scholarly journals An Oligoribonuclease Gene inStreptomyces griseus

2000 ◽  
Vol 182 (16) ◽  
pp. 4647-4653 ◽  
Author(s):  
Yasuo Ohnishi ◽  
Yoko Nishiyama ◽  
Rie Sato ◽  
Shogo Kameyama ◽  
Sueharu Horinouchi
Keyword(s):  
Sequence Similarity ◽  
Streptomyces Griseus ◽  
Aerial Mycelium ◽  
Morphological Development ◽  
Developmentally Regulated ◽  
High Sequence Similarity ◽  
S1 Nuclease ◽  
Reading Frame ◽  
Reverse Transcription Pcr ◽  
Nuclease Mapping

ABSTRACT In Streptomyces griseus, A-factor (2-isocapryloyl-3R-hydroxymethyl-γ-butyrolactone) serves as a microbial hormone that switches on many genes required for streptomycin production and morphological development. An open reading frame (Orf1) showing high sequence similarity to oligoribonucleases of various origins is present just downstream of adpA, one of the A-factor-dependent genes. Orf1 was named OrnA (oligoribonuclease A) because it showed 3′-to-5′ exo-oligoribonuclease activity, releasing [32P]CMP from ApCpC[32P]pC used as a substrate. Reverse transcription-PCR and S1 nuclease mapping analyses revealed that ornA was transcribed from two promoters; one was a developmentally regulated, A-factor-dependent promoter in front of adpA, and the other was a constitutive promoter in front of the ornA coding sequence. Transcription ofornA was thus additively enhanced at the initiation stage for secondary metabolism and aerial mycelium formation.ornA-disrupted strains grew slowly and scarcely formed aerial mycelium. ornA homologues were distributed in a wide variety of Streptomyces species, including S. coelicolor A3(2), as determined by Southern hybridization analysis. Disruption of the ornA homologue in S. coelicolor A3(2) also caused phenotypes similar to those of theS. griseus ΔornA strains. The OrnA oligoribonucleases inStreptomyces species are therefore not essential but play an important role in vegetative growth and in the initiation of differentiation.

scholarly journals Isolation and characterization of the mouse liver/bone/kidney-type alkaline phosphatase gene

1990 ◽  
Vol 268 (3) ◽  
pp. 641-648 ◽  
Author(s):  
M Terao ◽  
M Studer ◽  
M Gianní ◽  
E Garattini
Keyword(s):  
Alkaline Phosphatase ◽  
Sequence Similarity ◽  
Mrna Level ◽  
Consensus Binding Site ◽  
S1 Nuclease ◽  
Ancestral Gene ◽  
3T3 Fibroblasts ◽  
Isolation And Characterization ◽  
S1 Nuclease Mapping ◽  
Nuclease Mapping

The gene coding for the mouse alkaline phosphatase expressed in liver, bone, kidney and placenta (liver/bone/kidney-type alkaline phosphatase, L/B/K-ALP) was isolated and characterized. This gene consists of 12 exons and it is at least 49 kb long. The first two exons are separated by a long intron which is at least 32 kb in size, whereas the other exons span within the remaining 17 kb. Primer extension and S1-nuclease mapping analyses with placental mRNA demonstrate a single major transcription start site, which is preceded by a G + C-rich region containing a TATA-like sequence and three copies of the consensus binding site for the transcription factor Sp1. Transfection experiments using two different reporter genes show that the 5′-flanking region of the gene is active as a promoter in undifferentiated F9 teratocarcinoma cells, but not in 3T3 fibroblasts, consistent with the L/B/K-ALP mRNA level in the two cell lines. As expected from the sequence similarity at the cDNA level, the structural organization of the mouse gene is similar to that of the human and rat L/B/K-ALP genes, suggesting that they all derive from a single ancestral gene.

