scholarly journals Independent regulation of H-NS-mediated silencing of the bgl operon at two levels: upstream by BglJ and LeuO and downstream by DnaKJ

Microbiology ◽  
2005 ◽  
Vol 151 (10) ◽  
pp. 3349-3359 ◽  
Author(s):  
S. Madhusudan ◽  
Andreas Paukner ◽  
Yvonne Klingen ◽  
Karin Schnetz
Keyword(s):  
Escherichia Coli ◽  
Transcription Initiation ◽  
Promoter Activity ◽  
Transposon Mutagenesis ◽  
Open Reading Frames ◽  
Wild Type ◽  
Coding Region ◽  
First Time ◽  
Insertion Mutations ◽  
Reading Frames

Silencing of the Escherichia coli bgl operon by the histone-like nucleoid-structuring protein H-NS occurs at two levels. Binding of H-NS upstream of the promoter represses transcription initiation, whilst binding within the coding region is also proposed to repress transcription elongation. The latter, downstream level of repression is counteracted by the protease Lon and, thus, silencing of the bgl operon is more effective in lon mutants. Transposon-mutagenesis screens for suppression of this lon phenotype on bgl were performed and insertion mutations disrupting rpoS and crl were obtained, as well as mutations mapping upstream of the open reading frames of bglJ, leuO and dnaK. In rpoS and crl mutants, bgl promoter activity is known to be higher. Likewise, as shown here, bgl promoter activity is increased in the bglJ and leuO mutants, which express BglJ and LeuO constitutively. However, BglJ and LeuO have no impact on downstream repression. A dnaKJ mutant was isolated for the first time in the context of the bgl operon. The mutant expresses lower levels of DnaK than the wild-type. Interestingly, in this dnaKJ : : miniTn10 mutant, downstream repression of bgl by H-NS is less effective, whilst upstream repression by H-NS remains unaffected. Together, the data show that the two levels of bgl silencing by H-NS are regulated independently.

scholarly journals Chromatin Remodeling around Nucleosome-Free Regions Leads to Repression of Noncoding RNA Transcription

2010 ◽  
Vol 30 (21) ◽  
pp. 5110-5122 ◽  
Author(s):  
Adam N. Yadon ◽  
Daniel Van de Mark ◽  
Ryan Basom ◽  
Jeffrey Delrow ◽  
Iestyn Whitehouse ◽  
...  
Keyword(s):  
Chromatin Remodeling ◽  
Transcription Initiation ◽  
Noncoding Rna ◽  
Open Reading Frames ◽  
Yeast Genome ◽  
Transcription Start Sites ◽  
Transcriptional Regulatory ◽  
First Time ◽  
Reading Frames

ABSTRACT Nucleosome-free regions (NFRs) at the 5′ and 3′ ends of genes are general sites of transcription initiation for mRNA and noncoding RNA (ncRNA). The presence of NFRs within transcriptional regulatory regions and the conserved location of transcription start sites at NFRs strongly suggest that the regulation of NFRs profoundly affects transcription initiation. To date, multiple factors are known to facilitate transcription initiation by positively regulating the formation and/or size of NFRs in vivo. However, mechanisms to repress transcription by negatively regulating the size of NFRs have not been identified. We identified four distinct classes of NFRs located at the 5′ and 3′ ends of genes, within open reading frames (ORFs), and far from ORFs. The ATP-dependent chromatin-remodeling enzyme Isw2 was found enriched at all classes of NFRs. Analysis of RNA levels also demonstrated Isw2 is required to repress ncRNA transcription from many of these NFRs. Thus, by the systematic annotation of NFRs across the yeast genome and analysis of ncRNA transcription, we established, for the first time, a mechanism by which NFR size is negatively regulated to repress ncRNA transcription from NFRs. Finally, we provide evidence suggesting that one biological consequence of repression of ncRNA, by Isw2 or by the exosome, is prevention of transcriptional interference of mRNA.

scholarly journals Two genes for ribosomal protein 51 of Saccharomyces cerevisiae complement and contribute to the ribosomes.

