scholarly journals Identification and characterization of a novel enhancer for the rat neu promoter.

1991 ◽  
Vol 11 (4) ◽  
pp. 1875-1882 ◽  
Author(s):  
D H Yan ◽  
M C Hung
Keyword(s):  
Binding Site ◽  
Specific Protein ◽  
Potential Candidate ◽  
Band Shift ◽  
Independent Manner ◽  
Kinase Gene ◽  
Enhancer Activity ◽  
Cis Acting ◽  
Protein Binding Sequence ◽  
Binding Sequence

We used chloramphenicol acetyltransferase (CAT) assays to identify and characterize cis-acting elements responsible for rat neu promoter function. Deletion of a region of the neu promoter (-504 to -312) resulted in a marked decrease in CAT activity, indicating that this promoter region corresponds to a positive cis-acting element. Using band shift assays and methylation interference analyses, we further identified a specific protein-binding sequence, AAGATAAAACC (-466 to -456), that binds a specific trans-acting factor termed RVF (for EcoRV factor on the neu promoter). The RVF-binding site is required for maximum transcriptional activity of the rat neu promoter. This same sequence is also found in the corresponding regions of both human and mouse neu promoters. Furthermore, this sequence can enhance the CAT activity driven by a minimum promoter of the thymidine kinase gene in an orientation-independent manner, and thus it behaves as an enhancer. Our results demonstrate that RVF is the major DNA-binding protein contributing to enhancer activity. In addition, Southwestern (DNA-protein) blot analysis using the RVF-binding site as a probe points to a 60-kDa polypeptide as a potential candidate for RVF.

scholarly journals Identification and characterization of a novel enhancer for the rat neu promoter

1991 ◽  
Vol 11 (4) ◽  
pp. 1875-1882
Author(s):  
D H Yan ◽  
M C Hung
Keyword(s):  
Binding Site ◽  
Specific Protein ◽  
Potential Candidate ◽  
Band Shift ◽  
Independent Manner ◽  
Kinase Gene ◽  
Enhancer Activity ◽  
Cis Acting ◽  
Protein Binding Sequence ◽  
Binding Sequence

We used chloramphenicol acetyltransferase (CAT) assays to identify and characterize cis-acting elements responsible for rat neu promoter function. Deletion of a region of the neu promoter (-504 to -312) resulted in a marked decrease in CAT activity, indicating that this promoter region corresponds to a positive cis-acting element. Using band shift assays and methylation interference analyses, we further identified a specific protein-binding sequence, AAGATAAAACC (-466 to -456), that binds a specific trans-acting factor termed RVF (for EcoRV factor on the neu promoter). The RVF-binding site is required for maximum transcriptional activity of the rat neu promoter. This same sequence is also found in the corresponding regions of both human and mouse neu promoters. Furthermore, this sequence can enhance the CAT activity driven by a minimum promoter of the thymidine kinase gene in an orientation-independent manner, and thus it behaves as an enhancer. Our results demonstrate that RVF is the major DNA-binding protein contributing to enhancer activity. In addition, Southwestern (DNA-protein) blot analysis using the RVF-binding site as a probe points to a 60-kDa polypeptide as a potential candidate for RVF.

A REB1-binding site is required for GCN4-independent ILV1 basal level transcription and can be functionally replaced by an ABF1-binding site

1992 ◽  
Vol 12 (12) ◽  
pp. 5516-5526
Author(s):  
J E Remacle ◽  
S Holmberg
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Amino Acid Biosynthesis ◽  
Promoter Element ◽  
General Control ◽  
Acid Biosynthesis ◽  
Cis Acting ◽  
Isoleucine Biosynthesis ◽  
Binding Sequence ◽  
Basal Level Expression

