scholarly journals YtxR, a Conserved LysR-Like Regulator That Induces Expression of Genes Encoding a Putative ADP-Ribosyltransferase Toxin Homologue in Yersinia enterocolitica

2006 ◽  
Vol 188 (23) ◽  
pp. 8033-8043 ◽  
Author(s):  
Grace L. Axler-DiPerte ◽  
Virginia L. Miller ◽  
Andrew J. Darwin
Keyword(s):  
Yersinia Enterocolitica ◽  
Transcription Initiation ◽  
Photorhabdus Luminescens ◽  
Expression Of Genes ◽  
Dnase I Footprinting ◽  
Genes Encoding ◽  
Adp Ribosyltransferase ◽  
Insight Into

ABSTRACT Yersinia enterocolitica causes human gastroenteritis, and many isolates have been classified as either “American” or “non-American” strains based on their geographic prevalence and virulence properties. In this study we describe identification of a transcriptional regulator that controls expression of the Y. enterocolitica ytxAB genes. The ytxAB genes have the potential to encode an ADP-ribosylating toxin with similarity to pertussis toxin. However, a ytxAB null mutation did not affect virulence in mice. Nevertheless, the ytxAB genes are conserved in many Y. enterocolitica strains. Interestingly, American and non-American strains have different ytxAB alleles encoding proteins that are only 50 to 60% identical. To obtain further insight into the ytxAB locus, we investigated whether it is regulated as part of a known or novel regulon. Transposon mutagenesis identified a LysR-like regulator, which we designated YtxR. Expression of ytxR from a nonnative promoter increased Φ(ytxA-lacZ) operon fusion expression up to 35-fold. YtxR also activated expression of its own promoter. DNase I footprinting showed that a His6-YtxR fusion protein directly interacted with the ytxA and ytxR control regions at similar distances upstream of their probable transcription initiation sites, identified by primer extension. Deletion analysis demonstrated that removal of the regions protected by His6-YtxR in vitro eliminated YtxR-dependent induction in vivo. The ytxAB locus is not present in most Yersinia species. In contrast, ytxR is conserved in multiple Yersinia species, as well as in the closely related organisms Photorhabdus luminescens and Photorhabdus asymbiotica. These observations suggest that YtxR may play a conserved role involving regulation of other genes besides ytxAB.

scholarly journals Nrg1 Is a Transcriptional Repressor for Glucose Repression of STA1 Gene Expression inSaccharomyces cerevisiae

1999 ◽  
Vol 19 (3) ◽  
pp. 2044-2050 ◽  
Author(s):  
Seok Hee Park ◽  
Sang Seok Koh ◽  
Jae Hwan Chun ◽  
Hye Jin Hwang ◽  
Hyen Sam Kang
Keyword(s):  
Gene Expression ◽  
Zinc Finger Protein ◽  
Glucose Repression ◽  
Transcriptional Repressor ◽  
Dependent Manner ◽  
Expression Of Genes ◽  
Dnase I Footprinting ◽  
Genes Encoding

ABSTRACT Expression of genes encoding starch-degrading enzymes is regulated by glucose repression in the yeast Saccharomyces cerevisiae. We have identified a transcriptional repressor, Nrg1, in a genetic screen designed to reveal negative factors involved in the expression of STA1, which encodes a glucoamylase. TheNRG1 gene encodes a 25-kDa C2H2zinc finger protein which specifically binds to two regions in the upstream activation sequence of the STA1 gene, as judged by gel retardation and DNase I footprinting analyses. Disruption of theNRG1 gene causes a fivefold increase in the level of theSTA1 transcript in the presence of glucose. The expression of NRG1 itself is inhibited in the absence of glucose. DNA-bound LexA-Nrg1 represses transcription of a target gene 10.7-fold in a glucose-dependent manner, and this repression is abolished in bothssn6 and tup1 mutants. Two-hybrid and glutathione S-transferase pull-down experiments show an interaction of Nrg1 with Ssn6 both in vivo and in vitro. These findings indicate that Nrg1 acts as a DNA-binding repressor and mediates glucose repression of the STA1 gene expression by recruiting the Ssn6-Tup1 complex.

