scholarly journals Transcriptional Analysis of the Bacillus anthracis Capsule Regulators

2005 ◽  
Vol 187 (15) ◽  
pp. 5108-5114 ◽  
Author(s):  
Melissa Drysdale ◽  
Agathe Bourgogne ◽  
Theresa M. Koehler
Keyword(s):  
Bacillus Anthracis ◽  
Double Mutant ◽  
Current Model ◽  
Positive Control ◽  
Transcriptional Analysis ◽  
Start Site ◽  
Transcription Start ◽  
Positive Regulation ◽  
Transcription Start Sites ◽  
Gene Transcripts

ABSTRACT The poly-d-glutamic acid capsule of Bacillus anthracis is essential for virulence. Control of capsule synthesis occurs at the level of transcription and involves positive regulation of the capsule biosynthetic operon capBCAD by a CO2/bicarbonate signal and three plasmid-borne regulators: atxA, acpA, and acpB. Although the molecular mechanism for control of cap transcription is unknown, atxA affects cap expression via positive control of acpA and acpB, two genes with partial functional similarity. Transcriptional analyses of a genetically complete strain indicate that capB expression is several hundred-fold higher during growth in 5% CO2 compared to growth in air. atxA was expressed appreciably during growth in air and induced only 2.5-fold by CO2. In contrast, expression of acpA and acpB was induced up to 23-fold and 59-fold, respectively, by CO2. The 5′-end mapping of gene transcripts revealed atxA-regulated and atxA-independent apparent transcription start sites for capB, acpA, and acpB. Transcripts mapping to all atxA-regulated start sites were increased during growth in elevated CO2. The acpA gene has one atxA-regulated and one atxA-independent start site. acpB lies downstream of capBCAD. A single atxA-independent start site maps immediately upstream of acpB. atxA-mediated control of acpB appears to occur via transcriptional read-through from atxA-dependent start sites 5′ of capB. One atxA-independent and two atxA-regulated start sites map upstream of capB. Transcription from the atxA-regulated start sites of capBCAD was reduced significantly in an acpA acpB double mutant but unaffected in mutants with deletion of only acpA or acpB, in agreement with the current model for epistatic relationships between the regulators.

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 Identification of a new promoter in the early region of the human papillomavirus type 16 genome

1999 ◽  
Vol 80 (12) ◽  
pp. 3241-3250 ◽  
Author(s):  
T. H. Braunstein ◽  
B. S. Madsen ◽  
B. Gavnholt ◽  
M. W. Rosenstierne ◽  
C. Koefoed Johnsen ◽  
...  
Keyword(s):  
Human Papillomavirus ◽  
Control Synthesis ◽  
Start Site ◽  
Late Gene Expression ◽  
Transcription Start ◽  
Transcription Start Sites ◽  
Polycistronic Mrna ◽  
Human Papillomavirus Type 16 ◽  
E6 And E7 ◽  
E7 Proteins

Transcription of the human papillomavirus type 16 (HPV-16) genome is controlled by several promoters; the P97 promoter is considered to be the main one. An additional promoter has been identified within the E7 ORF as well as an antisense promoter just upstream of the L2 ORF. The significance of these promoters for early and late gene expression and their activity related to cell differentiation is not known in detail. Identification of two new, previously undescribed transcription start sites at nt 542 just upstream of the E7 ORF and at nt 611 within the E7 ORF is reported. The promoter responsible for the start site at nt 542 (P542) was active in SiHa, HeLa and C33A cells. Very low promoter activity was found upstream of the nt 611 start site. The E7 protein has previously been shown to be synthesized from a polycistronic mRNA encoding both the E6 and E7 proteins under the control of the P97 promoter. The data reported in the present paper suggest that promoter P542 may control synthesis of the E7 oncoprotein from a monocistronic mRNA.

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 A catalog of transcription start sites across 115 human tissue and cell types

2021 ◽  
Author(s):  
Jill E Moore ◽  
Xiao-Ou Zhang ◽  
Shaimae I Elhajjajy ◽  
Kaili Fan ◽  
Fairlie Reese ◽  
...  
Keyword(s):  
Gene Expression ◽  
Disease Gene ◽  
Cell Types ◽  
Start Site ◽  
Valuable Resource ◽  
Cell Type ◽  
Transcription Start ◽  
Transcription Start Sites ◽  
Cell Type Specific ◽  
Gwas Catalog

Accurate transcription start site (TSS) annotations are essential for understanding transcriptional regulation and its role in human disease. Gene collections such as GENCODE contain annotations for tens of thousands of TSSs, but not all of these annotations are experimentally validated nor do they contain information on cell type-specific usage. Therefore, we sought to generate a collection of experimentally validated TSSs by integrating RNA Annotation and Mapping of Promoters for the Analysis of Gene Expression (RAMPAGE) data from 115 cell and tissue types, which resulted in a collection of approximately 50 thousand representative RAMPAGE peaks. These peaks were primarily proximal to GENCODE-annotated TSSs and were concordant with other transcription assays. Because RAMPAGE uses paired-end reads, we were then able to connect peaks to transcripts by analyzing the genomic positions of the 3' ends of read mates. Using this paired-end information, we classified the vast majority (37 thousand) of our RAMPAGE peaks as verified TSSs, updating TSS annotations for 20% of GENCODE genes. We also found that these updated TSS annotations were supported by epigenomic and other transcriptomic datasets. To demonstrate the utility of this RAMPAGE rPeak collection, we intersected it with the NHGRI/EBI GWAS catalog and identified new candidate GWAS genes. Overall, our work demonstrates the importance of integrating experimental data to further refine TSS annotations and provides a valuable resource for the biological community.

scholarly journals Properties of an Intergenic Terminator and Start Site Switch That Regulate IMD2 Transcription in Yeast

2008 ◽  
Vol 28 (12) ◽  
pp. 3883-3893 ◽  
Author(s):  
M. Harley Jenks ◽  
Thomas W. O'Rourke ◽  
Daniel Reines
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Initiation Site ◽  
Start Codon ◽  
Start Site ◽  
Transcription Start ◽  
Transcription Start Sites ◽  
Short Transcript ◽  
Alternative Transcription ◽  
Polyadenylation Sites

ABSTRACT The IMD2 gene in Saccharomyces cerevisiae is regulated by intracellular guanine nucleotides. Regulation is exerted through the choice of alternative transcription start sites that results in synthesis of either an unstable short transcript terminating upstream of the start codon or a full-length productive IMD2 mRNA. Start site selection is dictated by the intracellular guanine nucleotide levels. Here we have mapped the polyadenylation sites of the upstream, unstable short transcripts that form a heterogeneous family of RNAs of ≈200 nucleotides. The switch from the upstream to downstream start sites required the Rpb9 subunit of RNA polymerase II. The enzyme's ability to locate the downstream initiation site decreased exponentially as the start was moved downstream from the TATA box. This suggests that RNA polymerase II's pincer grip is important as it slides on DNA in search of a start site. Exosome degradation of the upstream transcripts was highly dependent upon the distance between the terminator and promoter. Similarly, termination was dependent upon the Sen1 helicase when close to the promoter. These findings extend the emerging concept that distinct modes of termination by RNA polymerase II exist and that the distance of the terminator from the promoter, as well as its sequence, is important for the pathway chosen.

scholarly journals In vivo transcriptional analysis of the TATA-less promoter of the Drosophila melanogaster vermilion gene

1992 ◽  
Vol 12 (10) ◽  
pp. 4571-4577
Author(s):  
Y W Fridell ◽  
L L Searles
Keyword(s):  
Drosophila Melanogaster ◽  
Transcription Start Site ◽  
Fat Body ◽  
Germ Line ◽  
Transcriptional Analysis ◽  
Start Site ◽  
Transcription Start ◽  
Tata Element ◽  
Type V

Transcriptional regulation of the TATA-less promoter of the Drosophila melanogaster vermilion (v) gene was investigated. Developmental Northern (RNA) blot analysis showed that v transcripts accumulate during late embryo, larval, and adult stages. Sequences that control expression in adults were delineated by analyzing a series of 5' and 3' deletion constructions after germ line transformation. These studies defined two regions, -300 to -600 and -60 to -160, relative to the major transcription start site, as important for maximal levels of expression. Analysis of transformants bearing v-lacZ promoter fusions showed that larval expression is fat body specific and that expression depends on sequences located between +19 and +36 downstream of transcription start site. This downstream element can be functionally replaced by a TATA box in vivo. Furthermore, when added to the wild-type v promoter, a TATA element augments the level of v transcription by three- to fivefold.

scholarly journals Multiple Alternate Transcripts Direct the Biosynthesis of Microcystin, a Cyanobacterial

2002 ◽  
Vol 68 (2) ◽  
pp. 449-455 ◽  
Author(s):  
Melanie Kaebernick ◽  
Elke Dittmann ◽  
Thomas B�rner ◽  
Brett A. Neilan
Keyword(s):  
Gene Cluster ◽  
Polyketide Synthase ◽  
Bidirectional Promoter ◽  
Transcriptional Analysis ◽  
Transcription Start ◽  
Transcription Start Sites ◽  
Reverse Transcription Pcr ◽  
Peptide Synthetase ◽  
Rapid Amplification ◽  
Translational Start

ABSTRACT The mcyABCDEFGHIJ gene cluster of Microcystis aeruginosa encodes the mixed polyketide synthase/nonribosomal peptide synthetase (microcystin synthetase) which is responsible for biosynthesis of the potent liver toxin microcystin. The sequence and orientation of the mcy genes have previously been reported, but no transcriptional analysis had been performed prior to this study. The mcyABCDEFGHIJ genes are transcribed as two polycistronic operons, mcyABC and mcyDEFGHIJ, from a central bidirectional promoter between mcyA and mcyD. Two transcription start sites were detected for both mcyA and mcyD when cells were exposed to light intensities of 68 and 16 μmol of photons m−2 s−1. The start sites, located 206 and 254 bp upstream of the translational start for mcyD under high and low light conditions, respectively, indicate long untranslated leader regions. Putative transcription start sites were also identified for mcyE, mcyF, mcyG, mcyH, mcyI, and mcyJ but not for mcyB and mcyC. A combination of reverse transcription-PCR and rapid amplification of cDNA ends was employed throughout this work, which may have been one of the first transcriptional analyses of a large nonribosomal polyketide gene cluster.

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 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.

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.

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