Analysis of signals controlling expression of the Chinese hamster ovary aprt gene

1988 ◽  
Vol 8 (6) ◽  
pp. 2536-2544
Author(s):  
J H Park ◽  
M W Taylor
Keyword(s):  
Transcription Start Site ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster ◽  
Gene Transcript ◽  
Start Site ◽  
Transcription Start ◽  
S1 Nuclease ◽  
Deletion Mutations ◽  
Transcription Start Sites ◽  
Aprt Gene

The 5' end of the Chinese hamster ovary aprt gene was sequenced and transcription start sites were determined by both S1 nuclease protection and primer extension assays. Deletion mutants covering the same area were constructed, and adenine phosphoribosyltransferase (APRT) or chloramphenicol acetyltransferase (CAT) activity was measured by transient-expression assays. The aprt gene uses a single cluster of transcription start sites and lacks consensus sequences such as TATA and CCAAT, which are general components of eucaryotic promoters. The 5' deletion mutations of the promoter sequences demonstrated that (i) there is no decrease in either APRT activity or transcription extending to position -89 (relative to the main transcription start site); (ii) an additional 29-base-pair (bp) deletion decreases APRT activity and transcription twofold; and (iii) a deletion past the transcription start sites (P5' delta +27) abolishes both APRT activity and transcription, indicating that a 60-bp fragment immediately upstream of the main transcription start site is involved in basic transcription and a 29-bp fragment just upstream of the 60 bp-fragment stimulates transcription twofold. The 3' deletion mutations showed that a deletion of a 61-bp fragment in the 5' leader and coding sequence abolishes the efficient translation of an aprt-CAT gene transcript. In addition, there are two polyadenylation signals at the genomic 3' end, with the proximal one being sufficient for functional polyadenylation.

scholarly journals Analysis of signals controlling expression of the Chinese hamster ovary aprt gene.

1988 ◽  
Vol 8 (6) ◽  
pp. 2536-2544 ◽  
Author(s):  
J H Park ◽  
M W Taylor
Keyword(s):  
Transcription Start Site ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster ◽  
Gene Transcript ◽  
Start Site ◽  
Transcription Start ◽  
S1 Nuclease ◽  
Deletion Mutations ◽  
Transcription Start Sites ◽  
Aprt Gene

The 5' end of the Chinese hamster ovary aprt gene was sequenced and transcription start sites were determined by both S1 nuclease protection and primer extension assays. Deletion mutants covering the same area were constructed, and adenine phosphoribosyltransferase (APRT) or chloramphenicol acetyltransferase (CAT) activity was measured by transient-expression assays. The aprt gene uses a single cluster of transcription start sites and lacks consensus sequences such as TATA and CCAAT, which are general components of eucaryotic promoters. The 5' deletion mutations of the promoter sequences demonstrated that (i) there is no decrease in either APRT activity or transcription extending to position -89 (relative to the main transcription start site); (ii) an additional 29-base-pair (bp) deletion decreases APRT activity and transcription twofold; and (iii) a deletion past the transcription start sites (P5' delta +27) abolishes both APRT activity and transcription, indicating that a 60-bp fragment immediately upstream of the main transcription start site is involved in basic transcription and a 29-bp fragment just upstream of the 60 bp-fragment stimulates transcription twofold. The 3' deletion mutations showed that a deletion of a 61-bp fragment in the 5' leader and coding sequence abolishes the efficient translation of an aprt-CAT gene transcript. In addition, there are two polyadenylation signals at the genomic 3' end, with the proximal one being sufficient for functional polyadenylation.

scholarly journals A Chinese hamster transcription start site atlas that enables targeted editing of CHO cells

2021 ◽  
Vol 3 (3) ◽  
Author(s):  
Isaac Shamie ◽  
Sascha H Duttke ◽  
Karen J la Cour Karottki ◽  
Claudia Z Han ◽  
Anders H Hansen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Start Site ◽  
Cho Cells ◽  
Genome Engineering ◽  
Chinese Hamster ◽  
Regulatory Elements ◽  
Start Site ◽  
Transcription Start ◽  
Transcription Start Sites ◽  
Silent Genes

