scholarly journals Biochemical and Mutational Analysis of Glutamine Synthetase Type III from the Rumen Anaerobe Ruminococcus albus 8

2005 ◽  
Vol 187 (21) ◽  
pp. 7481-7491 ◽  
Author(s):  
Kensey R. Amaya ◽  
Svetlana A. Kocherginskaya ◽  
Roderick I. Mackie ◽  
Isaac K. O. Cann
Keyword(s):  
Glutamine Synthetase ◽  
Mutational Analysis ◽  
Sufficient Conditions ◽  
Kinetic Studies ◽  
Type I ◽  
Gene Encoding ◽  
Genes Encoding ◽  
Subunit Organization ◽  
Glutamyl Transferase ◽  
First Time

ABSTRACT Two different genes encoding glutamine synthetase type I (GSI) and GSIII were identified in the genome sequence of R. albus 8. The identity of the GSIII protein was confirmed by the presence of its associated conserved motifs. The glnN gene, encoding the GSIII, was cloned and expressed in Escherichia coli BL21 cells. The recombinant protein was purified and subjected to biochemical and physical analyses. Subunit organization suggested a protein present in solution as both monomers and oligomers. Kinetic studies using the forward and the γ-glutamyl transferase (γ-GT) assays were carried out. Mutations that changed conserved glutamic acid residues to alanine in the four GSIII motifs resulted in drastic decreases in GS activity using both assays, except for an E380A mutation, which rather resulted in an increase in activity in the forward assay compared to the wild-type protein. Reduced GSIII activity was also exhibited by mutating, individually, two lysines (K308 and K318) located in the putative nucleotide-binding site to alanine. Most importantly, the presence of mRNA transcripts of the glnN gene in R. albus 8 cells grown under ammonia limiting conditions, whereas little or no transcript was detected in cells grown under ammonia sufficient conditions, suggested an important role for the GSIII in the nitrogen metabolism of R. albus 8. Furthermore, the mutational studies on the conserved GSIII motifs demonstrated, for the first time, their importance in the structure and/or function of a GSIII protein.

scholarly journals Non-Syndromic Dentinogenesis Imperfecta Caused by Mild Mutations in COL1A2

2021 ◽  
Vol 11 (6) ◽  
pp. 526
Author(s):  
Yejin Lee ◽  
Youn Jung Kim ◽  
Hong-Keun Hyun ◽  
Jae-Cheoun Lee ◽  
Zang Hee Lee ◽  
...  
Keyword(s):  
Collagen Type ◽  
Molecular Genetic ◽  
Collagen Type I ◽  
Type I ◽  
Dentinogenesis Imperfecta ◽  
Gene Encoding ◽  
Korean Families ◽  
Whole Exome ◽  
Genes Encoding ◽  
Candidate Gene Sequencing

Hereditary dentin defects can be categorized as a syndromic form predominantly related to osteogenesis imperfecta (OI) or isolated forms without other non-oral phenotypes. Mutations in the gene encoding dentin sialophosphoprotein (DSPP) have been identified to cause dentinogenesis imperfecta (DGI) Types II and III and dentin dysplasia (DD) Type II. While DGI Type I is an OI-related syndromic phenotype caused mostly by monoallelic mutations in the genes encoding collagen type I alpha 1 chain (COL1A1) and collagen type I alpha 2 chain (COL1A2). In this study, we recruited families with non-syndromic dentin defects and performed candidate gene sequencing for DSPP exons and exon/intron boundaries. Three unrelated Korean families were further analyzed by whole-exome sequencing due to the lack of the DSPP mutation, and heterozygous COL1A2 mutations were identified: c.3233G>A, p.(Gly1078Asp) in Family 1 and c.1171G>A, p.(Gly391Ser) in Family 2 and 3. Haplotype analysis revealed different disease alleles in Families 2 and 3, suggesting a mutational hotspot. We suggest expanding the molecular genetic etiology to include COL1A2 for isolated dentin defects in addition to DSPP.

scholarly journals lmo1273, a novel gene involved in Listeria monocytogenes virulence

