relative to initiation, falling within a 42-base-pair sequence which is homologous to the 60/81-base-pair repeated elements which occur outside of the promoter in the spacer. Protected region III is upstream of region II, from nucleotide -120 to nucleotide -140. All three regions correlate with sequences known from deletion studies to be important for promoter function. Deletion mutants which retain either region I or regions II and III together footprint normally. Deletion of region III, however, reduces but does not eliminate footprinting on region II, suggesting either that one protein binds to both regions or that the proteins which bind to these sites interact with each other.

DNase I footprinting shows three protected regions in the promoter of the rRNA genes of Xenopus laevis

1985 ◽  
Vol 5 (2) ◽  
pp. 313-319
Author(s):  
M Dunaway ◽  
R H Reeder
Keyword(s):  
Xenopus Laevis ◽  
Base Pair ◽  
Transcription Initiation ◽  
Protein S ◽  
Dnase I ◽  
Rrna Genes ◽  
Xenopus Laevis Oocytes ◽  
Dnase I Footprinting ◽  
Region I ◽  
Region Ii

Extracts prepared from Xenopus laevis oocytes contain a protein(s) which specifically protects three discrete regions of the RNA polymerase I promoter from digestion by DNase I. Protected region I, from nucleotide +15 to nucleotide -10, spans the site of transcription initiation. Protected region II extends from nucleotide -70 to nucleotide -100 relative to initiation, falling within a 42-base-pair sequence which is homologous to the 60/81-base-pair repeated elements which occur outside of the promoter in the spacer. Protected region III is upstream of region II, from nucleotide -120 to nucleotide -140. All three regions correlate with sequences known from deletion studies to be important for promoter function. Deletion mutants which retain either region I or regions II and III together footprint normally. Deletion of region III, however, reduces but does not eliminate footprinting on region II, suggesting either that one protein binds to both regions or that the proteins which bind to these sites interact with each other.

Both upstream and intron sequence elements are required for elevated expression of the rat somatic cytochrome c gene in COS-1 cells

1988 ◽  
Vol 8 (1) ◽  
pp. 35-41
Author(s):  
M J Evans ◽  
R C Scarpulla
Keyword(s):  
Cytochrome C ◽  
Cell Lines ◽  
Transcription Initiation ◽  
Promoter Activity ◽  
Physiological Role ◽  
Regulatory Elements ◽  
T Antigen ◽  
Regulatory Sequences ◽  
Region I ◽  
Region Ii

To investigate the transcriptional control of nuclear-encoded respiratory genes in mammals, we have performed a deletional analysis of cis-acting regulatory sequences in the rat somatic cytochrome c gene. Three major regions are required for maximal expression of the transfected gene in kidney cell lines CV-1 and COS-1. One of these, region III (+71 to +115 from the transcription initiation site), is an unusual intragenic controlling element found in the 5' end of the first intron, while the other two, region I (-191 to -165) and region II (-139 to -84), define the upstream promoter. Region II contains two consensus CCAAT boxes and mediates a constitutive level of expression in both cell lines. In contrast, regions I and III are both required for the increased promoter activity observed in COS-1 cells compared with promoter activity observed in CV-1 cells, and the regions function individually as competitors with the full promoter for trans-acting factors or complexes. Region III contains a perfect octanucleotide homology with region I in addition to a consensus Sp1-transcription-factor-binding site. Promoter stimulation in COS-1 cells can be duplicated in CV-1 cells by cotransfecting with a T-antigen-producing vector, but purified T antigen does not bind anywhere in the cytochrome c promoter. A control promoter from the mouse metallothionein I gene is similarly activated in T-antigen-producing cells only in the presence of zinc, which activates its upstream regulatory sites. We conclude that T antigen stimulates these cellular promoters through the activation or induction of cellular factors or complexes that mediate their effects through promoter-specific regulatory elements. Cytochrome c promoter regions activated in this system may play a physiological role in controlling gene expression.