Genetic characteristics of bla NDM-1-positive plasmid in Citrobacter freundii isolate separated from a clinical infectious patient

2013 ◽  
Vol 62 (9) ◽  
pp. 1332-1337 ◽  
Author(s):  
Xiao-Xing Du ◽  
Jian-Feng Wang ◽  
Ying Fu ◽  
Feng Zhao ◽  
Yan Chen ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Sequence Similarity ◽  
Blot Hybridization ◽  
Variable Region ◽  
Southern Blot Hybridization ◽  
Potential Threat ◽  
High Sequence Similarity ◽  
S1 Nuclease ◽  
Genetic Characteristics ◽  
Clinical Patient

This study reports an infectious case involving an NDM-1-producing Citrobacter freundii and further explored the potential threat of the bla NDM-1 gene by analysing the characteristics of the NDM-1-encoding plasmid sequence. A bla NDM-1-positive C. freundii with high resistance to carbapenems was separated from a clinical patient suffering from a urinary tract infection. S1 nuclease-based plasmid analysis followed by Southern blot hybridization, a conjugation experiment and electrotransformation confirmed that the bla NDM-1 gene was located on a plasmid. High-throughput sequencing of the bla NDM-1-postive plasmid (pCFNDM-CN) showed that it was a 54 kb IncX-type plasmid and contained a backbone region and a variable region with two β-lactamase genes (bla NDM-1 and bla SHV-12). The NDM-1 composite transposon in the variable region was surrounded by IS26 and IS5-truncated ISAba125, and shared a high sequence similarity to the bla NDM-1 surrounding structure in Acinetobacter spp. Our research suggested that the NDM-1 composite transposon might play an essential role in mobilization of the bla NDM-1 gene from Acinetobacter spp. to Enterobacteriaceae.

scholarly journals Serine and threonine catabolism in Saccharomyces cerevisiae: the CHA1 polypeptide is homologous with other serine and threonine dehydratases.

Genetics ◽  
1992 ◽  
Vol 131 (3) ◽  
pp. 531-539 ◽  
Author(s):  
C Bornaes ◽  
J G Petersen ◽  
S Holmberg
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Transcription Initiation ◽  
Open Reading Frame ◽  
Predicted Amino Acid Sequence ◽  
S1 Nuclease ◽  
Reading Frame ◽  
Threonine Dehydratase ◽  
Nuclease Mapping

Abstract The catabolic L-serine (L-threonine) dehydratase of Saccharomyces cerevisiae allows the yeast to grow on media with L-serine or L-threonine as sole nitrogen source. Previously we have cloned the CHA1 gene by complementation of a mutant, cha1, lacking the dehydratase activity. Here we present the DNA sequence of a 1,766-bp fragment of the CHA1 region encompassing an open reading frame of 1080 bp. Comparison of the predicted amino acid sequence of the CHA1 polypeptide with that of other serine/threonine dehydratases revealed several blocks of sequence homology. Thus, the amino acid sequence of rat liver serine dehydratase (SDH2) and the CHA1 polypeptide are 44% homologous allowing for conservative substitutions, while 36% similarity is found between the catabolic threonine dehydratase (tdcB) of Escherichia coli and the CHA1 protein. This strongly suggests that CHA1 is the structural gene for the yeast catabolic serine (threonine) dehydratase. S1-nuclease mapping of the CHA1 mRNA ends showed a major transcription initiation site corresponding to an untranslated leader of about 19 nucleotides, while a major polyadenylation site was located about 86 nucleotides downstream from the open reading frame. Furthermore, we have mapped the chromosomal position of the CHA1 gene to less than 0.5 kb centromere proximal to HML on the left arm of chromosome III.

scholarly journals Slow myosin in developing rat skeletal muscle.

