Molecular characterization of a gene encoding N-myristoyl transferase (NMT) from Triticum aestivum (bread wheat)

Genome ◽  
2004 ◽  
Vol 47 (6) ◽  
pp. 1036-1042 ◽  
Author(s):  
Tim Dumonceaux ◽  
Raju V.S Rajala ◽  
Rajendra Sharma ◽  
Gopalan Selvaraj ◽  
Raju Datla
Keyword(s):  
Triticum Aestivum ◽  
Protein Modification ◽  
Wheat Plant ◽  
Single Copy ◽  
Gene Encoding ◽  
Growth And Survival ◽  
Reading Frame ◽  
Eukaryotic Proteins ◽  
Taxonomic Groups

Myristoyl-CoA:protein N-myristoyl transferase (NMT; EC 2.3.1.97) acylates the Gly residue abutting the N-terminal Met with a myristic acid following the removal of the Met residue in certain eukaryotic proteins, and in some cases myristoylation is essential to cell growth and survival. We report the cloning of a full-length cDNA encoding NMT from Triticum aestivum (TaNMT). The cDNA included a predicted open reading frame of 1317 nucleotides, which encoded a predicted protein of 438 amino acids containing all of the residues that are important for NMT activity. The TaNMT amino acid and nucleotide sequences were compared with NMTs from 14 other species encompassing a wide array of taxonomic groups. Among the experimentally validated NMTs, TaNMT was most similar to that of Arabidopsis thaliana. Southern blot analysis of wheat genomic DNA showed that TaNMT is encoded by a single copy gene, with one copy per haploid genome. We expressed TaNMT in Escherichia coli cells and determined that the recombinant protein possessed NMT activity, catalyzing the N-myristoylation of peptides from known or putatively myristoylated proteins from plants and animals without a strong preference for the plant peptides. TaNMT is the second experimentally validated plant NMT sequence and the first from a monocotyledonous species.Key words: N-myristoyl transferase, myristoylation, protein modification, wheat, plant development.

scholarly journals Cloning, Nucleotide Sequence, and Expression of the Gene Encoding the Bacteriocin Boticin B from Clostridium botulinum Strain 213B

2000 ◽  
Vol 66 (12) ◽  
pp. 5480-5483 ◽  
Author(s):  
Sean S. Dineen ◽  
Marite Bradshaw ◽  
Eric A. Johnson
Keyword(s):  
Amino Acids ◽  
Nucleotide Sequence ◽  
Clostridium Botulinum ◽  
Shuttle Vector ◽  
Open Reading Frame ◽  
Gene Encoding ◽  
Reading Frame ◽  
Heat Stable ◽  
Group I

ABSTRACT Boticin B is a heat-stable bacteriocin produced byClostridium botulinum strain 213B that has inhibitory activity against various strains of C. botulinum and related clostridia. The gene encoding the bacteriocin was localized to a 3.0-kb HindIII fragment of an 18.8-kb plasmid, cloned, and sequenced. DNA sequencing revealed the boticin B structural gene,btcB, to be an open reading frame encoding 50 amino acids. A C. botulinum strain 62A transconjugant containing theHindIII fragment inserted into a clostridial shuttle vector expressed boticin B, although at much lower levels than those observed in C. botulinum 213B. To our knowledge, this is the first demonstration and characterization of a bacteriocin from toxigenic group I C. botulinum.

scholarly journals Characterization of RPR1, an essential gene encoding the RNA component of Saccharomyces cerevisiae nuclear RNase P.

1991 ◽  
Vol 11 (2) ◽  
pp. 721-730 ◽  
Author(s):  
J Y Lee ◽  
C E Rohlman ◽  
L A Molony ◽  
D R Engelke
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Essential Gene ◽  
Rnase P ◽  
Single Copy ◽  
Temperature Sensitive ◽  
Coding Region ◽  
Gene Encoding ◽  
Processing Steps ◽  
Potential Precursor

RNA components have been identified in preparations of RNase P from a number of eucaryotic sources, but final proof that these RNAs are true RNase P subunits has been elusive because the eucaryotic RNAs, unlike the procaryotic RNase P ribozymes, have not been shown to have catalytic activity in the absence of protein. We previously identified such an RNA component in Saccharomyces cerevisiae nuclear RNase P preparations and have now characterized the corresponding, chromosomal gene, called RPR1 (RNase P ribonucleoprotein 1). Gene disruption experiments showed RPR1 to be single copy and essential. Characterization of the gene region located RPR1 600 bp downstream of the URA3 coding region on chromosome V. We have sequenced 400 bp upstream and 550 bp downstream of the region encoding the major 369-nucleotide RPR1 RNA. The presence of less abundant, potential precursor RNAs with an extra 84 nucleotides of 5' leader and up to 30 nucleotides of 3' trailing sequences suggests that the primary RPR1 transcript is subjected to multiple processing steps to obtain the 369-nucleotide form. Complementation of RPR1-disrupted haploids with one variant of RPR1 gave a slow-growth and temperature-sensitive phenotype. This strain accumulates tRNA precursors that lack the 5' end maturation performed by RNase P, providing direct evidence that RPR1 RNA is an essential component of this enzyme.

