scholarly journals Independent 5' and 3'-end determination of multiple dihydrofolate reductase transcripts.

1987 ◽  
Vol 7 (10) ◽  
pp. 3732-3739 ◽  
Author(s):  
J Y Yen ◽  
R E Kellems
Keyword(s):  
Dihydrofolate Reductase ◽  
Relative Abundance ◽  
Transcription Initiation ◽  
Promoter Region ◽  
Overlapping Genes ◽  
Cytoplasmic Rna ◽  
Upstream Promoter ◽  
Upstream Promoter Region ◽  
Polyadenylation Sites

Multiple dihydrofolate reductase (dhfr) mRNAs, differing substantially in abundance, are produced as a result of the utilization of multiple transcription initiation sites and multiple polyadenylation sites. We have shown that dhfr mRNAs initiating from an upstream promoter region utilize the same collection of six polyadenylation sites and generate multiple dhfr mRNAs at the same relative abundance as do the mRNAs initiating from the major transcription promoter region. These results indicate that the 5' and 3' ends of dhfr mRNAs are independently determined. We show that the relative abundance of steady-state dhfr mRNAs was the same in nuclear and cytoplasmic RNA fractions. This finding makes it unlikely that differences in mRNA stability account for differences in the relative abundance of the multiple dhfr mRNAs in the cytoplasm. Our analysis of the dhfr promoter region revealed the existence of stable cytoplasmic polyadenylated transcripts complementary to the first 300 nucleotides of the dhfr transcripts initiating from the upstream promoter region. Therefore, the dhfr locus hosts two divergent and partially overlapping genes which share the same promoter region.

Independent 5' and 3'-end determination of multiple dihydrofolate reductase transcripts

1987 ◽  
Vol 7 (10) ◽  
pp. 3732-3739
Author(s):  
J Y Yen ◽  
R E Kellems
Keyword(s):  
Dihydrofolate Reductase ◽  
Relative Abundance ◽  
Transcription Initiation ◽  
Promoter Region ◽  
Overlapping Genes ◽  
Cytoplasmic Rna ◽  
Upstream Promoter ◽  
Upstream Promoter Region ◽  
Polyadenylation Sites

Multiple dihydrofolate reductase (dhfr) mRNAs, differing substantially in abundance, are produced as a result of the utilization of multiple transcription initiation sites and multiple polyadenylation sites. We have shown that dhfr mRNAs initiating from an upstream promoter region utilize the same collection of six polyadenylation sites and generate multiple dhfr mRNAs at the same relative abundance as do the mRNAs initiating from the major transcription promoter region. These results indicate that the 5' and 3' ends of dhfr mRNAs are independently determined. We show that the relative abundance of steady-state dhfr mRNAs was the same in nuclear and cytoplasmic RNA fractions. This finding makes it unlikely that differences in mRNA stability account for differences in the relative abundance of the multiple dhfr mRNAs in the cytoplasm. Our analysis of the dhfr promoter region revealed the existence of stable cytoplasmic polyadenylated transcripts complementary to the first 300 nucleotides of the dhfr transcripts initiating from the upstream promoter region. Therefore, the dhfr locus hosts two divergent and partially overlapping genes which share the same promoter region.

scholarly journals The miR-203a Regulatory Network Affects the Proliferation of Chronic Myeloid Leukemia K562 Cells

2021 ◽  
Vol 9 ◽  
Author(s):  
Jinhua He ◽  
Zeping Han ◽  
Ziyi An ◽  
Yumin Li ◽  
Xingyi Xie ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Transcription Initiation ◽  
Promoter Region ◽  
Target Genes ◽  
Mononuclear Cells ◽  
K562 Cells ◽  
Luciferase Reporter ◽  
Upstream Promoter ◽  
Upstream Promoter Region

