scholarly journals Multiple regulatory elements in the intergenic region between the alpha-fetoprotein and albumin genes.

1986 ◽  
Vol 6 (2) ◽  
pp. 477-487 ◽  
Author(s):  
R Godbout ◽  
R Ingram ◽  
S M Tilghman
Keyword(s):  
Thymidine Kinase ◽  
Hela Cells ◽  
Hepatoma Cell ◽  
Regulatory Elements ◽  
Alpha Fetoprotein ◽  
Hepatoma Cell Line ◽  
Thymidine Kinase Gene ◽  
Hep G2 ◽  
Transient Expression Assay ◽  
Kinase Gene

Three enhancer elements spanning a distance of 7 kilobases have been found at the 5' end of the alpha-fetoprotein (AFP) gene. These elements were identified by transient expression assay after the introduction of a modified mouse AFP gene with variable amounts of 5' flanking sequence into a human hepatoma cell line, Hep G2. These regulatory elements function in a position-independent and orientation-independent manner that is typical of enhancers. All three elements will stimulate transcription from the promoter of the herpes simplex virus thymidine kinase gene. In Hep G2 cells, transcriptional activation from the heterologous promoter was approximately 25- to 50-fold higher than the basal levels obtained in the absence of AFP enhancer elements. In HeLa cells, the increase in thymidine kinase gene transcription varied from 6- to 14-fold, indicating that the enhancer elements exhibit some cell type specificity. Deletion analysis of the region proximal to the AFP transcription initiation site identified an essential region between 85 and 52 bases upstream of the site of initiation of transcription whose removal resulted in almost complete extinction of transcriptional activity. This region, which has been shown to be dispensable for transcription in HeLa cells, defines a second tissue-specific regulatory region in the gene.

Multiple regulatory elements in the intergenic region between the alpha-fetoprotein and albumin genes

1986 ◽  
Vol 6 (2) ◽  
pp. 477-487
Author(s):  
R Godbout ◽  
R Ingram ◽  
S M Tilghman
Keyword(s):  
Thymidine Kinase ◽  
Hela Cells ◽  
Hepatoma Cell ◽  
Regulatory Elements ◽  
Alpha Fetoprotein ◽  
Hepatoma Cell Line ◽  
Thymidine Kinase Gene ◽  
Hep G2 ◽  
Transient Expression Assay ◽  
Kinase Gene

Three enhancer elements spanning a distance of 7 kilobases have been found at the 5' end of the alpha-fetoprotein (AFP) gene. These elements were identified by transient expression assay after the introduction of a modified mouse AFP gene with variable amounts of 5' flanking sequence into a human hepatoma cell line, Hep G2. These regulatory elements function in a position-independent and orientation-independent manner that is typical of enhancers. All three elements will stimulate transcription from the promoter of the herpes simplex virus thymidine kinase gene. In Hep G2 cells, transcriptional activation from the heterologous promoter was approximately 25- to 50-fold higher than the basal levels obtained in the absence of AFP enhancer elements. In HeLa cells, the increase in thymidine kinase gene transcription varied from 6- to 14-fold, indicating that the enhancer elements exhibit some cell type specificity. Deletion analysis of the region proximal to the AFP transcription initiation site identified an essential region between 85 and 52 bases upstream of the site of initiation of transcription whose removal resulted in almost complete extinction of transcriptional activity. This region, which has been shown to be dispensable for transcription in HeLa cells, defines a second tissue-specific regulatory region in the gene.

Building a metal-responsive promoter with synthetic regulatory elements

1985 ◽  
Vol 5 (6) ◽  
pp. 1480-1489
Author(s):  
P F Searle ◽  
G W Stuart ◽  
R D Palmiter
Keyword(s):  
Thymidine Kinase ◽  
Promoter Region ◽  
Fusion Gene ◽  
Regulatory Elements ◽  
Regulatory Sequence ◽  
Thymidine Kinase Gene ◽  
Coding Region ◽  
Base Pairs ◽  
Promoter Elements ◽  
Kinase Gene

A fusion gene consisting of the promoter region from the mouse metallothionein-I gene joined to the coding region of the herpes simplex virus thymidine kinase gene is efficiently regulated by zinc in a transient assay when transfected into baby hamster kidney cells. Analysis of similar plasmids in which the metallothionein-I promoter region was mutated indicated the presence of multiple metal regulatory elements (MREs) between -176 and -44 base pairs from the cap site. To further investigate the function of MREs, we inserted a synthetic DNA fragment containing the sequence of MRE-a (the element between -55 and -44 base pairs) into the nonresponsive promoter of the thymidine kinase gene in various positions and configurations. Little or no induction by zinc was observed with single insertions of the regulatory sequence, whereas many different constructions having two copies of MRE-a were inducible. The precise position of the two MREs relative to each other or to the thymidine kinase promoter elements had a relatively small effect on the efficiency of induction, but the inducibility could be further increased by the introduction of more MRE-a sequences. MRE-a can function synergistically with the thymidine kinase distal promoter elements, but in the presence of the TATA box alone it functions as a positive, zinc-dependent promoter element.

scholarly journals Identification of a 70-base-pair cell cycle regulatory unit within the promoter of the human thymidine kinase gene and its interaction with cellular factors.