1984 ◽  
Vol 4 (9) ◽  
pp. 1871-1879 ◽  
Author(s):  
N Abovich ◽  
M Rosbash
Keyword(s):  
Ribosomal Protein ◽  
Dna Sequence ◽  
Open Reading Frames ◽  
Growth Defect ◽  
Wild Type ◽  
Coding Region ◽  
Insufficient Amount ◽  
The One ◽  
Carboxy Terminal ◽  
Reading Frames

We cloned and sequenced the second gene coding for yeast ribosomal protein 51 (RP51B). When the DNA sequence of this gene was compared with the DNA sequence of RP51A (J.L. Teem and M. Rosbash, Proc. Natl. Acad. Sci. U.S.A. 80:4403--4407, 1983), the following conclusions emerged: both genes code for a protein of 135 amino acids; both open reading frames are interrupted by a single intron which occurs directly after the initiating methionine; the open reading frames are 96% homologous and code for the same protein with the exception of the carboxy-terminal amino acid; DNA sequence homology outside of the coding region is extremely limited. The cloned genes, in combination with the one-step gene disruption techniques of Rothstein (R. J. Rothstein, Methods Enzymol. 101:202-211, 1983), were used to generate haploid strains containing mutations in the RP51A or RP51B genes or in both. Strains missing a normal RP51A gene grew poorly (180-min generation time versus 130 min for the wild type), whereas strains carrying a mutant RP51B were relatively normal. Strains carrying mutations in the two genes grew extremely poorly (6 to 9 h), which led us to conclude that RP51A and RP51B were both expressed. The results of Northern blot and primer extension experiments indicate that strains with a wild-type copy of the RP51B gene and a mutant (or deleted) RP51A gene grow slowly because of an insufficient amount of RP51 mRNA. The growth defect was completely rescued with additional copies of RP51B. The data suggest that RP51A contributes more RP51 mRNA (and more RP51 protein) than does RP51B and that intergenic dosage compensation, sufficient to rescue the growth defect of strains missing a wild-type RP51A gene, does not take place.

scholarly journals Regulation of dev, an Operon That Includes Genes Essential for Myxococcus xanthus Development and CRISPR-Associated Genes and Repeats

2007 ◽  
Vol 189 (10) ◽  
pp. 3738-3750 ◽  
Author(s):  
Poorna Viswanathan ◽  
Kimberly Murphy ◽  
Bryan Julien ◽  
Anthony G. Garza ◽  
Lee Kroos
Keyword(s):  
Escherichia Coli ◽  
Promoter Activity ◽  
Myxococcus Xanthus ◽  
Regulatory Elements ◽  
Fruiting Bodies ◽  
Wild Type ◽  
Coding Region ◽  
Upstream Gene ◽  
Negative Element ◽  
Growth Induction

ABSTRACT Expression of dev genes is important for triggering spore differentiation inside Myxococcus xanthus fruiting bodies. DNA sequence analysis suggested that dev and cas (CRISPR-associated) genes are cotranscribed at the dev locus, which is adjacent to CRISPR (clustered regularly interspaced short palindromic repeats). Analysis of RNA from developing M. xanthus confirmed that dev and cas genes are cotranscribed with a short upstream gene and at least two repeats of the downstream CRISPR, forming the dev operon. The operon is subject to strong, negative autoregulation during development by DevS. The dev promoter was identified. Its −35 and −10 regions resemble those recognized by M. xanthus σA RNA polymerase, the homolog of Escherichia coli σ70, but the spacer may be too long (20 bp); there is very little expression during growth. Induction during development relies on at least two positive regulatory elements located in the coding region of the next gene upstream. At least two positive regulatory elements and one negative element lie downstream of the dev promoter, such that the region controlling dev expression spans more than 1 kb. The results of testing different fragments for dev promoter activity in wild-type and devS mutant backgrounds strongly suggest that upstream and downstream regulatory elements interact functionally. Strikingly, the 37-bp sequence between the two CRISPR repeats that, minimally, are cotranscribed with dev and cas genes exactly matches a sequence in the bacteriophage Mx8 intP gene, which encodes a form of the integrase needed for lysogenization of M. xanthus.

Two genes for ribosomal protein 51 of Saccharomyces cerevisiae complement and contribute to the ribosomes

1984 ◽  
Vol 4 (9) ◽  
pp. 1871-1879
Author(s):  
N Abovich ◽  
M Rosbash
Keyword(s):  
Ribosomal Protein ◽  
Dna Sequence ◽  
Open Reading Frames ◽  
Growth Defect ◽  
Wild Type ◽  
Coding Region ◽  
Insufficient Amount ◽  
The One ◽  
Carboxy Terminal ◽  
Reading Frames