Microbiology ◽  
2009 ◽  
Vol 155 (3) ◽  
pp. 891-902 ◽  
Author(s):  
Armelle Bigot ◽  
Catherine Raynaud ◽  
Iharilalao Dubail ◽  
Marion Dupuis ◽  
Hamid Hossain ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Mammalian Species ◽  
Foodborne Pathogen ◽  
Kinetic Studies ◽  
Amino Acid Uptake ◽  
Rnase H ◽  
Type I ◽  
Acid Uptake ◽  
Genes Encoding ◽  
Culture Models

Listeria monocytogenes is a foodborne pathogen able to infect humans and many other mammalian species, leading to serious, often fatal disease. We have previously identified a five-gene locus in the genome of L. monocytogenes EGD-e which comprised three contiguous genes encoding paralogous type I signal peptidases. In the present study, we focused on the two distal genes of the locus (lmo1272 and lmo1273), encoding proteins sharing significant similarities with the YlqF and RnhB proteins, respectively, of Bacillus subtilis. lmo1273 could complement an Escherichia coli rnhA-rnhB thermosensitive growth phenotype, suggesting that it encodes a functional RNase H. Strikingly, inactivation of lmo1273 provoked a strong attenuation of virulence in the mouse model, and kinetic studies in infected mice revealed that multiplication of the lmo1273 mutant in target organs was significantly impaired. However, the mutation did not impair L. monocytogenes intracellular multiplication or cell-to-cell spread in cell culture models. Transcriptional profiles obtained with an lmo1273-overexpressing strain were compared to those of the wild-type strain, using microarray analyses. The data obtained suggest a pleiotropic regulatory role of Lmo1273 and possible links with amino acid uptake.

scholarly journals Three tightly linked genes encoding human type I keratins: conservation of sequence in the 5'-untranslated leader and 5'-upstream regions of coexpressed keratin genes.

1986 ◽  
Vol 6 (2) ◽  
pp. 539-548 ◽  
Author(s):  
A RayChaudhury ◽  
D Marchuk ◽  
M Lindhurst ◽  
E Fuchs
Keyword(s):  
Molecular Mechanisms ◽  
Type I ◽  
Differential Splicing ◽  
Gene Encoding ◽  
Linked Gene ◽  
Human Dna ◽  
Human Epidermal Cells ◽  
Genes Encoding ◽  
Positive Hybridization ◽  
Endonuclease Mapping

We have isolated and subcloned three separate segments of human DNA which share strong sequence homology with a previously sequenced gene encoding a type I keratin, K14 (50 kilodaltons). Restriction endonuclease mapping has demonstrated that these three genes are tightly linked chromosomally, whereas the K14 gene appears to be separate. As judged by positive hybridization-translation and Northern blot analyses, the central linked gene encodes a keratin, K17, which is expressed in abundance with K14 and two other type I keratins in cultured human epidermal cells. None of these other epidermal keratin mRNAs appears to be generated from the K17 gene through differential splicing of its transcript. The sequence of the K17 gene reveals striking homologies not only with the coding portions and intron positions of the K14 gene, but also with its 5'-noncoding and 5'-upstream sequences. These similarities may provide an important clue in elucidating the molecular mechanisms underlying the coexpression of the two genes.

scholarly journals The RpoH-Mediated Stress Response in Neisseria gonorrhoeae Is Regulated at the Level of Activity

2004 ◽  
Vol 186 (24) ◽  
pp. 8443-8452 ◽  
Author(s):  
Lina Laskos ◽  
Catherine S. Ryan ◽  
Janet A. M. Fyfe ◽  
John K. Davies
Keyword(s):  
Stress Response ◽  
Neisseria Gonorrhoeae ◽  
Molecular Chaperones ◽  
Sigma Factor ◽  
Mutational Analysis ◽  
Normal Growth ◽  
Gene Encoding ◽  
General Stress Response ◽  
Level Of Activity ◽  
Genes Encoding