To study the molecular mechanism by which miR-203a affects the development of CML, bioinformatics software was used to predict the upstream transcription factors and downstream target genes of miR-203a. A 5’-rapid amplification of cDNA ends assay was performed to detect gene transcription initiation sites. A chromatin immunoprecipitation assay was used to verify the binding of transcription factors and promoter regions. A double luciferase reporter gene vector was constructed to demonstrate the regulatory effect of miR-203a on target genes. Real-time PCR and western blotting were used to detect the relative expression levels of genes and proteins, respectively. The results showed that there was a binding site for the transcription factor EGR1 in the upstream promoter region of miR-203a. WT1, BMI1, and XIAP were identified as target genes regulated by miR-203a. EGR1 and miR-203a were downregulated in human peripheral blood mononuclear cells and the CML K562 cell line, while WT1, BMI1, and XIAP were upregulated. The transcription initiation site of miR-203a was identified in the upstream promoter region (G nucleotide at −339 bp), and the transcription factor EGR1 could bind to the promoter region (at −268 bp) of miR-203a and increase its expression. Over expression of miR-203a inhibited the proliferation of K562 cells. A rescue assay showed that overexpression of WT1, BMI1, and XIAP offset the antitumor effect of miR-203a. Conclusion, EGR1 positively regulated the expression of miR-203a, thus relieving the inhibition of miR-203a on the translation of its target genes (WT1, BMI1, and XIAP) and affecting the proliferation of K562 cells.

Murine dihydrofolate reductase transcripts through the cell cycle

1986 ◽  
Vol 6 (2) ◽  
pp. 365-371
Author(s):  
P J Farnham ◽  
R T Schimke
Keyword(s):  
Cell Cycle ◽  
Dihydrofolate Reductase ◽  
Transcription Initiation ◽  
Untranslated Region ◽  
Cell Cycle Regulation ◽  
Leader Sequence ◽  
Reductase Gene ◽  
Opposite Strand ◽  
Polyadenylation Sites ◽  
Cycle Regulation

The murine dihydrofolate reductase gene codes for mRNAs that differ in the length of their 3' untranslated region as well as in the length of their 5' leader sequence. In addition, the dihydrofolate reductase promoter functions bidirectionally, producing a series of RNAs from the opposite strand than the dihydrofolate reductase mRNAs. We have examined the production of these RNAs and their heterogeneous 5' and 3' termini as mouse 3T6 cells progress through a physiologically continuous cell cycle. We found that all of the transcripts traverse the cell cycle in a similar manner, increasing at the G1/S boundary without significantly changing their ratios relative to one another. We conclude that cell-cycle regulation of dihydrofolate reductase is achieved without recruiting new transcription initiation sites and without a change in polyadenylation sites. It appears that the mechanism responsible for the transcriptional cell-cycle regulation of the dihydrofolate reductase gene is manifested only by transiently increasing the efficiency of transcription at the dihydrofolate reductase promoter.

Identification of functional TTF-1 and Sp1/Sp3 sites in the upstream promoter region of rabbit SP-B gene

2000 ◽  
Vol 278 (3) ◽  
pp. L477-L484 ◽  
Author(s):  
Ramgopal Margana ◽  
Kiflu Berhane ◽  
M. Nurul Alam ◽  
Vijayakumar Boggaram
Keyword(s):  
Promoter Region ◽  
Promoter Activity ◽  
Mutational Analysis ◽  
Surfactant Protein ◽  
Alveolar Type ◽  
Deletion Mapping ◽  
Clara Cells ◽  
Mobility Shift ◽  
Upstream Promoter ◽  
Upstream Promoter Region

Surfactant protein B (SP-B) is essential for the maintenance of biophysical properties and physiological function of pulmonary surfactant. SP-B mRNA is expressed in a cell type-restricted manner in alveolar type II and bronchiolar (Clara) epithelial cells of the lung and is developmentally induced. In NCI-H441 cells, a lung cell line with characteristics of Clara cells, a minimal promoter region comprising −236 to +39 nucleotides supports high-level expression of chloramphenicol acetyltransferase reporter activity. In the present investigation, we characterized the upstream promoter region, −236 to −140 nucleotides, that is essential for promoter activity. Deletion mapping identified two segments, −236 to −170 and −170 to −140 nucleotides, that are important for promoter activity. Mutational analysis and gel mobility shift experiments identified thyroid transcription factor-1, Sp1, and Sp3 as important trans-acting factors that bind to sequences in the upstream promoter region. Our data suggest that SP-B promoter activity is dependent on interactions between factors bound to upstream and downstream regions of the promoter.

