Analysis of promoter activity from an ?-zein gene 5? flanking sequence in transient expression assays

1990 ◽  
Vol 15 (5) ◽  
pp. 755-764 ◽  
Author(s):  
Gary A. Thompson ◽  
Rebecca S. Boston ◽  
Leszek A. Lyznik ◽  
Thomas K. Hodges ◽  
Brian A. Larkins
Keyword(s):  
Transient Expression ◽  
Promoter Activity ◽  
Flanking Sequence ◽  
Zein Gene

The long terminal repeat of an endogenous intracisternal A-particle gene functions as a promoter when introduced into eucaryotic cells by transfection

1984 ◽  
Vol 4 (10) ◽  
pp. 2128-2135
Author(s):  
K K Lueders ◽  
J W Fewell ◽  
E L Kuff ◽  
T Koch
Keyword(s):  
Long Terminal Repeat ◽  
Promoter Activity ◽  
Terminal Repeat ◽  
Flanking Sequence ◽  
Murine Sarcoma Virus ◽  
Sarcoma Virus ◽  
Mouse Cells ◽  
Moloney Murine Sarcoma Virus ◽  
Intracisternal A Particle ◽  
Murine Sarcoma

We describe experiments designed to determine whether an endogenous intracisternal A-particle (IAP) gene randomly selected from a mouse embryo library has the potential to be transcriptionally active. Assays for IAP gene transcription were done with permanently transformed rat cells and transiently transfected monkey and mouse cells. The rat cells, which had integrated IAP gene copies, contained IAP RNA. A start site within the IAP 5' long terminal repeat (LTR) was localized by S1 mapping. The promoter activity of the IAP LTR was also measured in cells 48 h after the introduction of recombinant plasmids in which bacterial chloramphenicol acetyl transferase (CAT) encoding sequences were under the control of the LTR. The IAP LTR promoted CAT activity in mouse and monkey cells. In mouse L-cells, the levels of CAT activity were 10 to 25% of those promoted by an analogous recombinant containing the Moloney murine sarcoma virus LTR as the promoter. In contrast to the Moloney murine sarcoma virus LTR, the IAP LTR was five- to eightfold more active in monkey cells than in mouse cells. The 5' and 3' LTRs were equally active, and promoter activity was dependent on having the orientation of the LTRs with respect to the CAT gene the same as their orientation with respect to the IAP gene. A 5'-flanking sequence containing a member of the highly repetitive R-sequence family increased CAT activity in COS cells 11-fold when present along with the LTR. Our results indicate that the LTR of an endogenous mouse IAP gene can function as an efficient promoter in heterologous as well as homologous cells.

scholarly journals Enhancer and promoter elements directing activation and glucocorticoid repression of the alpha 1-fetoprotein gene in hepatocytes.

1988 ◽  
Vol 8 (4) ◽  
pp. 1398-1407 ◽  
Author(s):  
M Guertin ◽  
H LaRue ◽  
D Bernier ◽  
O Wrange ◽  
M Chevrette ◽  
...  
Keyword(s):  
Glucocorticoid Receptor ◽  
Transient Expression ◽  
Promoter Activity ◽  
Gene Activation ◽  
Point Mutations ◽  
Binding Assays ◽  
Recognition Sequence ◽  
Base Pairs ◽  
Receptor Recognition ◽  
Afp Promoter

Mutations were introduced in 7 kilobases of 5'-flanking rat alpha 1-fetoprotein (AFP) genomic DNA, linked to the chloramphenicol acetyltransferase gene. AFP promoter activity and its repression by a glucocorticoid hormone were assessed by stable and transient expression assays. Stable transfection assays were more sensitive and accurate than transient expression assays in a Morris 7777 rat hepatoma recipient (Hepa7.6), selected for its strong AFP repression by dexamethasone. The segment of DNA encompassing a hepatocyte-constitutive chromatin DNase I-hypersensitive site at -3.7 kilobases and a liver developmental stage-specific site at -2.5 kilobases contains interacting enhancer elements sufficient for high AFP promoter activity in Hepa7.6 or HepG2 cells. Deletions and point mutations define an upstream promoter domain of AFP gene activation, operating with at least three distinct promoter-activating elements, PEI at -65 base pairs, PEII at -120 base pairs, and DE at -160 base pairs. PEI and PEII share homologies with albumin promoter sequences, PEII is a near-consensus nuclear factor I recognition sequence, and DE overlaps a glucocorticoid receptor recognition sequence. An element conferring glucocorticoid repression of AFP gene activity is located in the upstream AFP promoter domain. Receptor-binding assays indicate that this element is the glucocorticoid receptor recognition sequence which overlaps with promoter-activating element DE.