The ILV1 gene of Saccharomyces cerevisiae encodes the first committed step in isoleucine biosynthesis and is regulated by general control of amino acid biosynthesis. Deletion analysis of the ILV1 promoter revealed a GC-rich element important for the basal level expression. This cis-acting element, called ILV1BAS, is functional independently of whether GCN4 protein is present. Furthermore, unlike the situation at HIS4, the magnitude of GCN4-mediated derepression is independent of ILV1BAS. The element has homology to the consensus REB1-binding sequence CGGGTARNNR. Gel retardation assays showed that REB1 binds specifically to this element. We show that REB1-binding sites normally situated in the SIN3 promoter and in the 35S rRNA promoter can substitute for the ILV1 REB1 site. Furthermore, a SIN3 REB1 site containing a point mutation that abolishes REB1 binding does not support ILV1 basal level expression, suggesting that binding of REB1 is important for the control of ILV1 basal level expression. Interestingly, an ABF1-binding site can also functionally replace the ILV1 REB1-binding site. A mutated ABF1 site that displays a very low affinity for ABF1 does not functionally replace the ILV1 REB1 site. This suggests that ABF1 and REB1 may have related functions within the cell. Although the REB1-binding site is required for the ILV1 basal level expression, the site on its own stimulates transcription only slightly when combined with the CYC1 downstream promoter elements, indicating that another ILV1 promoter element functions in combination with the REB1 site to control high basal level expression.

scholarly journals A REB1-binding site is required for GCN4-independent ILV1 basal level transcription and can be functionally replaced by an ABF1-binding site.

1992 ◽  
Vol 12 (12) ◽  
pp. 5516-5526 ◽  
Author(s):  
J E Remacle ◽  
S Holmberg
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Amino Acid Biosynthesis ◽  
Promoter Element ◽  
General Control ◽  
Acid Biosynthesis ◽  
Cis Acting ◽  
Isoleucine Biosynthesis ◽  
Binding Sequence ◽  
Basal Level Expression

The ILV1 gene of Saccharomyces cerevisiae encodes the first committed step in isoleucine biosynthesis and is regulated by general control of amino acid biosynthesis. Deletion analysis of the ILV1 promoter revealed a GC-rich element important for the basal level expression. This cis-acting element, called ILV1BAS, is functional independently of whether GCN4 protein is present. Furthermore, unlike the situation at HIS4, the magnitude of GCN4-mediated derepression is independent of ILV1BAS. The element has homology to the consensus REB1-binding sequence CGGGTARNNR. Gel retardation assays showed that REB1 binds specifically to this element. We show that REB1-binding sites normally situated in the SIN3 promoter and in the 35S rRNA promoter can substitute for the ILV1 REB1 site. Furthermore, a SIN3 REB1 site containing a point mutation that abolishes REB1 binding does not support ILV1 basal level expression, suggesting that binding of REB1 is important for the control of ILV1 basal level expression. Interestingly, an ABF1-binding site can also functionally replace the ILV1 REB1-binding site. A mutated ABF1 site that displays a very low affinity for ABF1 does not functionally replace the ILV1 REB1 site. This suggests that ABF1 and REB1 may have related functions within the cell. Although the REB1-binding site is required for the ILV1 basal level expression, the site on its own stimulates transcription only slightly when combined with the CYC1 downstream promoter elements, indicating that another ILV1 promoter element functions in combination with the REB1 site to control high basal level expression.

scholarly journals An 18-base-pair sequence in the mouse proalpha1(II) collagen gene is sufficient for expression in cartilage and binds nuclear proteins that are selectively expressed in chondrocytes.

1996 ◽  
Vol 16 (8) ◽  
pp. 4512-4523 ◽  
Author(s):  
V Lefebvre ◽  
G Zhou ◽  
K Mukhopadhyay ◽  
C N Smith ◽  
Z Zhang ◽  
...  
Keyword(s):  
Binding Site ◽  
Promoter Activity ◽  
Molecular Mechanisms ◽  
Specific Protein ◽  
Collagen Gene ◽  
Enhancer Activity ◽  
Primary Chondrocytes ◽  
Pou Domain ◽  
Intron 1 ◽  
Pair Sequence