scholarly journals In vivo transcriptome analysis provides insights into host-dependent expression of virulence factors by Yersinia entomophaga MH96, during infection of Galleria mellonella

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Amber R Paulson ◽  
Maureen O’Callaghan ◽  
Xue-Xian Zhang ◽  
Paul B Rainey ◽  
Mark R H Hurst
Keyword(s):  
Galleria Mellonella ◽  
Functional Enrichment ◽  
Natural Environments ◽  
Insecticidal Toxin ◽  
Heat Stable ◽  
Expression Of Genes ◽  
Genes Encoding ◽  
Cell Densities

Abstract The function of microbes can be inferred from knowledge of genes specifically expressed in natural environments. Here, we report the in vivo transcriptome of the entomopathogenic bacterium Yersinia entomophaga MH96, captured during initial, septicemic, and pre-cadaveric stages of intrahemocoelic infection in Galleria mellonella. A total of 1285 genes were significantly upregulated by MH96 during infection; 829 genes responded to in vivo conditions during at least one stage of infection, 289 responded during two stages of infection, and 167 transcripts responded throughout all three stages of infection compared to in vitro conditions at equivalent cell densities. Genes upregulated during the earliest infection stage included components of the insecticidal toxin complex Yen-TC (chi1, chi2, and yenC1), genes for rearrangement hotspot element containing protein yenC3, cytolethal distending toxin cdtAB, and vegetative insecticidal toxin vip2. Genes more highly expressed throughout the infection cycle included the putative heat-stable enterotoxin yenT and three adhesins (usher-chaperone fimbria, filamentous hemagglutinin, and an AidA-like secreted adhesin). Clustering and functional enrichment of gene expression data also revealed expression of genes encoding type III and VI secretion system-associated effectors. Together these data provide insight into the pathobiology of MH96 and serve as an important resource supporting efforts to identify novel insecticidal agents.

scholarly journals Reconstitution of the Human tRNA Splicing Endonuclease Complex: insight into the regulation of pre-tRNA cleavage

2019 ◽  
Author(s):  
Cassandra K. Hayne ◽  
Casey A. Schmidt ◽  
A. Gregory Matera ◽  
Robin E. Stanley
Keyword(s):  
Animal Cells ◽  
Trna Splicing ◽  
Polynucleotide Kinase ◽  
Genes Encoding ◽  
Intron Removal ◽  
And Function ◽  
Insight Into ◽  
Negative Modulator

ABSTRACTThe splicing of tRNA introns is a critical step in pre-tRNA maturation. In archaea and eukaryotes, tRNA intron removal is catalyzed by the tRNA splicing endonuclease (TSEN) complex. Eukaryotic TSEN is comprised of four core subunits (TSEN54, TSEN2, TSEN34, and TSEN15). The human TSEN complex additionally co-purifies with the polynucleotide kinase CLP1; however, CLP1’s role in tRNA splicing remains unclear. Mutations in genes encoding all four TSEN subunits, as well as CLP1, are known to cause neurodegenerative disorders, yet the mechanisms underlying the pathogenesis of these disorders are unknown. Here, we developed a recombinant system that produces active TSEN complex. Co-expression of all four TSEN subunits is required for efficient formation and function of the complex. We show that human CLP1 associates with the active TSEN complex, but is not required for tRNA intron cleavage in vitro. Moreover, RNAi knockdown of the Drosophila CLP1 orthologue, cbc, promotes biogenesis of mature tRNAs and circularized tRNA introns (tricRNAs) in vivo. Collectively, these and other findings suggest that CLP1/cbc plays a regulatory role in tRNA splicing by serving as a negative modulator of the direct tRNA ligation pathway in animal cells.

scholarly journals Reconstitution of the human tRNA splicing endonuclease complex: insight into the regulation of pre-tRNA cleavage