scholarly journals Augmentation of gamma-globin gene promoter activity by carboxylic acids and components of the human beta-globin locus control region

Blood ◽  
1994 ◽  
Vol 84 (11) ◽  
pp. 3929-3935 ◽  
Author(s):  
S Safaya ◽  
A Ibrahim ◽  
RF Rieder
Keyword(s):  
Gene Expression ◽  
Organic Acids ◽  
Carboxylic Acids ◽  
Transient Expression ◽  
Promoter Activity ◽  
Globin Gene ◽  
Gene Promoter ◽  
K562 Cells ◽  
Hemoglobin Synthesis ◽  
Gamma Globin

Butyric acid increases fetal hemoglobin synthesis in adult animals and in erythroid cells in culture and induces the gamma-globin gene promoter in transient expression experiments in K562 cells (McDonagh KT, Nienhuis AW, Blood 78:255a, 1992 [abstr, suppl 1]). We compared the effect of butyrate and other short-chain carboxylic acids in transient expression studies with K562 cells using an expression plasmid bearing a luciferase reporter gene driven by the normal human A gamma-globin gene promoter. Butyrate (4 carbons) increased the activity of the human A gamma-globin gene promoter up to 123 times. Marked augmentation of the normal gamma-promoter activity was also noted with 5-carbon valeric acid (up to 394 times) and 3-carbon propionic acid (up to 129 times). The branched isobutyric acid as well as phenylacetate showed less ability to increase promoter activity. Addition of the tandemly repeated AP-1/NF-E2 (AP) enhancer sequences from hypersensitive site 2 (HS2) of the locus control region (LCR) increased gamma-promoter activity up to 24 times. Addition of a nearby 16-bp conserved motif (CM) in HS2 (Safaya S, Rieder RF, Blood 78:146a, 1992 [abstr, suppl 1]) to the AP-containing plasmid construct further increased gamma-promoter activity. In the presence of butyrate, the plasmid bearing both the AP and CM sequences showed gene expression up to 477 times greater than that of the basal gamma-promoter-driven luciferase plasmid in the absence of inducer. A plasmid bearing the herpes simplex thymidine kinase promoter was also tested and gene expression was markedly increased by the same organic acids. MEL cells responded to butyrate, valerate, and propionate with induction of hemoglobin synthesis. Responses to isobutyrate and 6-carbon caproate required higher concentrations of the compounds. Thus, other short-chain organic acids as well as butyrate increase gamma-promoter activity in the transient expression system, and this activity can be further augmented by incorporating LCR elements into the expression vector. Nonglobin promoters also respond to the same carboxylic acids.

Glucocorticoid inhibition of SP-A gene expression in lung type II cells is mediated via the TTF-1-binding element

2004 ◽  
Vol 286 (4) ◽  
pp. L767-L776 ◽  
Author(s):  
Joseph L. Alcorn ◽  
Kazi N. Islam ◽  
Pampee P. Young ◽  
Carole R. Mendelson
Keyword(s):  
Gene Expression ◽  
Promoter Activity ◽  
Fusion Gene ◽  
Primary Cultures ◽  
Protein A ◽  
Fetal Lung ◽  
Binding Activity ◽  
Type Ii Cells ◽  
Type Ii ◽  
Flanking Sequence

