scholarly journals barS1, a Gene for Biosynthesis of a γ-Butyrolactone Autoregulator, a Microbial Signaling Molecule Eliciting Antibiotic Production in Streptomyces Species

2002 ◽  
Vol 184 (18) ◽  
pp. 5151-5157 ◽  
Author(s):  
Noriyasu Shikura ◽  
Junji Yamamura ◽  
Takuya Nihira
Keyword(s):  
Acyl Carrier Protein ◽  
Antibiotic Production ◽  
Receptor Gene ◽  
Streptomyces Species ◽  
Reading Frame ◽  
Streptomyces Virginiae ◽  
Acid Protein ◽  
Short Chain Alcohol ◽  
Natural Enantiomer

ABSTRACT From Streptomyces virginiae, in which production of streptogramin antibiotic virginiamycin M1 and S is tightly regulated by a low-molecular-weight Streptomyces hormone called virginiae butanolide (VB), which is a member of the γ-butyrolactone autoregulators, the hormone biosynthetic gene (barS1) was cloned and characterized by heterologous expression in Escherichia coli and by gene disruption in S. virginiae. The barS1 gene (a 774-bp open reading frame encoding a 257-amino-acid protein [M r, 27,095]) is situated in the 10-kb regulator island surrounding the VB-specific receptor gene, barA. The deduced BarS1 protein is weakly homologous to β-ketoacyl-acyl carrier protein/coenzyme A reductase and belongs to the superfamily of short-chain alcohol dehydrogenase. The function of the BarS1 protein in VB biosynthesis was confirmed by BarS1-dependent in vitro conversion of 6-dehydro-VB-A to VB-A, the last catalytic step in VB biosynthesis. Of the four possible enantiomeric products from racemic 6-dehydro-VB-A as a substrate, only the natural enantiomer of (2R,3R,6S)-VB-A was produced by the purified recombinant BarS1 (rBarS1), indicating that rBarS1 is the stereospecific reductase recognizing (3R)-isomer as a substrate and reducing it stereospecifically to the (6S) product. In the ΔbarS1 mutant created by homologous recombination, the production of VB as well as the production of virginiamycin was lost. The production of virginiamycin by the ΔbarS1 mutant was fully recovered by the external addition of VB to the culture, which indicates that the barS1 gene is essential in the biosynthesis of the autoregulator VBs in S. virginiae and that the failure of virginiamycin production was a result of the loss of VB production.

scholarly journals Cloning and expression of human liver dehydroepiandrosterone sulphotransferase

1993 ◽  
Vol 289 (1) ◽  
pp. 233-240 ◽  
Author(s):  
K A Comer ◽  
J L Falany ◽  
C N Falany
Keyword(s):  
Molecular Mass ◽  
Bile Acids ◽  
Blot Analysis ◽  
Human Liver ◽  
Molecular Size ◽  
Cloning And Expression ◽  
Reading Frame ◽  
Acid Protein ◽  
Liver Cdna Library

Dehydroepiandrosterone sulphotransferase (DHEA-ST) catalyses the 3′-phosphoadenosine 5′-phosphosulphate-dependent sulphation of a wide variety of steroids in human liver and adrenal tissue and is responsible for most, if not all, of the sulphation of bile acids in human liver. This report describes the isolation, characterization and expression of a cDNA which encodes human liver DHEA-ST. The DHEA-ST cDNA, designated DHEA-ST8, was isolated from a Uni-Zap XR human liver cDNA library and is composed of 1060 bp and contains an open reading frame encoding a 285-amino-acid protein with a molecular mass of approx. 33765 Da. Translation of DHEA-ST8 in vitro generated a protein identical in molecular size with that of DHEA-ST. Expression of DHEA-ST8 in COS-7 cells produces an active DHEA-ST protein which is capable of sulphating DHEA, has the same molecular mass as human liver DHEA-ST and is recognized by rabbit anti-(human liver DHEA-ST) antibodies. Northern-blot analysis of human liver RNA detects the presence of three different size transcripts; however, Southern-blot analysis of human DNA suggests that only one gene may be present in the genome. These results describe the cloning of a human ST which has an important role in the sulphation of steroids and bile acids in human liver and adrenals.

scholarly journals Expression of the bglH Gene ofLactobacillus plantarum Is Controlled by Carbon Catabolite Repression

1998 ◽  
Vol 180 (13) ◽  
pp. 3400-3404 ◽  
Author(s):  
Rosangela Marasco ◽  
Lidia Muscariello ◽  
Mario Varcamonti ◽  
Maurilio De Felice ◽  
Margherita Sacco
Keyword(s):  
Mutational Analysis ◽  
Glucose Repression ◽  
Transcriptional Unit ◽  
Calculated Molecular Mass ◽  
Reading Frame ◽  
Gene Coding ◽  
Acid Protein ◽  
Responsive Element