2020 ◽  
Vol 48 (14) ◽  
pp. 7609-7622 ◽  
Author(s):  
Cassandra K Hayne ◽  
Casey A Schmidt ◽  
Maira I Haque ◽  
A Gregory Matera ◽  
Robin E Stanley
Keyword(s):  
Animal Cells ◽  
Trna Splicing ◽  
Polynucleotide Kinase ◽  
Genes Encoding ◽  
Intron Removal ◽  
And Function ◽  
Insight Into ◽  
Negative Modulator

Abstract The splicing of tRNA introns is a critical step in pre-tRNA maturation. In archaea and eukaryotes, tRNA intron removal is catalyzed by the tRNA splicing endonuclease (TSEN) complex. Eukaryotic TSEN is comprised of four core subunits (TSEN54, TSEN2, TSEN34 and TSEN15). The human TSEN complex additionally co-purifies with the polynucleotide kinase CLP1; however, CLP1’s role in tRNA splicing remains unclear. Mutations in genes encoding all four TSEN subunits, as well as CLP1, are known to cause neurodegenerative disorders, yet the mechanisms underlying the pathogenesis of these disorders are unknown. Here, we developed a recombinant system that produces active TSEN complex. Co-expression of all four TSEN subunits is required for efficient formation and function of the complex. We show that human CLP1 associates with the active TSEN complex, but is not required for tRNA intron cleavage in vitro. Moreover, RNAi knockdown of the Drosophila CLP1 orthologue, cbc, promotes biogenesis of mature tRNAs and circularized tRNA introns (tricRNAs) in vivo. Collectively, these and other findings suggest that CLP1/cbc plays a regulatory role in tRNA splicing by serving as a negative modulator of the direct tRNA ligation pathway in animal cells.

scholarly journals RhoGDIα-dependent balance between RhoA and RhoC is a key regulator of cancer cell tumorigenesis

2011 ◽  
Vol 22 (17) ◽  
pp. 3263-3275 ◽  
Author(s):  
T. T. Giang Ho ◽  
Audrey Stultiens ◽  
Johanne Dubail ◽  
Charles M. Lapière ◽  
Betty V. Nusgens ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Cancer Progression ◽  
Dynamic Balance ◽  
Cellular Functions ◽  
Expression Of Genes ◽  
Genes Encoding ◽  
Interfering Rna

RhoGTPases are key signaling molecules regulating main cellular functions such as migration, proliferation, survival, and gene expression through interactions with various effectors. Within the RhoA-related subclass, RhoA and RhoC contribute to several steps of tumor growth, and the regulation of their expression affects cancer progression. Our aim is to investigate their respective contributions to the acquisition of an invasive phenotype by using models of reduced or forced expression. The silencing of RhoC, but not of RhoA, increased the expression of genes encoding tumor suppressors, such as nonsteroidal anti-inflammatory drug–activated gene 1 (NAG-1), and decreased migration and the anchorage-independent growth in vitro. In vivo, RhoC small interfering RNA (siRhoC) impaired tumor growth. Of interest, the simultaneous knockdown of RhoC and NAG-1 repressed most of the siRhoC-related effects, demonstrating the central role of NAG-1. In addition of being induced by RhoC silencing, NAG-1 was also largely up-regulated in cells overexpressing RhoA. The silencing of RhoGDP dissociation inhibitor α (RhoGDIα) and the overexpression of a RhoA mutant unable to bind RhoGDIα suggested that the effect of RhoC silencing is indirect and results from the up-regulation of the RhoA level through competition for RhoGDIα. This study demonstrates the dynamic balance inside the RhoGTPase network and illustrates its biological relevance in cancer progression.

scholarly journals Development of an in vitro transcription system for Neurospora crassa mitochondrial DNA and identification of transcription initiation sites.