Abstract Chinese hamster ovary (CHO) cells are widely used for producing biopharmaceuticals, and engineering gene expression in CHO is key to improving drug quality and affordability. However, engineering gene expression or activating silent genes requires accurate annotation of the underlying regulatory elements and transcription start sites (TSSs). Unfortunately, most TSSs in the published Chinese hamster genome sequence were computationally predicted and are frequently inaccurate. Here, we use nascent transcription start site sequencing methods to revise TSS annotations for 15 308 Chinese hamster genes and 3034 non-coding RNAs based on experimental data from CHO-K1 cells and 10 hamster tissues. We further capture tens of thousands of putative transcribed enhancer regions with this method. Our revised TSSs improves upon the RefSeq annotation by revealing core sequence features of gene regulation such as the TATA box and the Initiator and, as exemplified by targeting the glycosyltransferase gene Mgat3, facilitate activating silent genes by CRISPRa. Together, we envision our revised annotation and data will provide a rich resource for the CHO community, improve genome engineering efforts and aid comparative and evolutionary studies.

scholarly journals MAPCap: A fast and quantitative transcription start site profiling protocol

2019 ◽  
Author(s):  
Vivek Bhardwaj ◽  
Giuseppe Semplicio ◽  
Niyazi Umut Erdogdu ◽  
Asifa Akhtar
Keyword(s):  
High Resolution ◽  
Differential Expression ◽  
Transcription Start Site ◽  
Expression Analysis ◽  
Differential Expression Analysis ◽  
Start Site ◽  
Transcription Start ◽  
Transcription Start Sites

Abstract Below we present a simple and quick TSS quantification protocol, MAPCap (Multiplexed Affinity Purification of Capped RNA) that enables users to combine high-resolution detection of transcription start-sites and differential expression analysis. MAPCap can be used to profile TSS from dozens of samples in a multiplexed way, in 16-18 hours. MAPCap data can be analyzed using our easy-to-use software icetea (https://bioconductor.org/packages/icetea), which allows users to detect robust TSS using replicates, and perform differential TSS analysis.

scholarly journals Characterization of Binding Sequences for Butyrolactone Autoregulator Receptors in Streptomycetes

1999 ◽  
Vol 181 (16) ◽  
pp. 5075-5080 ◽  
Author(s):  
Hiroshi Kinoshita ◽  
Tomohiro Tsuji ◽  
Hiroomi Ipposhi ◽  
Takuya Nihira ◽  
Yasuhiro Yamada
Keyword(s):  
Transcription Start Site ◽  
Dna Sequences ◽  
Target Genes ◽  
Start Codon ◽  
Receptor Protein ◽  
Upstream Region ◽  
Start Site ◽  
Transcription Start ◽  
S1 Nuclease ◽  
Nuclease Mapping

ABSTRACT BarA of Streptomyces virginiae is a specific receptor protein for a member of butyrolactone autoregulators which binds to an upstream region of target genes to control transcription, leading to the production of the antibiotic virginiamycin M1 and S. BarA-binding DNA sequences (BarA-responsive elements [BAREs]), to which BarA binds for transcriptional control, were restricted to 26 to 29-nucleotide (nt) sequences on barA and barBupstream regions by the surface plasmon resonance technique, gel shift assay, and DNase I footprint analysis. Two BAREs (BARE-1 and BARE-2) on the barB upstream region were located 57 to 29 bp (BARE-1) and 268 to 241 bp (BARE-2) upstream from the barBtranslational start codon. The BARE located on the barAupstream region (BARE-3) was found 101 to 76 bp upstream of thebarA start codon. High-resolution S1 nuclease mapping analysis revealed that BARE-1 covered the barBtranscription start site and BARE-3 covered an autoregulator-dependent transcription start site of the barA gene. Deletion and mutation analysis of BARE-2 demonstrated that at least a 19-nt sequence was required for sufficient BarA binding, and A or T residues at the edge as well as internal conserved nucleotides were indispensable. The identified binding sequences for autoregulator receptor proteins were found to be highly conserved among Streptomyces species.

scholarly journals Cooperation of E2F-p130 and Sp1-pRb Complexes in Repression of the Chinese Hamster dhfr Gene

2001 ◽  
Vol 21 (4) ◽  
pp. 1121-1131 ◽  
Author(s):  
Young-Chae Chang ◽  
Sharon Illenye ◽  
Nicholas H. Heintz
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Transcription Start Site ◽  
Trichostatin A ◽  
Chinese Hamster ◽  
Serum Starvation ◽  
Start Site ◽  
Transcription Start ◽  
Serum Stimulation ◽  
Cell Extracts