ABSTRACT A newly identified bglH gene coding for a phospho-β-glucosidase of Lactobacillus plantarum was isolated and expressed in Escherichia coli. The sequence analysis of the cloned DNA fragment showed an open reading frame encoding a 480-amino-acid protein with a calculated molecular mass of 53 kDa. The bglH gene was shown to be expressed on a monocistronic transcriptional unit. Its transcription was repressed 10-fold in L. plantarum cells grown on glucose compared to the β-glucoside salicin as a sole carbon source. A catabolite-responsive element (CRE) spanning from −3 to +11 with respect to the transcriptional start point was found, and its functionality was assessed by mutational analysis. In vitro and in vivo DNA binding experiments suggested the occurrence of a DNA-protein complex at the CRE site, which would mediate glucose repression ofbglH expression.

Molecular Cloning, Sequencing, and Expression of a Chemoreceptor Gene from Leptospirillum ferrooxidans

1998 ◽  
Vol 64 (7) ◽  
pp. 2380-2385 ◽  
Author(s):  
Mónica Delgado ◽  
Héctor Toledo ◽  
Carlos A. Jerez
Keyword(s):  
Chromosomal Dna ◽  
Reading Frame ◽  
Leptospirillum Ferrooxidans ◽  
E Coli ◽  
Acid Protein ◽  
Chemotaxis Proteins ◽  
Acidophilic Bacterium ◽  
Sequencing And Expression ◽  
The Right

ABSTRACT We have cloned and sequenced a 2,262-bp chromosomal DNA fragment from the chemolithoautotrophic acidophilic bacteriumLeptospirillum ferrooxidans. This DNA contained an open reading frame for a 577-amino-acid protein showing several characteristics of the bacterial chemoreceptors and, therefore, we named this gene lcrI for Leptospirillumchemotaxis receptor I. This is the first sequence reported for a gene from L. ferrooxidans encoding a protein. ThelcrI gene showed both ς28-like and ς70-like putative promoters. The LcrI deduced protein contained two hydrophobic regions most likely corresponding to the two transmembrane regions present in all of the methyl-accepting chemotaxis proteins (MCPs) which make them fold with both periplasmic and cytoplasmic domains. We have proposed a cytoplasmic domain for LcrI, which also contains the highly conserved domain (HCD region), present in all of the chemotactic receptors, and two probable methylation sites. The in vitro expression of a DNA plasmid containing the 2,262-bp fragment showed the synthesis of a 58-kDa protein which was immunoprecipitated by antibodies against the Tar protein (an MCP fromEscherichia coli), confirming some degree of antigenic conservation. In addition, this 58-kDa protein was expressed inE. coli, being associated with its cytoplasmic membrane fraction. It was not possible to determine a chemotactic receptor function for LcrI expressed in E. coli. This was most likely due to the fact that the periplasmic pH of E. coli, which differs by 3 to 4 pH units from that of acidophilic chemolithotrophs, does not allow the right conformation for the LcrI periplasmic domain.

ASSOCIATION OF BDKRB2 AND ACE GENE POLYMORPHISM WITH ERYTHROCYTE ADRENOREACTIVITY IN YOUNG MEN WITH DIFFERENT MOTOR ACTIVITY

2020 ◽  
pp. 96-107
Author(s):  
A.Z. Dautova ◽  
E.A. Khazhieva ◽  
V.G. Shamratova ◽  
L.Z. Sadykova
Keyword(s):  
Gene Polymorphism ◽  
Angiotensin Converting Enzyme ◽  
Motor Activity ◽  
Receptor Gene ◽  
Young Men ◽  
Converting Enzyme ◽  
Ace Gene ◽  
Ace Gene Polymorphism ◽  
Physically Inactive