The molecular mechanisms by which mesenchymal cells differentiate into chondrocytes are still poorly understood. We have used the gene for a chondrocyte marker, the proalpha1(II) collagen gene (Col2a1), as a model to delineate a minimal sequence needed for chondrocyte expression and identify chondrocyte-specific proteins binding to this sequence. We previously localized a cartilage-specific enhancer to 156 bp of the mouse Col2a1 intron 1. We show here that four copies of a 48-bp subsegment strongly increased promoter activity in transiently transfected rat chondrosarcoma (RCS) cells and mouse primary chondrocytes but not in 10T1/2 fibroblasts. They also directed cartilage specificity in transgenic mouse embryos. These 48 bp include two 11-bp inverted repeats with only one mismatch. Tandem copies of an 18-bp element containing the 3' repeat strongly enhanced promoter activity in RCS cells and chondrocytes but not in fibroblasts. Transgenic mice harboring 12 copies of this 18-mer expressed luciferase in ribs and vertebrae and in isolated chondrocytes but not in noncartilaginous tissues except skin and brain. In gel retardation assays, an RCS cell-specific protein and another closely related protein expressed only in RCS cells and primary chondrocytes bound to a 10-bp sequence within the 18-mer. Mutations in these 10 bp abolished activity of the multimerized 18-bp enhancer, and deletion of these 10 bp abolished enhancer activity of 465- and 231-bp intron 1 segments. This sequence contains a low-affinity binding site for POU domain proteins, and competition experiments with a high-affinity POU domain binding site strongly suggested that the chondrocyte proteins belong to this family. Together, our results indicate that an 18-bp sequence in Col2a1 intron 1 controls chondrocyte expression and suggest that RCS cells and chondrocytes contain specific POU domain proteins involved in enhancer activity.

A regulatory element in the CHA1 promoter which confers inducibility by serine and threonine on Saccharomyces cerevisiae genes

1993 ◽  
Vol 13 (12) ◽  
pp. 7604-7611
Author(s):  
C Bornaes ◽  
M W Ignjatovic ◽  
P Schjerling ◽  
M C Kielland-Brandt ◽  
S Holmberg
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Binding Site ◽  
Regulatory Element ◽  
Consensus Sequence ◽  
Nitrogen Sources ◽  
Binding Activity ◽  
Band Shift ◽  
Cis Acting ◽  
Inducer Exclusion

CHA1 of Saccharomyces cerevisiae is the gene for the catabolic L-serine (L-threonine) dehydratase, which is responsible for biodegradation of serine and threonine. We have previously shown that expression of the CHA1 gene is transcriptionally induced by serine and threonine. Northern (RNA) analysis showed that the additional presence of good nitrogen sources affects induction. This may well be due to inducer exclusion. To identify interactions of cis-acting elements with trans activators of the CHA1 promoter, we performed band shift assays of nuclear protein extracts with CHA1 promoter fragments. By this approach, we identified a protein-binding site of the CHA1 promoter. The footprint of this protein contains the ABF1-binding site consensus sequence. This in vitro binding activity is present irrespectively of CHA1 induction. By deletion analysis, two other elements of the CHA1 promoter, UAS1CHA and UAS2CHA, which are needed for induction of the CHA1 gene were identified. Each of the two sequence elements is sufficient to confer serine and threonine induction upon the CYC1 promoter when substituting its upstream activating sequence. Further, in a cha4 mutant strain which is unable to grow with serine or threonine as the sole nitrogen source, the function of UAS1CHA, as well as that of UAS2CHA, is obstructed.

scholarly journals A regulatory element in the CHA1 promoter which confers inducibility by serine and threonine on Saccharomyces cerevisiae genes.

1993 ◽  
Vol 13 (12) ◽  
pp. 7604-7611 ◽  
Author(s):  
C Bornaes ◽  
M W Ignjatovic ◽  
P Schjerling ◽  
M C Kielland-Brandt ◽  
S Holmberg
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Binding Site ◽  
Regulatory Element ◽  
Consensus Sequence ◽  
Nitrogen Sources ◽  
Binding Activity ◽  
Band Shift ◽  
Cis Acting ◽  
Inducer Exclusion