ABSTRACT The general stress response in Neisseria gonorrhoeae was investigated. Transcriptional analyses of the genes encoding the molecular chaperones DnaK, DnaJ, and GrpE suggested that they are transcribed from σ32 (RpoH)-dependent promoters upon exposure to stress. This was confirmed by mutational analysis of the σ32 promoter of dnaK. The gene encoding the gonococcal RpoH sigma factor appears to be essential, as we could not isolate viable mutants. Deletion of an unusually long rpoH leader sequence resulted in elevated levels of transcription, suggesting that this region is involved in negative regulation of RpoH expression during normal growth. Transcriptional analyses and protein studies determined that regulation of the RpoH-mediated stress response is different from that observed in most other species, in which regulation occurs predominantly at the transcriptional and translational levels. We suggest that an increase in the activity of preformed RpoH is primarily responsible for induction of the stress response in N. gonorrhoeae.

scholarly journals Adaptive evolution of drug targets in producer and non-producer organisms

2011 ◽  
Vol 441 (1) ◽  
pp. 219-226 ◽  
Author(s):  
Bjarne G. Hansen ◽  
Xin E. Sun ◽  
Hans J. Genee ◽  
Christian S. Kaas ◽  
Jakob B. Nielsen ◽  
...  
Keyword(s):  
Drug Targets ◽  
Mutational Analysis ◽  
Terminal Segment ◽  
Immunosuppressive Drug ◽  
Toxic Metabolite ◽  
Gene Encoding ◽  
Genes Encoding ◽  
Km Value ◽  
Penicillium Brevicompactum ◽  
Structural Determinant

MPA (mycophenolic acid) is an immunosuppressive drug produced by several fungi in Penicillium subgenus Penicillium. This toxic metabolite is an inhibitor of IMPDH (IMP dehydrogenase). The MPA-biosynthetic cluster of Penicillium brevicompactum contains a gene encoding a B-type IMPDH, IMPDH-B, which confers MPA resistance. Surprisingly, all members of the subgenus Penicillium contain genes encoding IMPDHs of both the A and B types, regardless of their ability to produce MPA. Duplication of the IMPDH gene occurred before and independently of the acquisition of the MPAbiosynthetic cluster. Both P. brevicompactum IMPDHs are MPA-resistant, whereas the IMPDHs from a non-producer are MPA-sensitive. Resistance comes with a catalytic cost: whereas P. brevicompactum IMPDH-B is >1000-fold more resistant to MPA than a typical eukaryotic IMPDH, its kcat/Km value is 0.5% of ‘normal’. Curiously, IMPDH-B of Penicillium chrysogenum, which does not produce MPA, is also a very poor enzyme. The MPA-binding site is completely conserved among sensitive and resistant IMPDHs. Mutational analysis shows that the C-terminal segment is a major structural determinant of resistance. These observations suggest that the duplication of the IMPDH gene in the subgenus Penicillium was permissive for MPA production and that MPA production created a selective pressure on IMPDH evolution. Perhaps MPA production rescued IMPDH-B from deleterious genetic drift.

scholarly journals Understanding the Role of Prevotella Genus in the Digestion of Lignocellulose and Other Substrates in Vietnamese Native Goats’ Rumen by Metagenomic Deep Sequencing

Animals ◽  
2021 ◽  
Vol 11 (11) ◽  
pp. 3257
Author(s):  
Trong-Khoa Dao ◽  
Thi-Huyen Do ◽  
Ngoc-Giang Le ◽  
Hong-Duong Nguyen ◽  
Thi-Quy Nguyen ◽  
...  
Keyword(s):  
Deep Sequencing ◽  
Gene Encoding ◽  
Amylolytic Enzymes ◽  
E Coli ◽  
Lignocellulose Conversion ◽  
Genes Encoding ◽  
Abundant Genus ◽  
First Time ◽  
Goat Rumen