1989 ◽  
Vol 9 (9) ◽  
pp. 3603-3613 ◽  
Author(s):  
J C Kennell ◽  
A M Lambowitz
Keyword(s):  
Mitochondrial Dna ◽  
Neurospora Crassa ◽  
Transcription Initiation ◽  
Consensus Sequence ◽  
In Vitro Transcription ◽  
Transcription System ◽  
Group I ◽  
Genes Encoding

We have developed an in vitro transcription system for Neurospora crassa mitochondrial DNA (mtDNA) and used it to identify transcription initiation sites at the 5' ends of the genes encoding the mitochondrial small and large rRNA and cytochrome b (cob). The in vitro transcription start sites correspond to previously mapped 5' ends of major in vivo transcripts of these genes. Sequences around the three transcription initiation sites define a 15-nucleotide consensus sequence, 5'-TTAGARA(T/G)G(T/G)ARTRR-3', all or part of which appears to be an element of an N. crassa mtDNA promoter. A somewhat looser 11-nucleotide consensus sequence, 5'-TTAGARR(T/G)R(T/G)A-3', was derived by including two additional promoters identified recently. Group I extranuclear mutants, such as [poky] and [SG-3], have a 4-base-pair (bp) deletion in the consensus sequence at the 5' end of the mitochondrial small rRNA and are grossly deficient in mitochondrial small rRNA (R. A. Akins and A. M. Lambowitz, Proc. Natl. Acad. Sci. USA 81:3791-3795, 1984). We show here that the 4-bp deletion in the consensus sequence decreases in vitro transcription from this site by more than 99%. N. crassa mtDNA is similar to Saccharomyces cerevisiae mtDNA in having multiple promoters, including separate promoters for the genes encoding the mitochondrial small and large rRNAs. Our results suggest that the primary effect of the 4-bp deletion in group I extranuclear mutants is to inhibit transcription of the mitochondrial small rRNA, leading to severe deficiency of mitochondrial small rRNA and small ribosomal subunits.

scholarly journals Stigmasterol blocks cartilage degradation in rabbit model of osteoarthritis.

2012 ◽  
Vol 59 (4) ◽  
Author(s):  
Wei-Ping Chen ◽  
Chong Yu ◽  
Peng-Fei Hu ◽  
Jia-Peng Bao ◽  
Jing-Li Tang ◽  
...  
Keyword(s):  
Cruciate Ligament ◽  
Rabbit Model ◽  
Cartilage Degradation ◽  
Tissue Inhibitors Of Metalloproteinase ◽  
Expression Of Genes ◽  
Genes Encoding ◽  
Group 2 ◽  
Group 1

Stigmasterol has been shown exhibit anti-osteoarthritic properties in vitro studies. However, the in vivo effects of stigmasterol on cartilage are still unclear. This study investigated the anti-osteoarthritic properties of stigmasterol on cartilage degradation in a rabbit model of osteoarthritis (OA). Twenty rabbits underwent bilateral anterior cruciate ligament transection (ACLT) to induce OA. Five rabbits were used as normal control. Two weeks after operation, the rabbits were randomly divided into two groups. Each group of 10 rabbits received intra-articular injection with 0.3 ml of stigmasterol in left knees and vehicle in right knees, once weekly. Group 1 was killed 6 weeks after ACLT and 2 were sacrificed 9 weeks after ACLT. The knee joints were assessed by gross morphology, histology and gene expression analysis. We found that expression of genes encoding matrix metalloproteinases (MMPs) was significantly higher while tissue inhibitors of metalloproteinase (TIMP)-1 was significantly lower in the both joints of the two OA groups compared to normal controls. Stigmasterol reduced the cartilage degradation as assessed by histological analysis and markedly suppressed MMPs expression both in group 1 and group 2. Our results suggest that stigmasterol may be considered as a possible therapeutical agent in the treatment of OA.