1987 ◽  
Vol 104 (3) ◽  
pp. 447-459 ◽  
Author(s):  
M Narusawa ◽  
R B Fitzsimons ◽  
S Izumo ◽  
B Nadal-Ginard ◽  
N A Rubinstein ◽  
...  
Keyword(s):  
Cdna Probe ◽  
Early Event ◽  
Primary Cells ◽  
Limb Muscle ◽  
Developmentally Regulated ◽  
S1 Nuclease ◽  
Rat Fetus ◽  
Slow Myosin ◽  
Fashion Studies ◽  
Nuclease Mapping

Through S1 nuclease mapping using a specific cDNA probe, we demonstrate that the slow myosin heavy-chain (MHC) gene, characteristic of adult soleus, is expressed in bulk hind limb muscle obtained from the 18-d rat fetus. We support these results by use of a monoclonal antibody (mAb) which is highly specific to the adult slow MHC. Immunoblots of MHC peptide maps show the same peptides, uniquely recognized by this antibody in adult soleus, are also identified in 18-d fetal limb muscle. Thus synthesis of slow myosin is an early event in skeletal myogenesis and is expressed concurrently with embryonic myosin. By immunofluorescence we demonstrate that in the 16-d fetus all primary myotubes in future fast and future slow muscles homogeneously express slow as well as embryonic myosin. Fiber heterogeneity arises owing to a developmentally regulated inhibition of slow MHC accumulation as muscles are progressively assembled from successive orders of cells. Assembly involves addition of new, superficial areas of the anterior tibial muscle (AT) and extensor digitorum longus muscle (EDL) in which primary cells initially stain weakly or are unstained with the slow mAb. In the developing AT and EDL, expression of slow myosin is unstable and is progressively restricted as these muscles specialize more and more towards the fast phenotype. Slow fibers persisting in deep portions of the adult EDL and AT are interpreted as vestiges of the original muscle primordium. A comparable inhibition of slow MHC accumulation occurs in the developing soleus but involves secondary, not primary, cells. Our results show that the fate of secondary cells is flexible and is spatially determined. By RIA we show that the relative proportions of slow MHC are fivefold greater in the soleus than in the EDL or AT at birth. After neonatal denervation, concentrations of slow MHC in the soleus rapidly decline, and we hypothesize that, in this muscle, the nerve protects and amplifies initial programs of slow MHC synthesis. Conversely, the content of slow MHC rises in the neonatally denervated EDL. This suggests that as the nerve amplifies fast MHC accumulation in the developing EDL, accumulation of slow MHC is inhibited in an antithetic fashion. Studies with phenylthiouracil-induced hypothyroidism indicate that inhibition of slow MHC accumulation in the EDL and AT is not initially under thyroid regulation. At later stages, the development of thyroid function plays a role in inhibiting slow MHC accumulation in the differentiating EDL and AT.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals An A-Factor-Dependent Extracytoplasmic Function Sigma Factor (ςAdsA) That Is Essential for Morphological Development in Streptomyces griseus

2000 ◽  
Vol 182 (16) ◽  
pp. 4596-4605 ◽  
Author(s):  
Haruka Yamazaki ◽  
Yasuo Ohnishi ◽  
Sueharu Horinouchi
Keyword(s):  
Sigma Factor ◽  
Streptomyces Griseus ◽  
Transcriptional Activator ◽  
Start Codon ◽  
Morphological Development ◽  
Aerial Hyphae ◽  
Transcriptional Start Point ◽  
Translational Start Codon ◽  
Translational Start ◽  
Nuclease Mapping