scholarly journals Structure, clustering and functional insights of repeats configurations in the upstream promoter region of the human coding genes

BMC Genomics ◽  
2018 ◽  
Vol 19 (S8) ◽  
Author(s):  
Fabian Tobar-Tosse ◽  
Patricia E. Veléz ◽  
Eliana Ocampo-Toro ◽  
Pedro A. Moreno
Keyword(s):  
Promoter Region ◽  
Upstream Promoter ◽  
Upstream Promoter Region

scholarly journals PhoP-Responsive Expression of the Salmonella enterica Serovar Typhimurium slyA Gene

2003 ◽  
Vol 185 (12) ◽  
pp. 3508-3514 ◽  
Author(s):  
Valia A. Norte ◽  
Melanie R. Stapleton ◽  
Jeffrey Green
Keyword(s):  
Transcription Factors ◽  
Salmonella Enterica ◽  
Promoter Region ◽  
External Signal ◽  
Dependent Manner ◽  
Factors Analysis ◽  
Upstream Promoter ◽  
Serovar Typhimurium ◽  
Transcription Regulators ◽  
Upstream Promoter Region

ABSTRACT The SlyA protein of Salmonella enterica serovar Typhimurium is a member of the MarR family of transcription regulators and is required for virulence and survival in professional macrophages. Isolated SlyA protein was able to bind a specific DNA target without posttranslational modification. This suggested that SlyA might not be activated by directly sensing an external signal but rather that the intracellular concentration of SlyA is enhanced in appropriate environments through the action of other transcription factors. Analysis of slyA transcription reveals the presence of a promoter region located upstream of the previously recognized SlyA repressed promoter. The newly identified upstream promoter region did not respond to SlyA but was activated by Mg(II) starvation in a PhoP-dependent manner. We present here evidence for a direct link between two transcription factors (PhoP and SlyA) crucial for Salmonella virulence.

scholarly journals Cell type-specific protein-DNA interactions in the human zeta-globin upstream promoter region: displacement of Sp1 by the erythroid cell-specific factor NF-E1.

1990 ◽  
Vol 10 (1) ◽  
pp. 282-294 ◽  
Author(s):  
C Y Yu ◽  
J Chen ◽  
L I Lin ◽  
M Tam ◽  
C K Shen
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Promoter Region ◽  
Globin Gene ◽  
Cell Type ◽  
Upstream Promoter ◽  
Protein Dna Interactions ◽  
Cell Type Specific ◽  
Upstream Promoter Region ◽  
Zeta Globin Gene

The protein-DNA interactions of the upstream promoter region of the human embryonic zeta-globin gene in nuclear extracts of erythroid K562 cells and nonerythroid HeLa cells were analyzed by DNase I footprinting, gel mobility shift assay, methylation interference, and oligonucleotide competition experiments. There are mainly two clusters of nuclear factor-binding sites in the zeta promoter. The proximal cluster spans the DNA sequence from -110 to -60 and consists of binding sites for CP2, Sp1, and NF-E1. NF-E1 binding is K562 specific, whereas CP2 binding is common to both types of cells. Overlapping the NF-E1- and CP2-binding sites is a hidden Sp1-binding site or CAC box, as demonstrated by binding studies of affinity-purified Sp1. In the distal promoter region at -250 to -220, another NF-E1-binding site overlaps a CAC box or Sp1-binding site. Extract-mixing experiments demonstrated that the higher affinity of NF-E1 binding excluded the binding of Sp1 in the K562 extract. NF-E1 factors could also displace prebound Sp1 molecules. Between the two clusters of multiple-factor-binding sites are sequences recognized by other factors, including zeta-globin factors 1 and 2, that are present in both HeLa and K562 extracts. We discuss the cell type-specific, competitive binding of multiple nuclear factors in terms of functional implications in transcriptional regulation of the zeta-globin gene.

