scholarly journals Lack of an initiator element is responsible for multiple transcriptional initiation sites of the TATA-less mouse thymidylate synthase promoter.

1993 ◽  
Vol 13 (8) ◽  
pp. 4894-4903 ◽  
Author(s):  
Y Geng ◽  
L F Johnson
Keyword(s):  
Thymidylate Synthase ◽  
Cultured Cells ◽  
Transcriptional Start Site ◽  
Start Site ◽  
Promoter Elements ◽  
Transcriptional Initiation ◽  
Initiator Element ◽  
Insertion Mutations ◽  
Wild Type Promoter ◽  
Broad Region

The mouse thymidylate synthase promoter lacks a TATA box and initiates transcription at many sites across a 90-nucleotide initiation window. We showed previously that wild-type promoter activity is maintained with a promoter that extends only 13 nucleotides upstream of the first start site. G/A-rich and G/C-rich promoter elements were identified in the vicinity of the first transcriptional start site. The goals of the present study were to determine whether there are additional promoter elements in the initiation window and to determine why transcription initiates across such a broad region. Minigenes containing a variety of substitution, deletion, and insertion mutations in the promoter region were transfected into cultured cells, and the effects on expression and the pattern of start sites were determined. The results indicate that there are no additional promoter elements downstream of the G/C box. The boundaries of the transcription window are established by elements near the 5' end of the window, whereas the pattern of start sites is determined by sequences within the window. The promoter lacks an initiator element. When an initiator element was inserted, transcription initiated predominantly at the position directed by the initiator when it was inserted within the initiation window but not when it was inserted immediately upstream of the window.

Lack of an initiator element is responsible for multiple transcriptional initiation sites of the TATA-less mouse thymidylate synthase promoter

1993 ◽  
Vol 13 (8) ◽  
pp. 4894-4903
Author(s):  
Y Geng ◽  
L F Johnson
Keyword(s):  
Thymidylate Synthase ◽  
Cultured Cells ◽  
Transcriptional Start Site ◽  
Start Site ◽  
Promoter Elements ◽  
Transcriptional Initiation ◽  
Initiator Element ◽  
Insertion Mutations ◽  
Wild Type Promoter ◽  
Broad Region

The mouse thymidylate synthase promoter lacks a TATA box and initiates transcription at many sites across a 90-nucleotide initiation window. We showed previously that wild-type promoter activity is maintained with a promoter that extends only 13 nucleotides upstream of the first start site. G/A-rich and G/C-rich promoter elements were identified in the vicinity of the first transcriptional start site. The goals of the present study were to determine whether there are additional promoter elements in the initiation window and to determine why transcription initiates across such a broad region. Minigenes containing a variety of substitution, deletion, and insertion mutations in the promoter region were transfected into cultured cells, and the effects on expression and the pattern of start sites were determined. The results indicate that there are no additional promoter elements downstream of the G/C box. The boundaries of the transcription window are established by elements near the 5' end of the window, whereas the pattern of start sites is determined by sequences within the window. The promoter lacks an initiator element. When an initiator element was inserted, transcription initiated predominantly at the position directed by the initiator when it was inserted within the initiation window but not when it was inserted immediately upstream of the window.

The 5'-flanking region of the mouse thymidylate synthase gene is necessary but not sufficient for normal regulation in growth-stimulated cells

1991 ◽  
Vol 11 (2) ◽  
pp. 1023-1029
Author(s):  
Y Li ◽  
D Li ◽  
K Osborn ◽  
L F Johnson
Keyword(s):  
Thymidylate Synthase ◽  
Cultured Cells ◽  
Transcriptional Start Site ◽  
Simian Virus 40 ◽  
Normal Growth ◽  
Housekeeping Gene ◽  
Simian Virus ◽  
Coding Region ◽  
Proliferating Cells ◽  
Early Promoter