ABSTRACT A-factor (2-isocapryloyl-3R-hydroxymethyl-γ-butyrolactone) at an extremely low concentration triggers streptomycin production and aerial mycelium formation in Streptomyces griseus. A-factor induces the expression of an A-factor-dependent transcriptional activator, AdpA, essential for both morphological and physiological differentiation by binding to the A-factor receptor protein ArpA, which has bound and repressed the adpA promoter, and dissociating it from the promoter. Nine DNA fragments that were specifically recognized and bound by histidine-tagged AdpA were isolated by cycles of a gel mobility shift-PCR method. One of them was located in front of a gene encoding an extracytoplasmic function ς factor belonging to a subgroup of the primary ς70 family. The cloned gene was named AdpA-dependent sigma factor gene (adsA), and the gene product was named ςAdsA. Transcription ofadsA depended on A-factor and AdpA, since adsAwas transcribed at a very low and constant level in an A-factor-deficient mutant strain or in an adpA-disrupted strain. Consistent with this, transcription of adsA was greatly enhanced at or near the timing of aerial hyphae formation, as determined by low-resolution S1 nuclease mapping. High-resolution S1 mapping determined the transcriptional start point 82 nucleotides upstream of the translational start codon. DNase I footprinting showed that AdpA bound both strands symmetrically between the transcriptional start point and the translational start codon; AdpA protected the antisense strand from positions +7 to +41 with respect to the transcriptional start point and the sense strand from positions +12 to +46. A weak palindrome was found in the AdpA-binding site. The unusual position bound by AdpA as a transcriptional activator, in relation to the promoter, suggested the presence of a mechanism by which AdpA activates transcription of adsA in some unknown way. Disruption of the chromosomal adsA gene resulted in loss of aerial hyphae formation but not streptomycin or yellow pigment production, indicating that ςAdsA is involved only in morphological development and not in secondary metabolic function. The presence of a single copy in each of the Streptomycesspecies examined by Southern hybridization suggests a common role in morphogenesis in this genus.

scholarly journals The Synechocystis sp. PCC 6803 open reading frame slr0201 that is homologous to sdhC from Archaea codes for a [2Fe-2S] protein

2021 ◽  
Author(s):  
Fusheng Xiong ◽  
Russell LoBrutto ◽  
Wim F. J. Vermaas
Keyword(s):  
Sequence Similarity ◽  
Immunoblot Analysis ◽  
Open Reading Frame ◽  
Heterodisulfide Reductase ◽  
Redox Titration ◽  
High Sequence Similarity ◽  
Reading Frame ◽  
Midpoint Potential ◽  
Synechocystis Sp ◽  
Pcc 6803

A hypothetical protein encoded by Synechocystis sp. PCC 6803 open reading frame slr0201 shows high sequence similarity to the C subunit of a group of unusual succinate dehydrogenases found in some archaeal species. Slr0201 was originally annotated as HdrB, the B subunit of heterodisulfide reductase, but appears to be SdhC instead. This protein was overexpressed in E. coli by cloning the PCR-derived slr0201 open reading frame into a pET16b-based expression vector. The overproduced Slr0201 accumulated predominantly in inclusion bodies with an apparent molecular mass of 33 kDa. The protein contained at least one [2Fe-2S] cluster based on UV-visible absorbance and CD spectra and EPR spectroscopy, in conjunction with stoichiometric analysis of protein-bound iron and sulfur content. Redox titration showed a midpoint potential (Em) of + 17 mV at pH 7.0, which is consistent with Slr0201 serving a role in transferring electrons between succinate and plastoquinone. Slr0201 was also overproduced in Synechocystis sp. PCC 6803 by introducing an additional, His-tagged slr0201 into the Synechocystis genome replacing psbA3, creating the slr0201+-His overexpression strain. Immunoblot analysis shows that Slr0201 is membrane-associated in the wild type. However, in the Slr0201+-His strain, immunoreaction occurred in both the membrane and soluble fractions, possibly as a consequence of processing near the N-terminus. The results obtained with Slr0201 are discussed in the light of one of the cyanobacterial SdhB subunits, which shares redox commonalities with archaeal SdhB.

scholarly journals Phosphate-Controlled Regulator for the Biosynthesis of the Dalbavancin Precursor A40926

2007 ◽  
Vol 189 (22) ◽  
pp. 8120-8129 ◽  
Author(s):  
Rosa Alduina ◽  
Luca Lo Piccolo ◽  
Davide D'Alia ◽  
Clelia Ferraro ◽  
Nina Gunnarsson ◽  
...  
Keyword(s):  
Response Regulator ◽  
Sequence Similarity ◽  
Streptomyces Griseus ◽  
Phosphate Limitation ◽  
Pcr Analysis ◽  
Binding Motif ◽  
Glycopeptide Antibiotics ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Key Steps