A 12-base-pair sequence is an essential element of the ribosomal gene terminator in Xenopus laevis

1987 ◽  
Vol 7 (5) ◽  
pp. 1900-1905
Author(s):  
P Labhart ◽  
R H Reeder
Keyword(s):  
Xenopus Laevis ◽  
Base Pair ◽  
Transcription Initiation ◽  
Gene Promoter ◽  
Essential Element ◽  
Ribosomal Gene ◽  
Pulse Label ◽  
Conserved Sequence ◽  
Rrna Transcription ◽  
Pair Sequence

rRNA transcription in Xenopus laevis terminates near a 7-base-pair (bp) conserved sequence (T3 box) located 200 bp upstream of the site of transcription initiation for the adjacent gene promoter. We present evidence here that a 12-bp element containing the T3 box is an essential part of the terminator. Using an oocyte injection assay, we found that the 12-bp element (but not the T3 box alone) severely reduced the amount of RNA detectable at sites downstream from itself and that the T3 box within the 12-bp element was required to specify the formation of correct 3' ends. This requirement for the 12-bp element was also seen in pulse-label experiments by using a homogenate of oocyte nuclei, but the present data did not allow us to determine the exact mechanism by which the 12-bp element acts. Removal of the T3 region from its normal location allowed a significant amount of readthrough transcripts to accumulate, indicating that additional sequences may be required for complete terminator function.

scholarly journals Both upstream and intron sequence elements are required for elevated expression of the rat somatic cytochrome c gene in COS-1 cells.

1988 ◽  
Vol 8 (1) ◽  
pp. 35-41 ◽  
Author(s):  
M J Evans ◽  
R C Scarpulla
Keyword(s):  
Cytochrome C ◽  
Cell Lines ◽  
Transcription Initiation ◽  
Promoter Activity ◽  
Physiological Role ◽  
Regulatory Elements ◽  
T Antigen ◽  
Regulatory Sequences ◽  
Region I ◽  
Region Ii

To investigate the transcriptional control of nuclear-encoded respiratory genes in mammals, we have performed a deletional analysis of cis-acting regulatory sequences in the rat somatic cytochrome c gene. Three major regions are required for maximal expression of the transfected gene in kidney cell lines CV-1 and COS-1. One of these, region III (+71 to +115 from the transcription initiation site), is an unusual intragenic controlling element found in the 5' end of the first intron, while the other two, region I (-191 to -165) and region II (-139 to -84), define the upstream promoter. Region II contains two consensus CCAAT boxes and mediates a constitutive level of expression in both cell lines. In contrast, regions I and III are both required for the increased promoter activity observed in COS-1 cells compared with promoter activity observed in CV-1 cells, and the regions function individually as competitors with the full promoter for trans-acting factors or complexes. Region III contains a perfect octanucleotide homology with region I in addition to a consensus Sp1-transcription-factor-binding site. Promoter stimulation in COS-1 cells can be duplicated in CV-1 cells by cotransfecting with a T-antigen-producing vector, but purified T antigen does not bind anywhere in the cytochrome c promoter. A control promoter from the mouse metallothionein I gene is similarly activated in T-antigen-producing cells only in the presence of zinc, which activates its upstream regulatory sites. We conclude that T antigen stimulates these cellular promoters through the activation or induction of cellular factors or complexes that mediate their effects through promoter-specific regulatory elements. Cytochrome c promoter regions activated in this system may play a physiological role in controlling gene expression.

scholarly journals A 12-base-pair sequence is an essential element of the ribosomal gene terminator in Xenopus laevis.

1987 ◽  
Vol 7 (5) ◽  
pp. 1900-1905 ◽  
Author(s):  
P Labhart ◽  
R H Reeder
Keyword(s):  
Xenopus Laevis ◽  
Base Pair ◽  
Transcription Initiation ◽  
Gene Promoter ◽  
Essential Element ◽  
Ribosomal Gene ◽  
Pulse Label ◽  
Conserved Sequence ◽  
Rrna Transcription ◽  
Pair Sequence

rRNA transcription in Xenopus laevis terminates near a 7-base-pair (bp) conserved sequence (T3 box) located 200 bp upstream of the site of transcription initiation for the adjacent gene promoter. We present evidence here that a 12-bp element containing the T3 box is an essential part of the terminator. Using an oocyte injection assay, we found that the 12-bp element (but not the T3 box alone) severely reduced the amount of RNA detectable at sites downstream from itself and that the T3 box within the 12-bp element was required to specify the formation of correct 3' ends. This requirement for the 12-bp element was also seen in pulse-label experiments by using a homogenate of oocyte nuclei, but the present data did not allow us to determine the exact mechanism by which the 12-bp element acts. Removal of the T3 region from its normal location allowed a significant amount of readthrough transcripts to accumulate, indicating that additional sequences may be required for complete terminator function.