Induction of surfactant protein-A ( SP-A) gene expression in fetal lung type II cells by cAMP and IL-1 is mediated by increased binding of thyroid transcription factor-1 (TTF-1) and NF-κB proteins p50 and p65 to the TTF-1-binding element (TBE) at -183 bp. In type II cell transfections, dexamethasone (Dex) markedly inhibits cAMP-induced expression of rabbit SP-A:human growth hormone ( hGH) fusion genes containing as little as ∼300 bp of the SP-A 5′-flanking sequence. Dex inhibition is blocked by RU-486, suggesting a role of the glucocorticoid receptor (GR). The present study was undertaken to define the mechanisms for GR inhibition of SP-A expression. Cotransfection of primary cultures of type II cells with a GR expression vector abrogated cAMP induction of SP-A promoter activity while, at the same time, causing a 60-fold induction of cotransfected mouse mammary tumor virus ( MMTV) promoter. In lung cells transfected with a fusion gene containing three TBEs fused to the basal SP-A promoter, Dex prevented the stimulatory effect of IL-1 on TTF-1 induction of SP-A promoter activity, suggesting that the GR inhibits SP-A promoter activity through the TBE. In gel shift assays using nuclear extracts from human fetal type II cells cultured in the absence or presence of cAMP, Dex markedly reduced binding of nuclear proteins to the TBE and blocked the stimulatory effect of cAMP on TBE-binding activity. Our finding that Dex increased expression of the NF-κB inhibitory partner IκB-α suggests that the decrease in TBE-binding activity may be caused, in part, by GR inhibition of NF-κB interaction with this site.

scholarly journals The long terminal repeat of an endogenous intracisternal A-particle gene functions as a promoter when introduced into eucaryotic cells by transfection.

1984 ◽  
Vol 4 (10) ◽  
pp. 2128-2135 ◽  
Author(s):  
K K Lueders ◽  
J W Fewell ◽  
E L Kuff ◽  
T Koch
Keyword(s):  
Long Terminal Repeat ◽  
Promoter Activity ◽  
Terminal Repeat ◽  
Flanking Sequence ◽  
Murine Sarcoma Virus ◽  
Sarcoma Virus ◽  
Mouse Cells ◽  
Moloney Murine Sarcoma Virus ◽  
Intracisternal A Particle ◽  
Murine Sarcoma

We describe experiments designed to determine whether an endogenous intracisternal A-particle (IAP) gene randomly selected from a mouse embryo library has the potential to be transcriptionally active. Assays for IAP gene transcription were done with permanently transformed rat cells and transiently transfected monkey and mouse cells. The rat cells, which had integrated IAP gene copies, contained IAP RNA. A start site within the IAP 5' long terminal repeat (LTR) was localized by S1 mapping. The promoter activity of the IAP LTR was also measured in cells 48 h after the introduction of recombinant plasmids in which bacterial chloramphenicol acetyl transferase (CAT) encoding sequences were under the control of the LTR. The IAP LTR promoted CAT activity in mouse and monkey cells. In mouse L-cells, the levels of CAT activity were 10 to 25% of those promoted by an analogous recombinant containing the Moloney murine sarcoma virus LTR as the promoter. In contrast to the Moloney murine sarcoma virus LTR, the IAP LTR was five- to eightfold more active in monkey cells than in mouse cells. The 5' and 3' LTRs were equally active, and promoter activity was dependent on having the orientation of the LTRs with respect to the CAT gene the same as their orientation with respect to the IAP gene. A 5'-flanking sequence containing a member of the highly repetitive R-sequence family increased CAT activity in COS cells 11-fold when present along with the LTR. Our results indicate that the LTR of an endogenous mouse IAP gene can function as an efficient promoter in heterologous as well as homologous cells.