The aim of the paper was to study the association of polymorphic variants of rs4646994 (I/D) of the angiotensin converting enzyme gene (ACE) and rs5810761 (+9/-9) of the bradykinin B2 receptor gene (BDKRB2) with erythrocyte adrenoreactivity (ARE) in athletes and untrained young men. Materials and Methods. The study involved 61 young men (aged 21–23) with different levels of motor activity (MA). ARE was evaluated according to the erythrocyte sedimentation rate (ESR) change under adrenaline in vitro at final concentrations 10-5, 10-6, 10-7, 10-9, 10-11, 10-13 g/ml of venous blood. According to the effect observed and ESR shifts under adrenaline, we distinguished 3 ARE types: antiaggregative (AnAg), areactive (Ar) and aggregative (Ar). Results. The results of comparative and correlation analyses demonstrated that young athletes with +9/-9 (BDKRB2) genotype were characterized by a higher aggregative resistance of erythrocytes to the effects of both physiological (<10-9 g/ml) (physiological adrenaline concentration, PAC) and stressful doses (>10-9 g/ml) of adrenaline (stress adrenaline concentration, SAC), as well as by predominance of AnAg and Ar ARE types. In athletes, among the representatives of different genotypes of АСЕ gene I/D polymorphism, the erythrocyte response to adrenaline did not have any statistically significant differences. In physically inactive students, namely individuals with the D/D genotype, maximal ESR deviation under PAC was less than in those with I/D genotype. Conclusion. Athletes with *-9 allele (+9/-9 genotype) in their genotype can be considered more stress-resistant, which is provided by optimal adaptive and compensatory body mechanisms. Apparently, resistance of cells to the adrenaline contributes much to the work of these mechanisms. As for the ACE gene polymorphism, its effect on the suspension characteristics of erythrocytes is less pronounced not only in physically inactive young men, but in athletes as well. Keywords: erythrocyte adrenoreactivity (ARE), stress tolerance, β2 bradykinin receptor gene (BDKRB2), angiotensin converting enzyme (ACE) gene, motor activity. Цель работы – изучить ассоциацию полиморфных вариантов rs4646994 (I/D) гена ангиотензинпревращающего фермента (АСЕ) и rs5810761 (+9/-9) гена рецептора брадикинина 2 типа (BDKRB2) с адренореактивностью эритроцитов (АРЭ) у спортсменов и юношей, ведущих физически малоактивный образ жизни. Материалы и методы. В исследовании принял участие 61 юноша с разным уровнем двигательной активности (ДА) в возрасте 21–23 лет. Оценку АРЭ проводили по изменению скорости оседания эритроцитов (СОЭ) под действием адреналина in vitro в конечных концентрациях 10-5, 10-6, 10-7, 10-8, 10-9, 10-11, 10-13 г/мл венозной крови. По характеру наблюдаемого эффекта в соответствии с направленностью сдвигов СОЭ в присутствии адреналина мы выделили 3 типа АРЭ: антиагрегационный (АнАг), ареактивный (Ар) и агрегационный (Аг). Результаты. По результатам сравнительного и корреляционного анализа установлено, что юноши-спортсмены с генотипом +9/-9 (BDKRB2) характеризуются более высокой агрегативной устойчивостью эритроцитов к воздействию как физиологических (10-9 г/мл и ниже), так и повышенных (стрессовых) доз (выше 10-8 г/мл крови) адреналина, а также преобладанием АнАг- и Ар-типов АРЭ. У представителей разных генотипов полиморфизма I/D гена АСЕ реакция эритроцитов на адреналин не имела статистически значимых различий в группе спортсменов, тогда как в группе малоактивных студентов у лиц с генотипом D/D максимальное отклонение СОЭ при ФКА было меньше, чем при генотипе I/D. Выводы. Спортсменов, имеющих в своём генотипе аллель *-9 (+9/-9 генотип), можно считать более стрессоустойчивыми, что обеспечивается оптимальными адаптивно-компенсаторными механизмами организма, существенная роль в обеспечении которых, по-видимому, принадлежит устойчивости клеток к действию адреналина. Что касается полиморфизма гена АСЕ, то его влияние на суспензионные характеристики эритроцитов выражено слабее не только у физически малоактивных юношей, но и у спортсменов. Ключевые слова: адренореактивность эритроцитов (АРЭ), стрессоустойчивость, ген рецептора брадикинина β2 (BDKRB2), ген ангиотензинпревращающего фермента (АСЕ), двигательная активность.

scholarly journals Evidence for the interleukin-2 dependent expansion of leukemic cells in adult T cell leukemia

Blood ◽  
1987 ◽  
Vol 70 (5) ◽  
pp. 1407-1411 ◽  
Author(s):  
M Maeda ◽  
N Arima ◽  
Y Daitoku ◽  
M Kashihara ◽  
H Okamoto ◽  
...  
Keyword(s):  
T Cell ◽  
Receptor Gene ◽  
Interleukin 2 ◽  
Leukemic Cells ◽  
Type I ◽  
T Cell Leukemia ◽  
Cell Leukemia ◽  
In Vitro Proliferation ◽  
Adult T Cell Leukemia