The thymidylate synthase (TS) gene is a housekeeping gene that is expressed at much higher levels in proliferating cells than in quiescent cells. We have studied the role of the TS 5'-flanking sequences in regulating the level of expression of the mouse TS gene. A variety of chimeric TS minigenes that contain different promoters linked either to the TS coding region (with or without introns) or to the chloramphenicol acetyltransferase (CAT) coding region were constructed. The activities of the minigenes were determined by transfecting them into cultured cells and measuring the levels of mRNA or enzyme derived from the chimeric genes. We found that the mouse TS promoter had about the same strength as the simian virus 40 early promoter but was significantly stronger than the herpes simplex virus thymidine kinase promoter. Stable transfection studies revealed that minigenes consisting of the normal TS promoter (extending to -1 kb), coding region, and polyadenylation signal were regulated normally in response to growth stimulation. When the TS promoter was replaced by the simian virus 40 early promoter or by a TS promoter that retained only 60 nucleotides upstream of the first transcriptional start site, the minigene was expressed constitutively. A minigene consisting of the TS promoter (extending to -1 kb) linked to the CAT coding region was also expressed constitutively. These observations indicate that sequences upstream of the transcriptional start sites of the TS gene are necessary, although not sufficient, for normal growth-regulated expression of the mouse TS gene.

scholarly journals Characterization of Coding/Noncoding Variants for SHROOM3 in Patients with CKD

2018 ◽  
Vol 29 (5) ◽  
pp. 1525-1535 ◽  
Author(s):  
Jeremy W. Prokop ◽  
Nan Cher Yeo ◽  
Christian Ottmann ◽  
Surya B. Chhetri ◽  
Kacie L. Florus ◽  
...  
Keyword(s):  
Allele Frequency ◽  
Molecular Mechanisms ◽  
Rare Variants ◽  
Cultured Cells ◽  
Transcriptional Start Site ◽  
Hippo Pathway ◽  
Pdz Domain ◽  
Sequence Variant ◽  
Start Site ◽  
High Effect

Background Interpreting genetic variants is one of the greatest challenges impeding analysis of rapidly increasing volumes of genomic data from patients. For example, SHROOM3 is an associated risk gene for CKD, yet causative mechanism(s) of SHROOM3 allele(s) are unknown.Methods We used our analytic pipeline that integrates genetic, computational, biochemical, CRISPR/Cas9 editing, molecular, and physiologic data to characterize coding and noncoding variants to study the human SHROOM3 risk locus for CKD.Results We identified a novel SHROOM3 transcriptional start site, which results in a shorter isoform lacking the PDZ domain and is regulated by a common noncoding sequence variant associated with CKD (rs17319721, allele frequency: 0.35). This variant disrupted allele binding to the transcription factor TCF7L2 in podocyte cell nuclear extracts and altered transcription levels of SHROOM3 in cultured cells, potentially through the loss of repressive looping between rs17319721 and the novel start site. Although common variant mechanisms are of high utility, sequencing is beginning to identify rare variants involved in disease; therefore, we used our biophysical tools to analyze an average of 112,849 individual human genome sequences for rare SHROOM3 missense variants, revealing 35 high-effect variants. The high-effect alleles include a coding variant (P1244L) previously associated with CKD (P=0.01, odds ratio=7.95; 95% CI, 1.53 to 41.46) that we find to be present in East Asian individuals at an allele frequency of 0.0027. We determined that P1244L attenuates the interaction of SHROOM3 with 14–3-3, suggesting alterations to the Hippo pathway, a known mediator of CKD.Conclusions These data demonstrate multiple new SHROOM3-dependent genetic/molecular mechanisms that likely affect CKD.

scholarly journals The 5'-flanking region of the mouse thymidylate synthase gene is necessary but not sufficient for normal regulation in growth-stimulated cells.