Sequence of a Rhodococcus gene cluster encoding the subunits of ethanolamine ammonia-lyase and an APC-like permease

1994 ◽  
Vol 40 (5) ◽  
pp. 403-407 ◽  
Author(s):  
René De Mot ◽  
Istvan Nagy ◽  
Geert Schoofs ◽  
Jos Vanderleyden
Keyword(s):  
Open Reading Frame ◽  
Genomic Fragment ◽  
Positive Bacterium ◽  
Gram Positive ◽  
Gene Encoding ◽  
Reading Frame ◽  
Transporter Family ◽  
Rhodococcus Species ◽  
Rhodococcus Sp

Sequence analysis of a 5173-bp genomic fragment from the nocardioform actinomycete Rhodococcus sp. strain NI86/21 revealed the presence of two genes, eutB and eutC, encoding the putative homologues of the large and small subunits of the ethanolamine ammonia-lyase, respectively, from Salmonella typhimurium. This is the first report of the characterization of these genes in a Gram-positive species. Immediately upstream of eutB, a gene encoding a putative permease of the APC (amino acids, polyamines, choline) transporter family was located. At present, no other Gram-positive members of this permease family are known. The translational coupling of these eut genes suggests an operon-like organization of the ethanolamine genes in Rhodococcus species. A truncated open reading frame downstream of eutC contained an N-terminal motif characteristic of membrane-anchored lipoproteins.Key words: nocardioform actinomycete, cobalamin, APC transporter, membrane-anchored lipoprotein, Gram-positive bacterium.

scholarly journals Cloning and Expression of the algL Gene, Encoding the Azotobacter chroococcum Alginate Lyase: Purification and Characterization of the Enzyme

1999 ◽  
Vol 181 (5) ◽  
pp. 1409-1414 ◽  
Author(s):  
Ana Peciña ◽  
Alberto Pascual ◽  
Antonio Paneque
Keyword(s):  
Gene Sequence ◽  
Azotobacter Vinelandii ◽  
Alginate Lyase ◽  
Azotobacter Chroococcum ◽  
Open Reading Frame ◽  
Cloning And Expression ◽  
Gene Encoding ◽  
Reading Frame ◽  
Encoding Gene

ABSTRACT The alginate lyase-encoding gene (algL) ofAzotobacter chroococcum was localized to a 3.1-kbEcoRI DNA fragment that revealed an open reading frame of 1,116 bp. This open reading frame encodes a protein of 42.98 kDa, in agreement with the value previously reported by us for this protein. The deduced protein has a potential N-terminal signal peptide that is consistent with its proposed periplasmic location. The analysis of the deduced amino acid sequence indicated that the gene sequence has a high homology (90% identity) to the Azotobacter vinelandii gene sequence, which has very recently been deposited in the GenBank database, and that it has 64% identity to the Pseudomonas aeruginosa gene sequence but that it has rather low homology (15 to 22% identity) to the gene sequences encoding alginate lyase in other bacteria. The A. chroococcum AlgL protein was overproduced in Escherichia coli and purified to electrophoretic homogeneity in a two-step chromatography procedure on hydroxyapatite and phenyl-Sepharose. The kinetic and molecular parameters of the recombinant alginate lyase are similar to those found for the native enzyme.

scholarly journals Characterization of the chromosomal aminoglycoside 2'-N-acetyltransferase gene from Mycobacterium fortuitum.

1996 ◽  
Vol 40 (10) ◽  
pp. 2350-2355 ◽  
Author(s):  
J A Aínsa ◽  
C Martin ◽  
B Gicquel ◽  
R Gomez-Lus
Keyword(s):  
Dna Hybridization ◽  
Mycobacterium Smegmatis ◽  
Open Reading Frame ◽  
Mycobacterium Fortuitum ◽  
Aminoglycoside Resistance ◽  
Gene Encoding ◽  
Reading Frame ◽  
Putative Protein ◽  
Providencia Stuartii

A novel gene encoding an aminoglycoside 2'-N-acetyltransferase (AAC) was cloned from Mycobacterium fortuitum. DNA sequencing results identified an open reading frame that we have called aac(2')-Ib encoding a putative protein with a predicted molecular mass of 24,800 Da. The deduced AAC(2')-Ib protein showed homology to the AAC(2')-Ia from Providencia stuartii. This is the second member of a subfamily of AAC(2')-I enzymes to be identified. No homology was found with other acetyltransferases, including all of the AAC(3) and AAC(6') proteins. The aac(2')-Ib gene cloned in a mycobacterial plasmid and introduced in Mycobacterium smegmatis conferred resistance to gentamicin, tobramycin, dibekacin, netilmicin, and 6'-N-ethylnetilmicin. DNA hybridization with an intragenic probe of aac(2')-Ib showed that this gene was present in all 34 strains of M. fortuitum tested. The universal presence of the aac(2')-Ib gene in M. fortuitum was not correlated with any aminoglycoside resistance phenotype, suggesting that this gene may play a role in the secondary metabolism of the bacterium.