Abstract Interleukin 2 (IL-2) receptor/Tac antigen is abnormally expressed on cells of patients with adult T cell leukemia (ATL) caused by infection with human T lymphotropic virus type I (HTLV-I). Twenty-five patients with ATL were examined to determine whether their leukemic cells continued to show IL-2-dependent proliferation. In 21 patients, the in vitro proliferation of HTLV-I-infected nonleukemic T cell clones was found to be dependent on IL-2. However, clonality analysis based on T cell receptor gene rearrangement profiles and the site of HTLV-I provirus integration revealed IL-2-dependent growth in leukemic cells in four patients with ATL. These results provide evidence for the IL-2- dependent proliferation of leukemic cells in some ATL patients.

scholarly journals Structural comparison of Acinetobacter baumannii β-ketoacyl-acyl carrier protein reductases in fatty acid and aryl polyene biosynthesis

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Woo Cheol Lee ◽  
Sungjae Choi ◽  
Ahjin Jang ◽  
Kkabi Son ◽  
Yangmee Kim
Keyword(s):  
Fatty Acid ◽  
Acinetobacter Baumannii ◽  
Gene Cluster ◽  
Fatty Acid Synthase ◽  
Acyl Carrier Protein ◽  
Gene Clusters ◽  
Carrier Protein ◽  
Aryl Group ◽  
Visible Absorption

AbstractSome Gram-negative bacteria harbor lipids with aryl polyene (APE) moieties. Biosynthesis gene clusters (BGCs) for APE biosynthesis exhibit striking similarities with fatty acid synthase (FAS) genes. Despite their broad distribution among pathogenic and symbiotic bacteria, the detailed roles of the metabolic products of APE gene clusters are unclear. Here, we determined the crystal structures of the β-ketoacyl-acyl carrier protein (ACP) reductase ApeQ produced by an APE gene cluster from clinically isolated virulent Acinetobacter baumannii in two states (bound and unbound to NADPH). An in vitro visible absorption spectrum assay of the APE polyene moiety revealed that the β-ketoacyl-ACP reductase FabG from the A. baumannii FAS gene cluster cannot be substituted for ApeQ in APE biosynthesis. Comparison with the FabG structure exhibited distinct surface electrostatic potential profiles for ApeQ, suggesting a positively charged arginine patch as the cognate ACP-binding site. Binding modeling for the aryl group predicted that Leu185 (Phe183 in FabG) in ApeQ is responsible for 4-benzoyl moiety recognition. Isothermal titration and arginine patch mutagenesis experiments corroborated these results. These structure–function insights of a unique reductase in the APE BGC in comparison with FAS provide new directions for elucidating host–pathogen interaction mechanisms and novel antibiotics discovery.

scholarly journals Translational and Structural Requirements of the Early Nodulin Gene enod40, a Short-Open Reading Frame-Containing RNA, for Elicitation of a Cell-Specific Growth Response in the Alfalfa Root Cortex

2001 ◽  
Vol 21 (1) ◽  
pp. 354-366 ◽  
Author(s):  
Carolina Sousa ◽  
Christina Johansson ◽  
Celine Charon ◽  
Hamid Manyani ◽  
Christof Sautter ◽  
...  
Keyword(s):  
Amino Acids ◽  
Biological Activity ◽  
Rna Structure ◽  
Open Reading Frames ◽  
In Vitro Translation ◽  
Cortical Cells ◽  
Root Cortex ◽  
Reading Frame ◽  
Early Nodulin

ABSTRACT A diversity of mRNAs containing only short open reading frames (sORF-RNAs; encoding less than 30 amino acids) have been shown to be induced in growth and differentiation processes. The early nodulin geneenod40, coding for a 0.7-kb sORF-RNA, is expressed in the nodule primordium developing in the root cortex of leguminous plants after infection by symbiotic bacteria. Ballistic microtargeting of this gene into Medicago roots induced division of cortical cells. Translation of two sORFs (I and II, 13 and 27 amino acids, respectively) present in the conserved 5′ and 3′ regions ofenod40 was required for this biological activity. These sORFs may be translated in roots via a reinitiation mechanism. In vitro translation products starting from the ATG of sORF I were detectable by mutating enod40 to yield peptides larger than 38 amino acids. Deletion of a Medicago truncatula enod40 region between the sORFs, spanning a predicted RNA structure, did not affect their translation but resulted in significantly decreased biological activity. Our data reveal a complex regulation of enod40action, pointing to a role of sORF-encoded peptides and structured RNA signals in developmental processes involving sORF-RNAs.

scholarly journals LASS3 (longevity assurance homologue 3) is a mainly testis-specific (dihydro)ceramide synthase with relatively broad substrate specificity