CHA1 of Saccharomyces cerevisiae is the gene for the catabolic L-serine (L-threonine) dehydratase, which is responsible for biodegradation of serine and threonine. We have previously shown that expression of the CHA1 gene is transcriptionally induced by serine and threonine. Northern (RNA) analysis showed that the additional presence of good nitrogen sources affects induction. This may well be due to inducer exclusion. To identify interactions of cis-acting elements with trans activators of the CHA1 promoter, we performed band shift assays of nuclear protein extracts with CHA1 promoter fragments. By this approach, we identified a protein-binding site of the CHA1 promoter. The footprint of this protein contains the ABF1-binding site consensus sequence. This in vitro binding activity is present irrespectively of CHA1 induction. By deletion analysis, two other elements of the CHA1 promoter, UAS1CHA and UAS2CHA, which are needed for induction of the CHA1 gene were identified. Each of the two sequence elements is sufficient to confer serine and threonine induction upon the CYC1 promoter when substituting its upstream activating sequence. Further, in a cha4 mutant strain which is unable to grow with serine or threonine as the sole nitrogen source, the function of UAS1CHA, as well as that of UAS2CHA, is obstructed.

scholarly journals Role of the promoter in the sensitivity of human thymidine kinase to lack of Zn2+

1995 ◽  
Vol 308 (2) ◽  
pp. 659-664 ◽  
Author(s):  
J K Chesters ◽  
R Boyne ◽  
L Petrie ◽  
K E Lipson
Keyword(s):  
Binding Site ◽  
Thymidine Kinase ◽  
Transcription Initiation ◽  
Kinase Activity ◽  
Initiation Site ◽  
Specific Protein ◽  
Thymidine Kinase Gene ◽  
Thymidine Kinase Activity ◽  
Kinase Gene

Previous studies had indicated that lack of Zn2+ inhibits the expression of thymidine kinase activity and produces a corresponding reduction in the concentration of its mRNA. The present investigations have shown that with human thymidine kinase this is associated with increased binding of a specific protein to the gene's promoter in the region between -55 and -83 bp 5′ to the transcription initiation site. A second binding site for the protein is present within the sixth exon of the human thymidine kinase gene.

scholarly journals Embryonal Carcinoma P19 Cells Produce Erythropoietin Constitutively But Express Lactate Dehydrogenase in an Oxygen-Dependent Manner

Blood ◽  
1998 ◽  
Vol 91 (4) ◽  
pp. 1185-1195 ◽  
Author(s):  
Taiho Kambe ◽  
Junko Tada ◽  
Mariko Chikuma ◽  
Seiji Masuda ◽  
Masaya Nagao ◽  
...  
Keyword(s):  
Lactate Dehydrogenase ◽  
Binding Site ◽  
Embryonal Carcinoma ◽  
Hypoxia Inducible Factor ◽  
Embryonic Stem ◽  
Gene Promoter ◽  
Dependent Manner ◽  
P19 Cells ◽  
Independent Manner ◽  
Hep3b Cells

Abstract Embryonic stem cells and embryonal carcinoma P19 cells produce erythropoietin (Epo) in an oxygen-independent manner, although lactate dehydrogenase A (LDHA) is hypoxia-inducible. To explore this paradox, we studied the operation of cis-acting sequences from these genes in P19 and Hep3B cells. The Epo gene promoter and 3′ enhancer from P19 cells conveyed hypoxia-inducible responses in Hep3B cells but not in P19 cells. Together with DNA sequencing and the normal transcription start site of P19 Epo gene, this excluded the possibility that the noninducibility of Epo gene in P19 cells was due to mutation in these sequences or unusual initiation of transcription. In contrast, reporter constructs containing LDHA enhancer and promoter were hypoxia inducible in P19 and Hep3B cells, and mutation of a hypoxia- inducible factor 1 (HIF-1) binding site abolished the hypoxic inducibility in both cells, indicating that HIF-1 activation operates normally in P19 cells. Neither forced expression of hepatocyte nuclear factor 4 in P19 cells nor deletion of its binding site from the Epo enhancer was effective in restoring Epo enhancer function. P19 cells may lack an unidentified regulator(s) required for interaction of the Epo enhancer with Epo and LDHA promoters.

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