ABSTRACT The actinomycete Nonomuraea sp. strain ATCC 39727 produces the glycopeptide A40926, the precursor of the novel antibiotic dalbavancin. Previous studies have shown that phosphate limitation results in enhanced A40926 production. The A40926 biosynthetic gene (dbv) cluster, which consists of 37 genes, encodes two putative regulators, Dbv3 and Dbv4, as well as the response regulator (Dbv6) and the sensor-kinase (Dbv22) of a putative two-component system. Reverse transcription-PCR (RT-PCR) and real-time RT-PCR analysis revealed that the dbv14-dbv8 and the dbv30-dbv35 operons, as well as dbv4, were negatively influenced by phosphate. Dbv4 shows a putative helix-turn-helix DNA-binding motif and shares sequence similarity with StrR, the transcriptional activator of streptomycin biosynthesis in Streptomyces griseus. Dbv4 was expressed in Escherichia coli as an N-terminal His6-tagged protein. The purified protein bound the dbv14 and dbv30 upstream regions but not the region preceding dbv4. Bbr, a Dbv4 ortholog from the gene cluster for the synthesis of the glycopeptide balhimycin, also bound to the dbv14 and dbv30 upstream regions, while Dbv4 bound appropriate regions from the balhimycin cluster. Our results provide new insights into the regulation of glycopeptide antibiotics, indicating that the phosphate-controlled regulator Dbv4 governs two key steps in A40926 biosynthesis: the biosynthesis of the nonproteinogenic amino acid 3,5-dihydroxyphenylglycine and critical tailoring reactions on the heptapeptide backbone.

scholarly journals SSoNΔ andSsoNΔlong: two thermostable esterases from the same ORF in the archaeonSulfolobus solfataricus?

Archaea ◽  
2006 ◽  
Vol 2 (2) ◽  
pp. 109-115 ◽  
Author(s):  
Luigi Mandrich ◽  
Margherita Pezzullo ◽  
Mosè Rossi ◽  
Giuseppe Manco
Keyword(s):  
Sequence Similarity ◽  
Hormone Sensitive Lipase ◽  
Divergent Evolution ◽  
High Sequence Similarity ◽  
Ancestral Gene ◽  
Reading Frame ◽  
Family Analysis ◽  
N Terminus ◽  
Metabolic Conditions ◽  
Hormone Sensitive

Previously, we reported from theSulfolobus solfataricusopen reading frame (ORF) SSO2517 the cloning, overexpression and characterization of an esterase belonging to the hormone-sensitive lipase (HSL) family and apparently having a deletion at the N-terminus, which we namedSsoNΔ. Searching the recently reportedSulfolobus acidocaldariusgenome by sequence alignment, using SSO2517 as a query, allowed identity of a putative esterase (ORF SAC1105) sharing high sequence similarity (82%) with SSO2517. This esterase displays an N-terminus and total length similar to other known esterases of the HSL family. Analysis of the upstream DNA sequence of SS02517 revealed the possibility of expressing a longer version of the protein with an extended N-terminus; however, no clear translation signal consistent with a longer protein version was detected. This new version of SSO2517 was cloned, over-expressed, purified and characterized. The resulting protein, namedSsoNΔlong, was 15-fold more active with the substratep-nitrophenyl hexanoate thanSsoNΔ. Furthermore,SsoNΔlong andSsoNΔ displayed different substrate specificities for triacylglycerols. These results and the phylogenetic relationship betweenS. solfataricusandS. acidocaldariussuggest a common origin of SSO2517 and SAC1105 from an ancestral gene, followed by divergent evolution. Alternatively, a yet-to-be discovered mechanism of translation that directs the expression ofSsoNΔlong under specific metabolic conditions could be hypothesized.

scholarly journals Identification of the Promoter in the Intergenic Region betweenorf1andcry8Ea1Controlled by Sigma H Factor