scholarly journals The H2O2 Stress-Responsive Regulator PerR Positively Regulates srfA Expression in Bacillus subtilis

2005 ◽  
Vol 187 (19) ◽  
pp. 6659-6667 ◽  
Author(s):  
Kentaro Hayashi ◽  
Taku Ohsawa ◽  
Kazuo Kobayashi ◽  
Naotake Ogasawara ◽  
Mitsuo Ogura
Keyword(s):  
Bacillus Subtilis ◽  
Promoter Region ◽  
Feeding Assay ◽  
Mobility Shift ◽  
Genetic Competence ◽  
Upstream Promoter ◽  
Upstream Promoter Region ◽  
Fusion Analysis ◽  
Gel Mobility Shift

ABSTRACT srfA is an operon required for the synthesis of surfactin and the development of genetic competence in Bacillus subtilis. We observed that the expression of srfA is downregulated upon treatment with H2O2. Thus, we examined the involvement of several oxidative stress-responsive transcription factors in srfA expression. Our DNA microarray analysis revealed that the H2O2 stress-responsive regulator PerR is required for srfA expression. This was confirmed by lacZ fusion analysis. A ComX feeding assay and epistatic analyses revealed that the role of PerR in srfA expression is independent of other known regulators of srfA expression, namely, comQXP, rapC, and spx. Gel mobility shift and footprint assays revealed that PerR binds directly to two tandemly arranged noncanonical PerR boxes located in the upstream promoter region of srfA. A transcriptional srfA-lacZ fusion lacking both PerR boxes showed diminished and PerR-independent expression, indicating that the PerR boxes we identified function as positive cis elements for srfA transcription.

Cell type-specific protein-DNA interactions in the human zeta-globin upstream promoter region: displacement of Sp1 by the erythroid cell-specific factor NF-E1

1990 ◽  
Vol 10 (1) ◽  
pp. 282-294
Author(s):  
C Y Yu ◽  
J Chen ◽  
L I Lin ◽  
M Tam ◽  
C K Shen
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Promoter Region ◽  
Globin Gene ◽  
Cell Type ◽  
Upstream Promoter ◽  
Protein Dna Interactions ◽  
Cell Type Specific ◽  
Upstream Promoter Region ◽  
Zeta Globin Gene

The protein-DNA interactions of the upstream promoter region of the human embryonic zeta-globin gene in nuclear extracts of erythroid K562 cells and nonerythroid HeLa cells were analyzed by DNase I footprinting, gel mobility shift assay, methylation interference, and oligonucleotide competition experiments. There are mainly two clusters of nuclear factor-binding sites in the zeta promoter. The proximal cluster spans the DNA sequence from -110 to -60 and consists of binding sites for CP2, Sp1, and NF-E1. NF-E1 binding is K562 specific, whereas CP2 binding is common to both types of cells. Overlapping the NF-E1- and CP2-binding sites is a hidden Sp1-binding site or CAC box, as demonstrated by binding studies of affinity-purified Sp1. In the distal promoter region at -250 to -220, another NF-E1-binding site overlaps a CAC box or Sp1-binding site. Extract-mixing experiments demonstrated that the higher affinity of NF-E1 binding excluded the binding of Sp1 in the K562 extract. NF-E1 factors could also displace prebound Sp1 molecules. Between the two clusters of multiple-factor-binding sites are sequences recognized by other factors, including zeta-globin factors 1 and 2, that are present in both HeLa and K562 extracts. We discuss the cell type-specific, competitive binding of multiple nuclear factors in terms of functional implications in transcriptional regulation of the zeta-globin gene.

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