We cloned and sequenced the second gene coding for yeast ribosomal protein 51 (RP51B). When the DNA sequence of this gene was compared with the DNA sequence of RP51A (J.L. Teem and M. Rosbash, Proc. Natl. Acad. Sci. U.S.A. 80:4403--4407, 1983), the following conclusions emerged: both genes code for a protein of 135 amino acids; both open reading frames are interrupted by a single intron which occurs directly after the initiating methionine; the open reading frames are 96% homologous and code for the same protein with the exception of the carboxy-terminal amino acid; DNA sequence homology outside of the coding region is extremely limited. The cloned genes, in combination with the one-step gene disruption techniques of Rothstein (R. J. Rothstein, Methods Enzymol. 101:202-211, 1983), were used to generate haploid strains containing mutations in the RP51A or RP51B genes or in both. Strains missing a normal RP51A gene grew poorly (180-min generation time versus 130 min for the wild type), whereas strains carrying a mutant RP51B were relatively normal. Strains carrying mutations in the two genes grew extremely poorly (6 to 9 h), which led us to conclude that RP51A and RP51B were both expressed. The results of Northern blot and primer extension experiments indicate that strains with a wild-type copy of the RP51B gene and a mutant (or deleted) RP51A gene grow slowly because of an insufficient amount of RP51 mRNA. The growth defect was completely rescued with additional copies of RP51B. The data suggest that RP51A contributes more RP51 mRNA (and more RP51 protein) than does RP51B and that intergenic dosage compensation, sufficient to rescue the growth defect of strains missing a wild-type RP51A gene, does not take place.

scholarly journals Violaxanthin conversion by recombinant diatom and plant de-epoxidases, expressed in Escherichia coli – comparative analysis

2019 ◽  
Author(s):  
Monika Olchawa-Pajor ◽  
Monika Bojko ◽  
Wojciech Strzałka ◽  
Kazimierz Strzałka ◽  
Dariusz Latowski
Keyword(s):  
Escherichia Coli ◽  
Open Reading Frames ◽  
Amino Acid Residues ◽  
Metal Affinity ◽  
Comparable Activity ◽  
First Time ◽  
Kinetics Of ◽  
Reading Frames

The purpose of this research was to obtain recombinant violaxanthin de-epoxidases (VDEs) from two species. The first one was VDE of Arabidopsis thaliana (L.) Heynh. (WT Columbia strain) (AtVDE) which in vivo catalyzes conversion of violaxanthin (Vx) to zeaxanthin (Zx) via anteraxanthin (Ax). The second one was VDE of Phaeodactylum tricornutum Bohlin, 1897 (CCAP 1055/1 strain) (PtVDE) which is responsible for de-epoxidation of diadinoxanthin (Ddx) to diatoxanthin (Dtx). As the first step of our experiments, open reading frames coding for studied enzymes were amplified and subsequently cloned into pET-15b plasmid. For recombinant proteins production Escherichia coli Origami b strain was used. The molecular weight of the produced enzymes were estimated approximately at 45kDa and 50kDa for AtVDE and PtVDE, respectively. Both enzymes, purified under native conditions by immobilized metal affinity chromatography, displayed comparable activity in assay mixture and converted up to 90% Vx in 10 min in two steps enzymatic de-epoxidation, irrespective of enzyme origin. No statistically significant differences were observed when kinetics of the reactions catalyzed by these enzymes were compared. Putative role of selected amino-acid residues of AtVDE and PtVDE was also considered. The significance of the first time obtained recombinant PtVDE as a useful tool in various comparative investigations of de-epoxidation reactions in main types of xanthophyll cycles existing in nature are also indicated.

scholarly journals Analysis of the Gene Cluster Encoding Toluene/o-Xylene Monooxygenase from Pseudomonas stutzeri OX1

1998 ◽  
Vol 64 (10) ◽  
pp. 3626-3632 ◽  
Author(s):  
Giovanni Bertoni ◽  
Manuela Martino ◽  
Enrica Galli ◽  
Paola Barbieri
Keyword(s):  
Escherichia Coli ◽  
Gene Cluster ◽  
Dimethyl Sulfide ◽  
Pseudomonas Stutzeri ◽  
Open Reading Frames ◽  
Enzymatic System ◽  
Wild Type Allele ◽  
Wild Type ◽  
Bacterial Strains ◽  
Reading Frames

ABSTRACT The toluene/o-xylene monooxygenase cloned fromPseudomonas stutzeri OX1 displays a very broad range of substrates and a very peculiar regioselectivity, because it is able to hydroxylate more than one position on the aromatic ring of several hydrocarbons and phenols. The nucleotide sequence of the gene cluster coding for this enzymatic system has been determined. The sequence analysis revealed the presence of six open reading frames (ORFs) homologous to other genes clustered in operons coding for multicomponent monooxygenases found in benzene- and toluene-degradative pathways cloned from Pseudomonas strains. Significant similarities were also found with multicomponent monooxygenase systems for phenol, methane, alkene, and dimethyl sulfide cloned from different bacterial strains. The knockout of each ORF and complementation with the wild-type allele indicated that all six ORFs are essential for the full activity of the toluene/o-xylene monooxygenase inEscherichia coli. This analysis also shows that despite its activity on both hydrocarbons and phenols, toluene/ o-xylene monooxygenase belongs to a toluene multicomponent monooxygenase subfamily rather than to the monooxygenases active on phenols.