Bacteria in rumen play pivotal roles in the digestion of nutrients to support energy for the host. In this study, metagenomic deep sequencing of bacterial metagenome extracted from the goats’ rumen generated 48.66 GB of data with 3,411,867 contigs and 5,367,270 genes. The genes were mainly functionally annotated by Kyoto Encyclopedia of Genes and Genomes (KEGG) Carbohydrate-Active enZYmes (CAZy), and HMMER database, and taxonomically classified by MEGAN. As a result, 65,554 genes encoding for 30 enzymes/proteins related to lignocellulose conversion were exploited, in which nine enzymes were seen for the first time in goat rumen. Prevotella was the most abundant genus, contributing 30% hemicellulases and 36% enzymes/proteins for lignocellulose pretreatment, and supporting 98.8% of feruloyl esterases and 71.7% acetylxylan esterases. In addition, 18 of the 22 most lignocellulose digesting- potential contigs belonged to Prevotella. Besides, Prevotella possessed many genes coding for amylolytic enzymes. One gene encoding for endoxylanase was successfully expressed in E. coli. The recombinant enzyme had high Vmax, was tolerant to some salts and detergents, worked better at pH 5.5–6.5, temperature 40–50 °C, and was capable to be used in practices. Based on these findings, we confirm that Prevotella plays a pivotal role for hemicellulose digestion and significantly participates in starch, cellulose, hemicellulose, and pectin digestion in the goat rumen.

Hereditary Dentin Defects

2007 ◽  
Vol 86 (5) ◽  
pp. 392-399 ◽  
Author(s):  
J.-W. Kim ◽  
J.P. Simmer
Keyword(s):  
Matrix Protein ◽  
Type I Collagen ◽  
Rare Condition ◽  
Type I ◽  
Gene Encoding ◽  
Apparent Lack ◽  
Post Translational Modifications ◽  
Dentin Matrix Protein ◽  
Genes Encoding ◽  
Secreted Phosphoprotein 1

By the Shields classification, articulated over 30 years ago, inherited dentin defects are divided into 5 types: 3 types of dentinogenesis imperfecta (DGI), and 2 types of dentin dysplasia (DD). DGI type I is osteogenesis imperfecta (OI) with DGI. OI with DGI is caused, in most cases, by mutations in the 2 genes encoding type I collagen. Many genes are required to generate the enzymes that catalyze collagen’s diverse post-translational modifications and its assembly into fibers, fibrils, bundles, and networks. Rare inherited diseases of bone are caused by defects in these genes, and some are occasionally found to include DGI as a feature. Appreciation of the complicated genetic etiology of DGI associated with bony defects splintered the DGI type I description into a multitude of more precisely defined entities, all with their own designations. In contrast, DD-II, DGI-II, and DGI-III, each with its own pattern of inherited defects limited to the dentition, have been found to be caused by various defects in DSPP (dentin sialophosphoprotein), a gene encoding the major non-collagenous proteins of dentin. Only DD-I, an exceedingly rare condition featuring short, blunt roots with obliterated pulp chambers, remains untouched by the revolution in genetics, and its etiology is still a mystery. A major surprise in the characterization of genes underlying inherited dentin defects is the apparent lack of roles played by the genes encoding the less-abundant non-collagenous proteins in dentin, such as dentin matrix protein 1 ( DMP1), integrin-binding sialoprotein ( IBSP), matrix extracellular phosphoglycoprotein ( MEPE), and secreted phosphoprotein-1, or osteopontin ( SPP1, OPN). This review discusses the development of the dentin extracellular matrix in the context of its evolution, and discusses the phenotypes and clinical classifications of isolated hereditary defects of tooth dentin in the context of recent genetic data respecting their genetic etiologies.

Three tightly linked genes encoding human type I keratins: conservation of sequence in the 5'-untranslated leader and 5'-upstream regions of coexpressed keratin genes

1986 ◽  
Vol 6 (2) ◽  
pp. 539-548
Author(s):  
A RayChaudhury ◽  
D Marchuk ◽  
M Lindhurst ◽  
E Fuchs
Keyword(s):  
Molecular Mechanisms ◽  
Type I ◽  
Differential Splicing ◽  
Gene Encoding ◽  
Linked Gene ◽  
Human Dna ◽  
Human Epidermal Cells ◽  
Genes Encoding ◽  
Positive Hybridization ◽  
Endonuclease Mapping