2012 ◽  
Vol 78 (12) ◽  
pp. 4164-4168 ◽  
Author(s):  
Lixin Du ◽  
Lili Qiu ◽  
Qi Peng ◽  
Didier Lereclus ◽  
Jie Zhang ◽  
...  
Keyword(s):  
Intergenic Region ◽  
Transcriptional Activity ◽  
Sequence Similarity ◽  
High Sequence Similarity ◽  
Upstream Gene ◽  
Content Type ◽  
Transcription Start Sites ◽  
Reverse Transcription Pcr ◽  
Rapid Amplification ◽  
Sigma H

ABSTRACTBacillus thuringiensisCry8Ea toxin is specifically toxic to larvae of the Asian cockchafer,Holotrichia parallela. Here we investigated the mechanism of transcriptional regulation of thecry8Ea1gene. Reverse transcription-PCR (RT-PCR) results indicated thatcry8Ea1and an upstream gene (orf1) were cotranscribed. Transcriptional fusions with thelacZgene demonstrated that transcription of thecry8Ea1gene started from two promoters: Porf1, which is located upstream of theorf1gene, and Pcry8E, located in the intergenic region mapping betweenorf1andcry8Ea1. Of the known, similarorf1-cryoperons, this is the first report of the existence of a promoter in the intergenic region between theorf1andcrygenes. The transcriptional activity of Porf1was found during sporulation inB. thuringiensissubsp.kurstakiHD-73 and was almost abolished in thesigEmutant, while the transcriptional activity of Pcry8Ewas detected after the end of the exponential phase in HD-73 and was considerably lower in thesigHmutant. The transcription start sites generated by the twocry8Ea1promoters were determined by the 5′ -SMARTer rapid amplification of cDNA ends (RACE) method. The −35 and −10 regions of Porf1and Pcry8Eshowed high sequence similarity with the σEand σHpromoters, respectively. These results indicated that Porf1is controlled by the σEfactor and Pcry8Eby the σHfactor.

scholarly journals Analysis of Two Gene Clusters Involved in the Degradation of 4-Fluorophenol by Arthrobacter sp. Strain IF1

2009 ◽  
Vol 75 (24) ◽  
pp. 7767-7773 ◽  
Author(s):  
Maria Isabel M. Ferreira ◽  
Toshiya Iida ◽  
Syed A. Hasan ◽  
Kaoru Nakamura ◽  
Marco W. Fraaije ◽  
...  
Keyword(s):  
Sequence Similarity ◽  
Gene Clusters ◽  
Sole Source ◽  
Northern Blotting ◽  
Monooxygenase System ◽  
Flavin Reductase ◽  
Gene Encoding ◽  
High Sequence Similarity ◽  
Reverse Transcription Pcr ◽  
Two Component

ABSTRACT Arthrobacter sp. strain IF1 is able to grow on 4-fluorophenol (4-FP) as a sole source of carbon and energy. To clone the 4-FP degradation genes, DNA libraries were constructed and screened with a probe obtained by PCR using primers designed on the basis of conserved regions of aromatic two-component monooxygenases. Sequencing of positive clones yielded two gene clusters, each harboring a gene encoding a monooxygenase with high sequence similarity to the oxygenase component of 4-nitrophenol and 4-chlorophenol monooxygenase systems. Both these monooxygenase genes were differentially expressed during growth on 4-FP, as revealed by Northern blotting and reverse transcription-PCR. One cluster also contained a gene for a flavin reductase. The monooxygenase and reductase were purified from Escherichia coli cells expressing the corresponding genes, and together they catalyzed NADH-dependent hydroxylation and dehalogenation of 4-halophenols. The results indicate that strain IF1 transforms 4-FP to hydroquinone by a two-component monooxygenase system of which one component provides reduced flavin adenine dinucleotide at the expense of NADH and the other catalyzes para-hydroxylation of 4-FP and other 4-substituted phenols.

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