Role of AP2 consensus sites in regulation of rat Npt2 (sodium-phosphate cotransporter) promoter

2000 ◽  
Vol 278 (3) ◽  
pp. F406-F416 ◽  
Author(s):  
C. Shachaf ◽  
K. L. Skorecki ◽  
M. Tzukerman
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Proximal Tubule ◽  
Cell Lines ◽  
Promoter Activity ◽  
Promoter Sequence ◽  
Luciferase Reporter ◽  
Inverted Repeats ◽  
Proximal Tubule Cell ◽  
Flanking Sequence

Expression of the Npt2 gene, encoding the type II sodium-dependent phosphate cotransporter, is restricted to renal proximal tubule epithelium. We have isolated a 4,740-bp fragment of the 5′-flanking sequence of the rat Npt2 gene, identified the transcription initiation site, and demonstrated that this 5′-flanking sequence drives luciferase-reporter gene expression, following transfection in the proximal tubule cell-derived opossum kidney (OK) cell line but not in unrelated cell lines. Analysis of the promoter sequence revealed the presence of 10 consensus binding motifs for the AP2 transcription factor. Transient transfection assays revealed an important effect of the number of tandemly repeated AP2 sites in enhancing promoter activity. The promoter sequence also revealed a pair of inverted repeats enclosing 1,324 bp of intervening sequence and containing 8 of the total 10 AP2 consensus sites in the promoter sequence. Deletion or reversal of orientation of the distal inverted repeat resulted in marked enhancement of promoter activity. Electrophoretic mobility shift analysis revealed a distinct pattern of transcription factor binding to oligonucleotides containing AP2 sites, using nuclear extracts from OK cells, compared with unrelated cell lines. Taken together, these results suggest an important role for AP2 consensus binding sites in regulating Npt2 gene expression and suggest a mechanism of regulation mediated by the interaction of inverted repeats enclosing these sites.

scholarly journals Human dipeptidyl peptidase IV gene promoter: tissue-specific regulation from a TATA-less GC-rich sequence characteristic of a housekeeping gene promoter

1995 ◽  
Vol 311 (3) ◽  
pp. 835-843 ◽  
Author(s):  
S K Böhm ◽  
J R Gum ◽  
R H Erickson ◽  
J W Hicks ◽  
Y S Kim
Keyword(s):  
Hepg2 Cells ◽  
Promoter Activity ◽  
Gene Promoter ◽  
Housekeeping Gene ◽  
Dipeptidyl Peptidase ◽  
Dipeptidyl Peptidase Iv ◽  
Flanking Sequence ◽  
Specific Expression ◽  
Tissue Specific ◽  
Tissue Specific Expression

The dipeptidyl peptidase IV gene encodes a plasma-membrane exopeptidase that is highly expressed in small intestine, lung and kidney. In order to better understand the mechanisms responsible for this tissue-specific expression we cloned, sequenced and functionally characterized the 5′-flanking region of the human dipeptidyl peptidase IV gene. The first 500 bases of the 5′-flanking sequence constituted an unmethylated CpG island, contained several Sp1-binding sites and lacked a consensus TATA box, all characteristics of gene promoters lacking tissue-specific expression. RNase-protection analysis using both small intestinal and Caco2 cell RNA indicated that the dipeptidyl peptidase IV transcript was initiated from no fewer than six major and 12 minor start sites. The 5′-flanking sequence also exhibited functional promoter activity in transient transfection experiments. Here, various lengths of the sequence were cloned upstream of a luciferase gene and introduced into cultured cells using lipofectin. A region located between bases -150 and -109 relative to the start of translation was found to be important for high-level promoter activity in both Caco2 and HepG2 cells. Moreover, Caco2 cells and HepG2 cells, which express high levels of dipeptidyl peptidase IV activity, exhibited much higher normalized luciferase activity after transfection than did 3T3, Jurkat or COS-7 cells, which have low enzyme levels. Sodium butyrate was found to increase both enzyme activity and normalized luciferase in HepG2 cells. Thus the dipeptidyl peptidase IV promoter possesses the ability to initiate transcription in a tissue-specific fashion in spite of having the sequence characteristics of a housekeeping gene promoter.

Organization, 5′-flanking sequence and promoter activity of the rat GPC1 gene

Gene ◽  
1998 ◽  
Vol 206 (2) ◽  
pp. 255-261 ◽  
Author(s):  
Vinod K Asundi ◽  
Bonnie F Keister ◽  
David J Carey
Keyword(s):  
Promoter Activity ◽  
Flanking Sequence
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