scholarly journals Molecular Cloning and Nucleotide Sequence of the Superoxide Dismutase Gene and Characterization of Its Product fromBacillus subtilis

1998 ◽  
Vol 180 (14) ◽  
pp. 3697-3703 ◽  
Author(s):  
Takashi Inaoka ◽  
Yoshinobu Matsumura ◽  
Tetsuaki Tsuchido
Keyword(s):  
Superoxide Dismutase ◽  
Stationary Phase ◽  
Specific Activity ◽  
Upstream Region ◽  
Gene Encoding ◽  
Sod Activity ◽  
Vegetative Cells ◽  
Reading Frame ◽  
Superoxide Dismutase Gene

ABSTRACT Bacillus subtilis was found to possess one detectable superoxide dismutase (Sod) in both vegetative cells and spores. The Sod activity in vegetative cells was maximal at stationary phase. Manganese was necessary to sustain Sod activity at stationary phase, but paraquat, a superoxide generator, did not induce the expression of Sod. The specific activity of purified Sod was approximately 2,600 U/mg of protein, and the enzyme was a homodimer protein with a molecular mass of approximately 25,000 per monomer. The gene encoding Sod, designatedsodA, was cloned by the combination of several PCR methods and the Southern hybridization method. DNA sequence analysis revealed the presence of one open reading frame consisting of 606 bp. Several putative promoter sites were located in the upstream region ofsodA. The deduced amino acid sequence showed high homology with other bacterial manganese Sods. Conserved regions in bacterial manganese Sod could also be seen. The phenotype of double mutantEscherichia coli sodA sodB, which could not grow in minimal medium without supplemental amino acids, was complemented by the expression of B. subtilis sodA.

scholarly journals Menaquinone (Vitamin K2) Biosynthesis: Localization and Characterization of the menA Gene fromEscherichia coli

1998 ◽  
Vol 180 (10) ◽  
pp. 2782-2787 ◽  
Author(s):  
K. Suvarna ◽  
D. Stevenson ◽  
R. Meganathan ◽  
M. E. S. Hudspeth
Keyword(s):  
High Performance ◽  
Promoter Sequence ◽  
Anaerobic Growth ◽  
Gene Encoding ◽  
Reading Frame ◽  
Chromatography Analysis ◽  
Naphthoic Acid ◽  
Membrane Bound ◽  
Carbon Side Chain

ABSTRACT A key reaction in the biosynthesis of menaquinone involves the conversion of the soluble bicyclic naphthalenoid compound 1,4-dihydroxy-2-naphthoic acid (DHNA) to the membrane-bound demethylmenaquinone. The enzyme catalyzing this reaction, DHNA-octaprenyltransferase, attaches a 40-carbon side chain to DHNA. The menA gene encoding this enzyme has been cloned and localized to a 2.0-kb region of the Escherichia coli genome between cytR and glpK. DNA sequence analysis of the cloned insert revealed a 308-codon open reading frame (ORF), which by deletion analyses was shown to restore anaerobic growth of amenA mutant. Reverse-phase high-performance liquid chromatography analysis of quinones extracted from theorf-complemented cells independently confirmed the restoration of menaquinone biosynthesis, and similarly, analyses of isolated cell membranes for DHNA octaprenyltransferase activity confirmed the introduction of the menA product into theorf-complemented menA mutant. The validity of an ORF-associated putative promoter sequence was confirmed by primer extension analyses.

scholarly journals Overexpression of Trypanosoma rangeli trypanothione reductase increases parasite survival under oxidative stress

2016 ◽  
Vol 2 ◽  
Author(s):  
I.T. BELTRAME-BOTELHO ◽  
P.H. STOCO ◽  
M. STEINDEL ◽  
B. ANDERSSON ◽  
E.F. PELOSO ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Functional Characterization ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Single Copy ◽  
Trypanothione Reductase ◽  
Trypanosoma Rangeli ◽  
Reading Frame ◽  
Key Enzyme

SUMMARYThe infectivity and virulence of pathogenic trypanosomatids are directly associated with the efficacy of their antioxidant system. Among the molecules involved in the trypanosomatid response to reactive oxygen or nitrogen species, trypanothione reductase (TRed) is a key enzyme. In this study, we performed a molecular and functional characterization of the TRed enzyme fromTrypanosoma rangeli(TrTRed), an avirulent trypanosome of mammals. TheTrTRed gene has an open reading frame (ORF) of 1473 bp (~490 aa, 53 kDa) and occurs as a single-copy gene in the haploid genome. The predicted protein contains two oxidoreductase domains, which are equally expressed in the cytosol of epimastigotes and trypomastigotes. Nicotinamide adenine dinucleotide phosphate (NADPH) generation is reduced and endogenous H2O2production is elevated inT. rangeliChoachí strain compared withT. cruziY strain epimastigotes. Oxidative stress induced by H2O2does not induce significant alterations inTrTRed expression. Overexpression ofTrTRed did not influencein vitrogrowth or differentiation into trypomastigotes, but mutant parasites showed increased resistance to H2O2-induced stress. Our results indicate thatT. rangeliconstitutively expresses TRed during the entire life cycle, with reduced levels during infective and non-replicative trypomastigote stages.

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