scholarly journals Woot, an Active Gypsy-Class Retrotransposon in the Flour Beetle, Tribolium castaneum, is Associated With a Recent Mutation

Genetics ◽  
1996 ◽  
Vol 143 (1) ◽  
pp. 417-426
Author(s):  
Richard W Beeman ◽  
M Scott Thomson ◽  
John M Clark ◽  
Marco A DeCamillis ◽  
Susan J Brown ◽  
...  
Keyword(s):  
Sequence Analysis ◽  
Tribolium Castaneum ◽  
Frameshift Mutation ◽  
Open Reading Frames ◽  
Red Flour Beetle ◽  
Intron Sequence ◽  
Coding Region ◽  
Long Terminal Repeats ◽  
Flour Beetle ◽  
Reading Frames

Abstract A recently isolated, lethal mutation of the homeotic Abdominal gene of the red flour beetle Tribolium castaneum is associated with an insertion of a novel retrotransposon into an intron. Sequence analysis indicates that this retrotransposon, named Woot, is a member of the gypsy family of mobile elements. Most strains of T. castaneum appear to harbor ~25-35 copies of Woot per genome. Woot is composed of long terminal repeats of unprecedented length (3.6 kb each), flanking an internal coding region 5.0 kb in length. For most copies of Woot, the internal region includes two open reading frames (ORFs) that correspond to the gag and pol genes of previously described retrotransposons and retroviruses. The copy of Woot inserted into Abdominal bears an apparent single frameshift mutation that separates the normal second ORF into two. Woot does not appear to generate infectious virions by the criterion that no envelop gene is discernible. The association of Woot with a recent mutation suggests that this retroelement is currently transpositionally active in at least some strains.

scholarly journals Cloning and sequencing of a putative Escherichia coli [NiFe] hydrogenase-1 operon containing six open reading frames.

1990 ◽  
Vol 172 (4) ◽  
pp. 1969-1977 ◽  
Author(s):  
N K Menon ◽  
J Robbins ◽  
H D Peck ◽  
C Y Chatelus ◽  
E S Choi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Open Reading Frames ◽  
Cloning And Sequencing ◽  
Reading Frames

scholarly journals A Novel DNA Polymerase Family Found inArchaea

1998 ◽  
Vol 180 (8) ◽  
pp. 2232-2236 ◽  
Author(s):  
Yoshizumi Ishino ◽  
Kayoko Komori ◽  
Isaac K. O. Cann ◽  
Yosuke Koga
Keyword(s):  
Escherichia Coli ◽  
Dna Polymerase ◽  
Pyrococcus Furiosus ◽  
Dna Polymerases ◽  
Open Reading Frames ◽  
Gene Products ◽  
Polymerase Gene ◽  
Hyperthermophilic Archaeon ◽  
Dna Polymerase Ii ◽  
Reading Frames

ABSTRACT One of the most puzzling results from the complete genome sequence of the methanogenic archaeon Methanococcus jannaschii was that the organism may have only one DNA polymerase gene. This is because no other DNA polymerase-like open reading frames (ORFs) were found besides one ORF having the typical α-like DNA polymerase (family B). Recently, we identified the genes of DNA polymerase II (the second DNA polymerase) from the hyperthermophilic archaeonPyrococcus furiosus, which has also at least one α-like DNA polymerase (T. Uemori, Y. Sato, I. Kato, H. Doi, and Y. Ishino, Genes Cells 2:499–512, 1997). The genes in M. jannaschiiencoding the proteins that are homologous to the DNA polymerase II ofP. furiosus have been located and cloned. The gene products of M. jannaschii expressed in Escherichia colihad both DNA polymerizing and 3′→5′ exonuclease activities. We propose here a novel DNA polymerase family which is entirely different from other hitherto-described DNA polymerases.

scholarly journals Genome Sequence of a T4-like Phage, Escherichia Phage vB_EcoM-Sa45lw, Infecting Shiga Toxin-Producing Escherichia coli Strains

2019 ◽  
Vol 8 (32) ◽  
Author(s):  
Yen-Te Liao ◽  
Yujie Zhang ◽  
Alexandra Salvador ◽  
Vivian C. H. Wu
Keyword(s):  
Escherichia Coli ◽  
Surface Water ◽  
Genome Size ◽  
Shiga Toxin ◽  
Open Reading Frames ◽  
Content Type ◽  
E Coli ◽  
Double Stranded Dna ◽  
A Genome ◽  
Reading Frames

Escherichia phage vB_EcoM-Sa45lw, a new member of the T4-like phages, was isolated from surface water in a produce-growing area. The phage, containing double-stranded DNA with a genome size of 167,353 bp and 282 predicted open reading frames (ORFs), is able to infect generic Escherichia coli and Shiga toxin-producing E. coli O45 and O157 strains.

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