Development of an in vitro transcription system for Neurospora crassa mitochondrial DNA and identification of transcription initiation sites

1989 ◽  
Vol 9 (9) ◽  
pp. 3603-3613
Author(s):  
J C Kennell ◽  
A M Lambowitz
Keyword(s):  
Mitochondrial Dna ◽  
Neurospora Crassa ◽  
Transcription Initiation ◽  
Consensus Sequence ◽  
In Vitro Transcription ◽  
Transcription System ◽  
Group I ◽  
Genes Encoding

We have developed an in vitro transcription system for Neurospora crassa mitochondrial DNA (mtDNA) and used it to identify transcription initiation sites at the 5' ends of the genes encoding the mitochondrial small and large rRNA and cytochrome b (cob). The in vitro transcription start sites correspond to previously mapped 5' ends of major in vivo transcripts of these genes. Sequences around the three transcription initiation sites define a 15-nucleotide consensus sequence, 5'-TTAGARA(T/G)G(T/G)ARTRR-3', all or part of which appears to be an element of an N. crassa mtDNA promoter. A somewhat looser 11-nucleotide consensus sequence, 5'-TTAGARR(T/G)R(T/G)A-3', was derived by including two additional promoters identified recently. Group I extranuclear mutants, such as [poky] and [SG-3], have a 4-base-pair (bp) deletion in the consensus sequence at the 5' end of the mitochondrial small rRNA and are grossly deficient in mitochondrial small rRNA (R. A. Akins and A. M. Lambowitz, Proc. Natl. Acad. Sci. USA 81:3791-3795, 1984). We show here that the 4-bp deletion in the consensus sequence decreases in vitro transcription from this site by more than 99%. N. crassa mtDNA is similar to Saccharomyces cerevisiae mtDNA in having multiple promoters, including separate promoters for the genes encoding the mitochondrial small and large rRNAs. Our results suggest that the primary effect of the 4-bp deletion in group I extranuclear mutants is to inhibit transcription of the mitochondrial small rRNA, leading to severe deficiency of mitochondrial small rRNA and small ribosomal subunits.

scholarly journals Insight into the maintenance of odontogenic potential in mouse dental mesenchymal cells based on transcriptomic analysis

PeerJ ◽  
2016 ◽  
Vol 4 ◽  
pp. e1684 ◽  
Author(s):  
Yunfei Zheng ◽  
Lingfei Jia ◽  
Pengfei Liu ◽  
Dandan Yang ◽  
Waner Hu ◽  
...  
Keyword(s):  
Tooth Development ◽  
Differential Expression Analysis ◽  
Mesenchymal Cells ◽  
Genes Encoding ◽  
Dental Epithelium ◽  
Wnt Pathways ◽  
Tooth Germs ◽  
Insight Into

Background.Mouse dental mesenchymal cells (mDMCs) from tooth germs of cap or later stages are frequently used in the context of developmental biology or whole-tooth regeneration due to their odontogenic potential.In vitro-expanded mDMCs serve as an alternative cell source considering the difficulty in obtaining primary mDMCs; however, cultured mDMCs fail to support tooth development as a result of functional failures of specific genes or pathways. The goal of this study was to identify the genes that maintain the odontogenic potential of mDMCs in culture.Methods.We examined the odontogenic potential of freshly isolated versus cultured mDMCs from the lower first molars of embryonic day 14.5 mice. The transcriptome of mDMCs was detected using RNA sequencing and the data were validated by qRT-PCR. Differential expression analysis and pathway analysis were conducted to identify the genes that contribute to the loss of odontogenic potential.Results.Cultured mDMCs failed to develop into well-structured tooth when they were recombined with dental epithelium. Compared with freshly isolated mDMCs, we found that 1,004 genes were upregulated and 948 were downregulated in cultured mDMCs. The differentially expressed genes were clustered in the biological processes and signaling pathways associated with tooth development. Followingin vitroculture, genes encoding a wide array of components of MAPK, TGF-β/BMP, and Wnt pathways were significantly downregulated. Moreover, the activities ofBdnf,Vegfα,Bmp2, andBmp7were significantly inhibited in cultured mDMCs. Supplementation of VEGFα, BMP2, and BMP7 restored the expression of a subset of downregulated genes and induced mDMCs to form dentin-like structuresin vivo.Conclusions.Vegfα,Bmp2, andBmp7play a role in the maintenance of odontogenic potential in mDMCs.

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