We have isolated and subcloned three separate segments of human DNA which share strong sequence homology with a previously sequenced gene encoding a type I keratin, K14 (50 kilodaltons). Restriction endonuclease mapping has demonstrated that these three genes are tightly linked chromosomally, whereas the K14 gene appears to be separate. As judged by positive hybridization-translation and Northern blot analyses, the central linked gene encodes a keratin, K17, which is expressed in abundance with K14 and two other type I keratins in cultured human epidermal cells. None of these other epidermal keratin mRNAs appears to be generated from the K17 gene through differential splicing of its transcript. The sequence of the K17 gene reveals striking homologies not only with the coding portions and intron positions of the K14 gene, but also with its 5'-noncoding and 5'-upstream sequences. These similarities may provide an important clue in elucidating the molecular mechanisms underlying the coexpression of the two genes.

scholarly journals Characterization of catechol 2,3-dioxygenase from Planococcus sp. strain S5 induced by high phenol concentration.

2012 ◽  
Vol 59 (3) ◽  
Author(s):  
Katarzyna Hupert-Kocurek ◽  
Urszula Guzik ◽  
Danuta Wojcieszyńska
Keyword(s):  
Kinetic Studies ◽  
High Reactivity ◽  
Homology Search ◽  
Nucleotide Sequencing ◽  
Positive Bacterium ◽  
Gene Encoding ◽  
Genes Encoding ◽  
High Phenol Concentration ◽  
Dioxygenase Activity

This study aimed at characterization of a new catechol 2,3-dioxygenase isolated from a Gram-positive bacterium able to utilize phenol as the sole carbon and energy source. Planococcus sp. strain S5 grown on 1 or 2 mM phenol showed activity of both a catechol 1,2- and catechol 2,3-dioxygenase while at a higher concentrations of phenol only catechol 2,3-dioxygenase activity was observed. The enzyme was optimally active at 60°C and pH 8.0. Kinetic studies showed that the K(m) and V(max) of the enzyme were 42.70 µM and 329.96 mU, respectively. The catechol 2,3-dioxygenase showed the following relative meta-cleavage activities for various catechols tested: catechol (100%), 3-methylcatechol (13.67%), 4-methylcatechol (106.33%) and 4-chlorocatechol (203.80%). The high reactivity of this enzyme towards 4-chlorocatechol is different from that observed for other catechol 2,3-dioxygenases. Nucleotide sequencing and homology search revealed that the gene encoding the S5 catechol 2,3-dioxygenase shared the greatest homology with the known genes encoding isoenzymes from Gram-negative Pseudomonas strains.

scholarly journals Mycoplasma and host interaction: In vitro gene expression modulation in Mycoplasma synoviae and infected chicken chondrocytes

2016 ◽  
Vol 64 (1) ◽  
pp. 26-37 ◽  
Author(s):  
Ivanka Cizelj ◽  
Daliborka Dušanić ◽  
Dušan Benčina ◽  
Mojca Narat
Keyword(s):  
Gene Expression ◽  
Arginine Deiminase ◽  
Gene Encoding ◽  
Mycoplasma Synoviae ◽  
Host Interaction ◽  
Expression Of Genes ◽  
Cartilage Extracellular Matrix ◽  
Genes Encoding ◽  
First Time

The complex interplay between Mycoplasma synoviae and chicken chondrocytes (CCH), which come into direct contact during infectious synovitis, has been examined at the level of gene expression. Our previous studies demonstrated a significant influence of M. synoviae on the level of CCH gene expression. Here, we show for the first time that in vitro co-cultivation of M. synoviae and CCH also induces upregulation of gene expression in this mycoplasma. We observed significantly increased expression of genes important for M. synoviae pathogenicity, including cysteine protease cysP, neuraminidase nanH, haemagglutinin vlhA, and the putative nuclease MS53_0284. Moreover, the pattern of gene expression was dependent on the infection environment. In CCH, significant changes in the expression of genes encoding catabolic enzymes of the cartilage extracellular matrix (cathepsins B, K and L, aggrecanase ADAM10, and matrix metalloproteinase MMP2) were demonstrated. Infection of CCH with M. synoviae also elevated the expression of the gene encoding peptidyl arginine deiminase, type III (PADI3), which is responsible for the post-translational citrullination of proteins.

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