1991 ◽  
Vol 11 (4) ◽  
pp. 2296-2302 ◽  
Author(s):  
Y K Kim ◽  
A S Lee
Keyword(s):  
Cell Cycle ◽  
Thymidine Kinase ◽  
Consensus Sequence ◽  
Regulatory Elements ◽  
Thymidine Kinase Gene ◽  
Heterologous Promoter ◽  
Kinase Gene ◽  
Cellular Factors ◽  
Regulated Expression ◽  
Cycle Regulation

The promoter of the human thymidine kinase gene contains cis-regulatory elements responsible for its cell-cycle-regulated expression. We report here that a 70-bp region between -133 and -64 is sufficient to confer cell cycle regulation on a heterologous promoter. The 20-bp region between -64 and -83, which contains an inverted CCAAT motif, is important for transcriptional stimulation of this functional unit. The sequence of this CCAAT motif is nearly identical to the consensus sequence for the transcriptional factor CP1. We also examined the specificity and binding activities of cellular factors interacting with the 70-bp fragment. We showed that the cellular factors binding to the 70-bp region are similar during the G1, S, and G2 phases, suggesting that the cell cycle regulatory activity observed must involve processes other than factor binding to the DNA.

Identification of a 70-base-pair cell cycle regulatory unit within the promoter of the human thymidine kinase gene and its interaction with cellular factors

1991 ◽  
Vol 11 (4) ◽  
pp. 2296-2302
Author(s):  
Y K Kim ◽  
A S Lee
Keyword(s):  
Cell Cycle ◽  
Thymidine Kinase ◽  
Consensus Sequence ◽  
Regulatory Elements ◽  
Thymidine Kinase Gene ◽  
Heterologous Promoter ◽  
Kinase Gene ◽  
Cellular Factors ◽  
Regulated Expression ◽  
Cycle Regulation

The promoter of the human thymidine kinase gene contains cis-regulatory elements responsible for its cell-cycle-regulated expression. We report here that a 70-bp region between -133 and -64 is sufficient to confer cell cycle regulation on a heterologous promoter. The 20-bp region between -64 and -83, which contains an inverted CCAAT motif, is important for transcriptional stimulation of this functional unit. The sequence of this CCAAT motif is nearly identical to the consensus sequence for the transcriptional factor CP1. We also examined the specificity and binding activities of cellular factors interacting with the 70-bp fragment. We showed that the cellular factors binding to the 70-bp region are similar during the G1, S, and G2 phases, suggesting that the cell cycle regulatory activity observed must involve processes other than factor binding to the DNA.

scholarly journals Building a metal-responsive promoter with synthetic regulatory elements.

1985 ◽  
Vol 5 (6) ◽  
pp. 1480-1489 ◽  
Author(s):  
P F Searle ◽  
G W Stuart ◽  
R D Palmiter
Keyword(s):  
Thymidine Kinase ◽  
Promoter Region ◽  
Fusion Gene ◽  
Regulatory Elements ◽  
Regulatory Sequence ◽  
Thymidine Kinase Gene ◽  
Coding Region ◽  
Base Pairs ◽  
Promoter Elements ◽  
Kinase Gene

A fusion gene consisting of the promoter region from the mouse metallothionein-I gene joined to the coding region of the herpes simplex virus thymidine kinase gene is efficiently regulated by zinc in a transient assay when transfected into baby hamster kidney cells. Analysis of similar plasmids in which the metallothionein-I promoter region was mutated indicated the presence of multiple metal regulatory elements (MREs) between -176 and -44 base pairs from the cap site. To further investigate the function of MREs, we inserted a synthetic DNA fragment containing the sequence of MRE-a (the element between -55 and -44 base pairs) into the nonresponsive promoter of the thymidine kinase gene in various positions and configurations. Little or no induction by zinc was observed with single insertions of the regulatory sequence, whereas many different constructions having two copies of MRE-a were inducible. The precise position of the two MREs relative to each other or to the thymidine kinase promoter elements had a relatively small effect on the efficiency of induction, but the inducibility could be further increased by the introduction of more MRE-a sequences. MRE-a can function synergistically with the thymidine kinase distal promoter elements, but in the presence of the TATA box alone it functions as a positive, zinc-dependent promoter element.

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