1991 ◽  
Vol 11 (2) ◽  
pp. 1023-1029 ◽  
Author(s):  
Y Li ◽  
D Li ◽  
K Osborn ◽  
L F Johnson
Keyword(s):  
Thymidylate Synthase ◽  
Cultured Cells ◽  
Transcriptional Start Site ◽  
Simian Virus 40 ◽  
Normal Growth ◽  
Housekeeping Gene ◽  
Simian Virus ◽  
Coding Region ◽  
Proliferating Cells ◽  
Early Promoter

The thymidylate synthase (TS) gene is a housekeeping gene that is expressed at much higher levels in proliferating cells than in quiescent cells. We have studied the role of the TS 5'-flanking sequences in regulating the level of expression of the mouse TS gene. A variety of chimeric TS minigenes that contain different promoters linked either to the TS coding region (with or without introns) or to the chloramphenicol acetyltransferase (CAT) coding region were constructed. The activities of the minigenes were determined by transfecting them into cultured cells and measuring the levels of mRNA or enzyme derived from the chimeric genes. We found that the mouse TS promoter had about the same strength as the simian virus 40 early promoter but was significantly stronger than the herpes simplex virus thymidine kinase promoter. Stable transfection studies revealed that minigenes consisting of the normal TS promoter (extending to -1 kb), coding region, and polyadenylation signal were regulated normally in response to growth stimulation. When the TS promoter was replaced by the simian virus 40 early promoter or by a TS promoter that retained only 60 nucleotides upstream of the first transcriptional start site, the minigene was expressed constitutively. A minigene consisting of the TS promoter (extending to -1 kb) linked to the CAT coding region was also expressed constitutively. These observations indicate that sequences upstream of the transcriptional start sites of the TS gene are necessary, although not sufficient, for normal growth-regulated expression of the mouse TS gene.

scholarly journals Functional Studies of CCAAT/Enhancer Binding Protein Site Located Downstream of the Transcriptional Start Site

2017 ◽  
Vol 10 ◽  
pp. 117955571769455 ◽  
Author(s):  
Yujie Liu ◽  
Michael R Nonnemacher ◽  
Aikaterini Alexaki ◽  
Vanessa Pirrone ◽  
Anupam Banerjee ◽  
...  
Keyword(s):  
Binding Protein ◽  
Transcriptional Start Site ◽  
Replication Capacity ◽  
Start Site ◽  
Transcriptional Initiation ◽  
Functional Studies ◽  
Enhancer Binding Protein ◽  
Ccaat Enhancer Binding Protein ◽  
Hiv 1

Previous studies have identified a CCAAT/enhancer binding protein (C/EBP) site located downstream of the transcriptional start site (DS3). The role of the DS3 element with respect to HIV-1 transactivation by Tat and viral replication has not been characterized. We have demonstrated that DS3 was a functional C/EBPβ binding site and mutation of this site to the C/EBP knockout DS3-9C variant showed lower HIV-1 long terminal repeat (LTR) transactivation by C/EBPβ. However, it was able to exhibit similar or even higher transcription levels by Tat compared to the parental LTR. C/EBPβ and Tat together further enhanced the transcription level of the parental LAI-LTR and DS3-9C LTR, with higher levels in the DS3-9C LTR. HIV molecular clone viruses carrying the DS3-9C variant LTR demonstrated a decreased replication capacity and delayed rate of replication. These results suggest that DS3 plays a role in virus transcriptional initiation and provides new insight into C/EBP regulation of HIV-1.

scholarly journals Promoter elements of the mouse complement C4 gene critical for transcription activation and start site location.

1994 ◽  
Vol 269 (11) ◽  
pp. 8268-8279
Author(s):  
Y. Miyagoe ◽  
M.D. Galibert ◽  
E. Georgatsou ◽  
G. Fourel ◽  
T. Meo
Keyword(s):  
Transcription Activation ◽  
Start Site ◽  
Site Location ◽  
Promoter Elements ◽  
Complement C4

Competitive and cooperative functioning of the anterior and posterior promoter elements of an Alu family repeat

1986 ◽  
Vol 6 (6) ◽  
pp. 2041-2052
Author(s):  
C Perez-Stable ◽  
C K Shen
Keyword(s):  
Hela Cell ◽  
Rna Polymerase Iii ◽  
Trna Genes ◽  
Promoter Element ◽  
Class Iii ◽  
Promoter Elements ◽  
Transcriptional Initiation ◽  
Cell Extracts ◽  
Alu Repeat ◽  
Factor C