2006 ◽  
Vol 398 (3) ◽  
pp. 531-538 ◽  
Author(s):  
Yukiko Mizutani ◽  
Akio Kihara ◽  
Yasuyuki Igarashi
Keyword(s):  
Amino Acid ◽  
Substrate Specificity ◽  
Family Members ◽  
Fatty Acyl ◽  
Ceramide Synthase ◽  
Broad Substrate Specificity ◽  
Acid Protein ◽  
Amino Acid Protein

The LASS (longevity assurance homologue) family members are highly conserved from yeasts to mammals. Five mouse and human LASS family members, namely LASS1, LASS2, LASS4, LASS5 and LASS6, have been identified and characterized. In the present study we cloned two transcriptional variants of hitherto-uncharacterized mouse LASS3 cDNA, which encode a 384-amino-acid protein (LASS3) and a 419-amino-acid protein (LASS3-long). In vivo, [3H]dihydrosphingosine labelling and electrospray-ionization MS revealed that overproduction of either LASS3 isoform results in increases in several ceramide species, with some preference toward those having middle- to long-chain-fatty acyl-CoAs. A similar substrate preference was observed in an in vitro (dihydro)ceramide synthase assay. These results indicate that LASS3 possesses (dihydro)ceramide synthesis activity with relatively broad substrate specificity. We also found that, except for a weak display in skin, LASS3 mRNA expression is limited almost solely to testis, implying that LASS3 plays an important role in this gland.

scholarly journals Identification of NAD+ Synthetase from Streptococcus sobrinus as a B-Cell-Stimulatory Protein

2004 ◽  
Vol 186 (2) ◽  
pp. 419-426 ◽  
Author(s):  
Isabel Veiga-Malta ◽  
Margarida Duarte ◽  
Márcia Dinis ◽  
Pedro Madureira ◽  
Paula Ferreira ◽  
...  
Keyword(s):  
B Cell ◽  
Active Form ◽  
Lymphocyte Activation ◽  
Amino Acid Residues ◽  
Streptococcus Sobrinus ◽  
Reading Frame ◽  
High Sequence Homology ◽  
Culture Supernatants ◽  
Nad Synthetase

ABSTRACT Streptococcus sobrinus, one agent of dental caries, secretes a protein that induces lymphocyte polyclonal activation of the host as a mechanism of immune evasion. We have isolated from culture supernatants of this bacterium a protein with murine B-cell-stimulatory properties and subsequently cloned the relevant gene. It contains an open reading frame of 825 bp encoding a polypeptide with 275 amino acid residues and a molecular mass of 30 kDa. The protein displays high sequence homology with NAD+ synthetases from several organisms, including a conserved fingerprint sequence (SGGXD) characteristic of ATP pyrophosphatases. The polypeptide was expressed in Escherichia coli as a hexahistidine-tagged protein and purified in an enzymatically active form. The recombinant NAD+ synthetase stimulates murine B cells after in vitro treatment of spleen cell cultures, as demonstrated by its ability to induce up-regulation of the expression of CD69, an early marker of lymphocyte activation. Stimulation with the recombinant NAD+ synthetase was also observed with other B-cell markers, such as CD19+, B220+, and CD21+. Cell proliferation follows the activation induced by the recombinant NAD+ synthetase.

scholarly journals Plant holo-(acyl carrier protein) synthase

1988 ◽  
Vol 252 (1) ◽  
pp. 39-45 ◽  
Author(s):  
S A Elhussein ◽  
J A Miernyk ◽  
J B Ohlrogge
Keyword(s):  
Spinacia Oleracea ◽  
Acyl Carrier Protein ◽  
Ricinus Communis ◽  
Plant Cells ◽  
Gel Permeation Chromatography ◽  
Carrier Protein ◽  
Improved Method ◽  
Ph Optimum ◽  
Spinach Leaf

1. An improved method was developed for the assay of plant holo-(acyl carrier protein) synthase activity, using Escherichia coli acyl-(acyl carrier protein) synthetase as a coupling enzyme. 2. Holo-(acyl carrier protein) synthase was partially purified from spinach (Spinacia oleracea) leaves by a combination of (NH4)2SO4 fractionation and anion-exchange and gel-permeation chromatography. 3. The partially purified enzyme had a pH optimum of 8.2 and Km values of 2 microM, 72 microM and 3 mM for apo-(acyl carrier protein), CoA and Mg2+ respectively. Synthase activity was inhibited in vitro by the reaction product 3′,5′-ADP. 4. Results from the fractionation of spinach leaf and developing castor-oil-seed (Ricinus communis) endosperm cells were consistent with a cytosolic localization of holo-(acyl carrier protein) synthase activity in plant cells.

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