ABSTRACT In mammalian cells reiterated binding sites for Sp1 and two overlapping and inverted E2F sites at the transcription start site regulate the dhfr promoter during the cell growth cycle. Here we have examined the contributions of the dhfr Sp1 and E2F sites in the repression of dhfr gene expression. In serum-starved cells or during serum stimulation, the Chinese hamsterdhfr gene was not derepressed by trichostatin A (TSA), an inhibitor of histone deacetylases (HDAC). Immunoprecipitation experiments showed that HDAC1 and hypophosphorylated retinoblastoma protein (pRb) are associated with Sp1 in serum-starved CHOC400 cells. In transfection experiments, reporter plasmids containing the reiterated dhfr Sp1 sites were stimulated 10-fold by TSA, while a promoter containing four dhfr E2F sites and a TATA box was responsive to E2F but was completely unaffected by TSA. HDAC1 did not coprecipitate with p130-E2F DNA binding complexes, the predominant E2F binding activity in cell extracts after serum starvation, suggesting that p130 imposes a TSA-insensitive state on thedhfr promoter. In support of this notion, recruitment of GAL4-p130 to a dihydrofolate reductase-GAL4 reporter rendered the promoter insensitive to TSA, while repression by GAL4-pRb was sensitive to TSA. Upon phosphorylation of pRb and p130 after serum stimulation, the Sp1-pRb and p130-E2F interactions were lost while the Sp1-HDAC1 interaction persisted into S phase. Together these studies suggest a dynamic model for the cooperation of pRb and p130 in repression ofdhfr gene expression during withdrawal from the cell cycle. We propose that, during initial phases of cell cycle withdrawal, the binding of dephosphorylated pRb to Sp1-HDAC1 complexes and complexes of E2F-1 -to -3 with DP results in transient, HDAC-dependent suppression of dhfr transcription. Upon withdrawal of cells into G0, recruitment of p130 to E2F-4–DP-1 complexes at the transcription start site results in a TSA-insensitive complex that cooperates with Sp1-HDAC-pRb complexes to stably repressdhfr promoter activity in quiescent cells.

scholarly journals Transcriptional promoter of the human α1(V) collagen gene (COL5A1)

1995 ◽  
Vol 310 (1) ◽  
pp. 15-22 ◽  
Author(s):  
S Lee ◽  
D S Greenspan
Keyword(s):  
Transcription Start Site ◽  
Cpg Island ◽  
Growth Control ◽  
Gc Content ◽  
Collagen Gene ◽  
Start Site ◽  
Transcription Start ◽  
Mobility Shift ◽  
Broad Distribution ◽  
S1 Nuclease

We have characterized the 5′ region of the human alpha 1(V) collagen gene (COL5A1). The transcriptional promoter is shown to have a number of features characteristic of the promoters of ‘housekeeping’ and growth-control-related genes. It lacks obvious TATA and CAAT boxes, has multiple transcription start sites, has a high GC content, lies within a well-defined CpG island and has a number of consensus sites for the potential binding of transcription factor Sp1. This type of promoter structure, while unusual for a collagen gene, is consistent with the broad distribution of expression of COL5A1 and is reminiscent of the promoter structures of the genes encoding type VI collagen, which has a similarly broad distribution of expression. Stepwise deletion of COL5A1 5′ sequences, placed upstream of a heterologous reporter gene, yielded a gradual decrease in promoter activity, indicating that the COL5A1 promoter is composed of an array of cis-acting elements. A minimal promoter region contained within the 212 bp immediately upstream of the major transcription start site contained no consensus sequences for the binding of known transcription factors, but gel mobility shift assays showed this region to bind nuclear factors, including Sp1, at a number of sites. The major transcription start site is flanked by an upstream 34-bp oligopurine/oligopyrimidine stretch, or ‘GAGA’ box, and a downstream 56-bp GAGA box which contains a 10-bp mirror repeat and is sensitive to cleavage with S1 nuclease.

scholarly journals Wdr82 Is a C-Terminal Domain-Binding Protein That Recruits the Setd1A Histone H3-Lys4 Methyltransferase Complex to Transcription Start Sites of Transcribed Human Genes