Similar to tRNA genes and the VAI gene, the Alu family repeats are transcribed by RNA polymerase III and contain a split intragenic promoter. Results of our previous studies have shown that when the anterior, box A-containing promoter element (5'-Pu-Pu-Py-N-N-Pu-Pu-Py-G-G-3' in which Pu is any purine, Py is any pyrimidine, and N is any nucleotide) of a human Alu family repeat is deleted, the remaining box B-containing promoter element (5'-G-A/T-T-C-Pu-A-N-N-C-3') is still capable of directing weak transcriptional initiation at approximately 70 base pairs (bp) upstream from the box B sequence. This is different from the tRNA genes in which the box A-containing promoter element plays the major role in the positioning of the transcriptional initiation site(s). To account for this difference, we first carried out competition experiments in which we show that the posterior element of the Alu repeat competes with the VAI gene effectively for the transcription factor C in HeLa cell extracts. We then constructed a series of contraction and expansion mutants of the Alu repeat promoter in which the spacing between boxes A and B was systematically varied by molecular cloning. In vitro transcription of these clones in HeLa cell extracts was analyzed by RNA gel electrophoresis and primer extension mapping. We show that when the box A and box B promoter sequences are separated by 47 to 298 bp, the transcriptional initiation sites remain 4 to 5 bp upstream from box A. However, this positioning function by the box A-containing promoter element was lost when the spacing was shortened to only 26 bp or increased to longer than 600 bp. Instead, transcriptional initiation occurred approximately 70 bp upstream from box B, similar to that in the clones containing only the box B promoter element. All the mutant clones were transcribed less efficiently than was the wild type. An increase in the distance between boxes A and B also activated a second box A-like element within the Alu family repeat. We compare these results with the results of tRNA gene studies. We also discuss this comparison in terms of the positioning function of the split class III promoter elements and the evolutionary conservation of the spacing between the two promoter elements for optimum transcriptional efficiency.

The two isozymes of rat intestinal alkaline phosphatase are products of two distinct genes

2000 ◽  
Vol 3 (1) ◽  
pp. 1-8 ◽  
Author(s):  
Q. XIE ◽  
D. H. ALPERS
Keyword(s):  
Alkaline Phosphatase ◽  
Transcriptional Start Site ◽  
In Vivo Studies ◽  
Peroxisome Proliferator ◽  
Start Site ◽  
Intestinal Alkaline Phosphatase ◽  
Promoter Regions ◽  
Flanking Regions

Xie, Q., and D. H. Alpers. The two isozymes of rat intestinal alkaline phosphatase are products of two distinct genes. Physiol Genomics 3: 1–8, 2000.—Rat intestinal alkaline phosphatases (IAP-I and -II) differ in primary structure, substrate specificity, tissue localization, and response to fat feeding. This study identifies two distinct genes (∼5–6 kb) corresponding to each isozyme and containing 11 exons of nearly identical size. The exon-intron junctions are identical with those found in IAP genes from other species. The 1.7 and 1.2 bp of 5′ flanking regions isolated from each gene, respectively, contain Sp1 and gut-enriched Kruppel-like factor (GKLF) binding sites, but otherwise show little identity. There is a potential CAAT-box 14 bp 5′ to the transcriptional start site, 36 bp upstream from IAP-I, and a TATA-box 31 bp 5′ to the transcriptional start site, 55 bp upstream from IAP-II. Transfection of these promoter regions (linked to luciferase as a reporter gene) into a kidney cell line, COS-7, produced the differential response to oleic acid expected from in vivo studies, i.e., threefold increase using the 5′ flanking region of IAP-II, but not IAP-I. This response was not reproduced by 5,8,11,14-eicosatetraynoic acid (ETYA) or clofibrate, suggesting that peroxisome proliferator response elements are not involved. Isolation of the IAP-II gene will allow determination of the sequences responsible for dietary fat response in the enterocyte.

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