scholarly journals cDNA cloning of human and rat brain myo-inositol monophosphatase. Expression and characterization of the human recombinant enzyme

1992 ◽  
Vol 284 (3) ