scholarly journals Cloning and characterization of the DIR1 promoter from Eucommia ulmoides Oliv and its response to hormonal and abiotic stress.

2021 ◽  
Author(s):  
Li Ziyun ◽  
Li Biao ◽  
Zhao yichen ◽  
Zhao Degang
Keyword(s):  
Transgenic Tobacco ◽  
Transcription Initiation ◽  
Core Promoter ◽  
Promoter Sequence ◽  
Inhibitory Effect ◽  
Initiation Site ◽  
Histochemical Staining ◽  
Eucommia Ulmoides ◽  
Tobacco Seedlings ◽  
High Concentrations

Abstract The lignans of Eucommia ulmoides have been extensively studied and shown to have a dual mechanism of regulating blood pressure. Studies have shown that DIR1 is a key gene in the biosynthetic pathway of lignans in Eucommia ulmoides Oliv. In this study, a 2000 bp upstream promoter sequence was cloned, and part of the sequence (1495 bp) and its 5'-end truncated segment were constructed into the pCAMBIA1391Z expression plasmid upstream of ß-glucuronidase (GUS). Agrobacterium-mediated genetic transformation produced stable transgenic tobacco lines. The results showed that although both full-length and truncated promoters could initiate GUS expression, their levels were influenced by the degree of deletion at the 5' end. GUS histochemical staining showed that the core promoter region was located in the region containing the transcription initiation site (TIS) within 212 bp. In addition, the DIR1 promoter responded to environmental and hormonal stressors, such as jasmonic acid (MeJA), abscisic acid (ABA), D-mannitol (drought mimic), and high concentrations of NaCl. In transgenic tobacco seedlings, MeJA, D-mannitol, and ABA could activate the DIR1 promoter, whereas high concentrations of NaCl could inhibit it. In E. ulmoides Oliv seedlings, MeJA, NaCl, and D-mannitol activated the DIR1 promoter, whereas ABA had an inhibitory effect. In summary, our findings provide a theoretical basis for the use of the DIR1 promoter in plant genetic engineering, indicating its potential. Our study also presents novel insights for lignan biosynthesis and sheds light on the mechanisms of E. ulmoides Oliv in response to stress.

pGR71 plasmid promotor sequence temporally regulated in Bacillus subtilis

1998 ◽  
Vol 44 (12) ◽  
pp. 1186-1192
Author(s):  
Guy Daxhelet ◽  
Philippe Gilot ◽  
Etienne Nyssen ◽  
Philippe Hoet
Keyword(s):  
Bacillus Subtilis ◽  
Binding Site ◽  
Transcription Initiation ◽  
Promoter Sequence ◽  
Initiation Site ◽  
Chloramphenicol Acetyltransferase ◽  
Ribosome Binding Site ◽  
Chloramphenicol Resistance ◽  
E Coli ◽  
Ribosome Binding

pGR71, a composite of plasmids pUB110 and pBR322, replicates in Escherichia coli and in Bacillus subtilis. It carries the chloramphenicol resistance gene (cat) from Tn9, which is not transcribed in either host by lack of a promoter. The cat gene is preceded by a Shine-Dalgarno sequence functional in E. coli but not in B. subtilis. Deleted pGR71 plasmids were obtained in B. subtilis when cloning foreign viral DNA upstream of this cat sequence, as well as by BAL31 exonuclease deletions extending upstream from the cat into the pUB110 moiety. These mutant plasmids expressed chloramphenicol acetyltransferase (CAT), conferring on B. subtilis resistance to high chloramphenicol concentrations. CAT expression peaked at the early postexponential phase of B. subtilis growth. The transcription initiation site of cat, determined by primer extension, was located downstream of a putative promoter sequence within the pUB110 moiety. N-terminal amino acid sequencing showed that native CAT was produced by these mutant plasmids. The cat ribosome-binding site, functional in E. coli, was repositioned within the pUB110 moiety and had consequently an extended homology with B. subtilis 16S rRNA, explaining the production of native enzyme.Key words: chloramphenicol acetyltransferase, Bacillus subtilis, postexponential gene expression, plasmid pUB110, ribosome-binding site, transcriptional promoter.

scholarly journals Np4A alarmones function in bacteria as precursors to RNA caps

2020 ◽  
Vol 117 (7) ◽  
pp. 3560-3567 ◽  
Author(s):  
Daniel J. Luciano ◽  
Joel G. Belasco
Keyword(s):  
Rna Polymerase ◽  
Transcription Initiation ◽  
Promoter Sequence ◽  
Rna Degradation ◽  
Initiation Site ◽  
Bacterial Cells ◽  
E Coli ◽  
N Incorporation ◽  
Nascent Transcripts

Stresses that increase the cellular concentration of dinucleoside tetraphosphates (Np4Ns) have recently been shown to impact RNA degradation by inducing nucleoside tetraphosphate (Np4) capping of bacterial transcripts. However, neither the mechanism by which such caps are acquired nor the function of Np4Ns in bacteria is known. Here we report that promoter sequence changes upstream of the site of transcription initiation similarly affect both the efficiency with which Escherichia coli RNA polymerase incorporates dinucleoside polyphosphates at the 5′ end of nascent transcripts in vitro and the percentage of transcripts that are Np4-capped in E. coli, clear evidence for Np4 cap acquisition by Np4N incorporation during transcription initiation in bacterial cells. E. coli RNA polymerase initiates transcription more efficiently with Np4As than with ATP, particularly when the coding strand nucleotide that immediately precedes the initiation site is a purine. Together, these findings indicate that Np4Ns function in bacteria as precursors to Np4 caps and that RNA polymerase has evolved a predilection for synthesizing capped RNA whenever such precursors are abundant.

scholarly journals The gene structure and promoter sequence of mouse hyaluronan synthase 1 (mHAS1)

1998 ◽  
Vol 330 (3) ◽  
pp. 1223-1227 ◽  
Author(s):  
Yoichi YAMADA ◽  
Naoki ITANO ◽  
Masahiro ZAKO ◽  
Mamoru YOSHIDA ◽  
Petros LENAS ◽  
...  
Keyword(s):  
Gene Structure ◽  
Transcription Initiation ◽  
Direct Sequencing ◽  
Promoter Sequence ◽  
Initiation Site ◽  
Transcription Initiation Site ◽  
Hyaluronan Synthase ◽  
Pcr Method ◽  
Rapid Amplification ◽  
Ccaat Box

The structure and organization of mouse hyaluronan synthase 1 gene, HAS1 were determined by direct sequencing of λ phage clones carrying the entire gene and by application of the long and accurate (LA)-PCR method to amplify regions encompassing the exon-intron boundaries and all of the exons. This gene spans about 11 kb of genomic DNA and consists of 5 exons and 4 introns. A similarity in the exon-intron organization was found between the genes of mouse HAS1 and Xenopus laevis DG42 which was recently identified as Xenopus hyaluronan synthase. The transcription initiation site was determined by rapid amplification of the cDNA ends (5ʹ-RACE). Position +1 is located 55 nucleotides upstream of the ATG initiation codon. The promoter region of the HAS1 gene has no typical TATA box, but contains a CCAAT box located 190 nucleotides upstream of the transcription initiation site. Further analysis of 1.4 kb of the 5ʹ flanking region revealed several potential binding motifs for transcription factors. This information about the gene structure may be useful for further studies on the promoter activity.

scholarly journals Genomic organization and transcriptional analysis of the human l-glutaminase gene

2003 ◽  
Vol 370 (3) ◽  
pp. 771-784 ◽  
Author(s):  
Cristina PÉREZ-GÓMEZ ◽  
José M. MATÉS ◽  
Pedro M. GÓMEZ-FABRE ◽  
Antonio del CASTILLO-OLIVARES ◽  
Francisco J. ALONSO ◽  
...  
Keyword(s):  
Transcription Initiation ◽  
Genomic Organization ◽  
Core Promoter ◽  
Regulatory Elements ◽  
Initiation Site ◽  
Genome Project ◽  
Transcriptional Analysis ◽  
Cdna Clones ◽  
Mobility Shift ◽  
Specific Expression

In mammals, glutaminase (GA) is expressed in most tissues, but the regulation of organ-specific expression is largely unknown. Therefore, as an essential step towards studying the regulation of GA expression, the human liver-type GA (hLGA) gene has been characterized. LGA genomic sequences were isolated using the genome walking technique. Analysis and comparison of these sequences with two LGA cDNA clones and the Human Genome Project database, allowed the determination of the genomic organization of the LGA gene. The gene has 18 exons and is approx. 18kb long. All exon/intron junction sequences conform to the GT/AG rule. Progressive deletion analysis of LGA promoter—luciferase constructs indicated that the core promoter is located between nt −141 and +410, with several potential regulatory elements: CAAT, GC, TATA-like, Ras-responsive element binding protein and specificity protein 1 (Sp1) sites. The minimal promoter was mapped within +107 and +410, where only an Sp1 binding site is present. Mutation experiments suggested that two CAAT recognition elements near the transcription-initiation site (-138 and −87), play a crucial role for optimal promoter activity. Electrophoretic mobility-shift assays confirmed the importance of CAAT- and TATA-like boxes to enhance basal transcription, and demonstrated that HNF-1 motif is a significant distal element for transcriptional regulation of the hLGA gene.

scholarly journals Core Promoter-Dependent TFIIB Conformation and a Role for TFIIB Conformation in Transcription Start Site Selection

2002 ◽  
Vol 22 (19) ◽  
pp. 6697-6705 ◽  
Author(s):  
Jennifer A. Fairley ◽  
Rachel Evans ◽  
Nicola A. Hawkes ◽  
Stefan G. E. Roberts
Keyword(s):  
Transcription Start Site ◽  
Site Selection ◽  
Transcription Initiation ◽  
Core Promoter ◽  
Initiation Site ◽  
Start Site ◽  
Wild Type ◽  
Transcription Start ◽  
General Transcription Factor ◽  
Selection Of

ABSTRACT The general transcription factor TFIIB plays a central role in the selection of the transcription initiation site. The mechanisms involved are not clear, however. In this study, we analyze core promoter features that are responsible for the susceptibility to mutations in TFIIB and cause a shift in the transcription start site. We show that TFIIB can modulate both the 5′ and 3′ parameters of transcription start site selection in a manner dependent upon the sequence of the initiator. Mutations in TFIIB that cause aberrant transcription start site selection concentrate in a region that plays a pivotal role in modulating TFIIB conformation. Using epitope-specific antibody probes, we show that a TFIIB mutant that causes aberrant transcription start site selection assembles at the promoter in a conformation different from that for wild-type TFIIB. In addition, we uncover a core promoter-dependent effect on TFIIB conformation and provide evidence for novel sequence-specific TFIIB promoter contacts.

scholarly journals The Membrane-Bound H+-ATPase Complex Is Essential for Growth of Lactococcus lactis

2000 ◽  
Vol 182 (17) ◽  
pp. 4738-4743 ◽  
Author(s):  
Brian J. Koebmann ◽  
Dan Nilsson ◽  
Oscar P. Kuipers ◽  
Peter R. Jensen
Keyword(s):  
Amino Acid ◽  
Lactococcus Lactis ◽  
Transcription Initiation ◽  
Northern Blot Analysis ◽  
Promoter Sequence ◽  
Initiation Site ◽  
Amino Acid Sequences ◽  
Significant Homology ◽  
Atpase Subunits ◽  
Membrane Bound

ABSTRACT The eight genes which encode the (F1Fo) H+-ATPase in Lactococcus lactis subsp.cremoris MG1363 were cloned and sequenced. The genes were organized in an operon with the gene order atpEBFHAGDC; i.e., the order of atpE and atpB is reversed with respect to the more typical bacterial organization. The deduced amino acid sequences of the corresponding H+-ATPase subunits showed significant homology with the subunits from other organisms. Results of Northern blot analysis showed a transcript at approximately 7 kb, which corresponds to the size of theatp operon. The transcription initiation site was mapped by primer extension and coincided with a standard promoter sequence. In order to analyze the importance of the H+-ATPase forL. lactis physiology, a mutant strain was constructed in which the original atp promoter on the chromosome was replaced with an inducible nisin promoter. When grown on GM17 plates the resulting strain was completely dependent on the presence of nisin for growth. These data demonstrate that the H+-ATPase is essential for growth of L. lactis under these conditions.

Core promoter-selective RNA polymerase II transcription

2006 ◽  
Vol 73 ◽  
pp. 225-236 ◽  
Author(s):  
Petra Gross ◽  
Thomas Oelgeschläger
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Core Promoter ◽  
Promoter Sequence ◽  
Tata Box ◽  
Promoter Elements ◽  
The Core ◽  
Core Promoter Sequence ◽  
Rnap Ii

The initiation of mRNA synthesis in eukaryotic cells is a complex and highly regulated process that requires the assembly of general transcription factors and RNAP II (RNA polymerase II; also abbreviated as Pol II) into a pre-initiation complex at the core promoter. The core promoter is defined as the minimal DNA region that is sufficient to direct low levels of activator-independent (basal) transcription by RNAP II in vitro. The core promoter typically extends approx. 40 bp up- and down-stream of the start site of transcription and can contain several distinct core promoter sequence elements. Core promoters in higher eukaryotes are highly diverse in structure, and each core promoter sequence element is only found in a subset of genes. So far, only TATA box and INR (initiator) element have been shown to be capable of directing accurate RNAP II transcription initiation independent of other core promoter elements. Computational analysis of metazoan genomes suggests that the prevalence of the TATA box has been overestimated in the past and that the majority of human genes are TATA-less. While TATA-mediated transcription initiation has been studied in great detail and is very well understood, very little is known about the factors and mechanisms involved in the function of the INR and other core promoter elements. Here we summarize our current understanding of the factors and mechanisms involved in core promoter-selective transcription and discuss possible pathways through which diversity in core promoter architecture might contribute to combinatorial gene regulation in metazoan cells.

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