Transcription of Xenopus borealis rRNA genes in nuclei of Xenopus laevis oocytes

1982 ◽  
Vol 93 (2) ◽  
pp. 471-477 ◽  
Author(s):  
Garry T. Morgan ◽  
Aimee H. Bakken ◽  
Ronald H. Reeder
Keyword(s):  
Xenopus Laevis ◽  
Rrna Genes ◽  
Xenopus Laevis Oocytes ◽  
Xenopus Borealis

scholarly journals Store-activated Ca2+ inflow in Xenopus laevis oocytes: inhibition by primaquine and evaluation of the role of membrane fusion

1996 ◽  
Vol 319 (3) ◽  
pp. 755-760 ◽  
Author(s):  
Roland B GREGORY ◽  
Greg J BARRITT
Keyword(s):  
Endoplasmic Reticulum ◽  
Xenopus Laevis ◽  
Membrane Fusion ◽  
Direct Interaction ◽  
Protein S ◽  
Detectable Effect ◽  
Xenopus Laevis Oocytes ◽  
Endoplasmic Reticulum Membrane ◽  
Rate Of Increase

The role of membrane fusion in the activation of store-activated Ca2+ channels (SACCs) in the plasma membrane of Xenopus laevis oocytes was investigated with primaquine, an inhibitor of vesicle trafficking, reagents that disrupt the cytoskeleton, and reagents that activate or inhibit the functions of monomeric and trimeric GTP-binding regulatory proteins. Ca2+ inflow was assessed by measuring the rate of increase in the fluorescence of the intracellular Ca2+ chelator fluo-3 after the addition of extracellular Ca2+ to oocytes previously incubated in the absence of added Ca2+. Primaquine inhibited the 3-deoxy-3-fluoro-Ins(1,4,5)P3 (Ins(1,4,5)P3F)-stimulated increase in Ca2+o-induced fluo-3 fluorescence with no detectable effect on the release of Ca2+ from intracellular stores. The effect of primaquine was observed within 1.5 min, showed similarity to the inhibition induced by Gd3+, was reversible, and was observed when primaquine was added either before or after activation of the SACCs. The degree of inhibition of Ca2+ inflow by primaquine was halved when the extracellular concentration of Ca2+ was increased from 3.1 to 12.5 mM. Primaquine also inhibited Ca2+ inflow through cholera toxin-activated divalent cation channels and Drosophila Trpl channels (expressed in oocytes after injection of trpl cRNA). These results indicate that primaquine inhibits open SACCs, possibly by directly inhibiting Ca2+ flow through the channel pore. Colchicine plus cytochalasin B, Brefeldin A, the peptide Arf-1 (2–17) (introduced by microinjection), lovastatin or pertussis toxin did not inhibit the Ins(1,4,5)P3F-stimulated increase in fluo-3 fluorescence. In contrast, guanosine 5´-[γ-thio]triphosphate (GTP[S]), guanosine 5´-[β,γ-imido]triphosphate (p[NH]ppG) and AlF4-, but not guanosine 5´-[β-thio]diphosphate, inhibited the Ins(1,4,5)P3F-stimulated increase in fluo-3 fluorescence. Co-administration of GTP did not prevent the inhibition by GTP[S] or AlF4-. Staurosporine largely prevented the inhibition of store-activated Ca2+ inflow by GTP[S]. It is concluded that membrane fusion processes are unlikely to be involved in the link between the release of Ca2+ from the endoplasmic reticulum and activation of SACCs. The idea that this link is achieved by direct interaction of a protein(s) in the endoplasmic reticulum membrane with the SACC protein is briefly discussed.

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