2007 ◽  
Vol 28 (2) ◽  
pp. 609-618 ◽  
Author(s):  
Jeong-Heon Lee ◽  
David G. Skalnik
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Start Site ◽  
Histone H3 ◽  
Rna Recognition Motif ◽  
Start Site ◽  
Transcription Start ◽  
Transcription Start Sites ◽  
Terminal Domain ◽  
Rnap Ii

ABSTRACT Histone H3-Lys4 trimethylation is associated with the transcription start site of transcribed genes, but the molecular mechanisms that control this distribution in mammals are unclear. The human Setd1A histone H3-Lys4 methyltransferase complex was found to physically associate with the RNA polymerase II large subunit. The Wdr82 component of the Setd1A complex interacts with the RNA recognition motif of Setd1A and additionally binds to the Ser5-phosphorylated C-terminal domain of RNA polymerase II, which is involved in initiation of transcription, but does not bind to an unphosphorylated or Ser2-phosphorylated C-terminal domain. Chromatin immunoprecipitation analysis revealed that Setd1A is localized near the transcription start site of expressed genes. Small interfering RNA-mediated depletion of Wdr82 leads to decreased Setd1A expression and occupancy at transcription start sites and reduced histone H3-Lys4 trimethylation at these sites. However, neither RNA polymerase II (RNAP II) occupancy nor target gene expression levels are altered following Wdr82 depletion. Hence, Wdr82 is required for the targeting of Setd1A-mediated histone H3-Lys4 trimethylation near transcription start sites via tethering to RNA polymerase II, an event that is a consequence of transcription initiation. These results suggest a model for how the mammalian RNAP II machinery is linked with histone H3-Lys4 histone methyltransferase complexes at transcriptionally active genes.

The Role of XPB/Ssl2 dsDNA Translocase Processivity in Transcription Start-site Scanning

2021 ◽  
pp. 166813
Author(s):  
Eric J. Tomko ◽  
Olivia Luyties ◽  
Jenna K. Rimel ◽  
Chi-Lin Tsai ◽  
Jill O. Fuss ◽  
...  
Keyword(s):  
Transcription Start Site ◽  
Start Site ◽  
Transcription Start

scholarly journals Interactions between RNA polymerase and the core recognition element are a determinant of transcription start site selection

2016 ◽  
Vol 113 (21) ◽  
pp. E2899-E2905 ◽  
Author(s):  
Irina O. Vvedenskaya ◽  
Hanif Vahedian-Movahed ◽  
Yuanchao Zhang ◽  
Deanne M. Taylor ◽  
Richard H. Ebright ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Transcription Start Site ◽  
Transcription Initiation ◽  
Specific Protein ◽  
Start Site ◽  
Transcription Start ◽  
Transcription Bubble ◽  
Recognition Element

During transcription initiation, RNA polymerase (RNAP) holoenzyme unwinds ∼13 bp of promoter DNA, forming an RNAP-promoter open complex (RPo) containing a single-stranded transcription bubble, and selects a template-strand nucleotide to serve as the transcription start site (TSS). In RPo, RNAP core enzyme makes sequence-specific protein–DNA interactions with the downstream part of the nontemplate strand of the transcription bubble (“core recognition element,” CRE). Here, we investigated whether sequence-specific RNAP–CRE interactions affect TSS selection. To do this, we used two next-generation sequencing-based approaches to compare the TSS profile of WT RNAP to that of an RNAP derivative defective in sequence-specific RNAP–CRE interactions. First, using massively systematic transcript end readout, MASTER, we assessed effects of RNAP–CRE interactions on TSS selection in vitro and in vivo for a library of 47 (∼16,000) consensus promoters containing different TSS region sequences, and we observed that the TSS profile of the RNAP derivative defective in RNAP–CRE interactions differed from that of WT RNAP, in a manner that correlated with the presence of consensus CRE sequences in the TSS region. Second, using 5′ merodiploid native-elongating-transcript sequencing, 5′ mNET-seq, we assessed effects of RNAP–CRE interactions at natural promoters in Escherichia coli, and we identified 39 promoters at which RNAP–CRE interactions determine TSS selection. Our findings establish RNAP–CRE interactions are a functional determinant of TSS selection. We propose that RNAP–CRE interactions modulate the position of the downstream end of the transcription bubble in RPo, and thereby modulate TSS selection, which involves transcription bubble expansion or transcription bubble contraction (scrunching or antiscrunching).

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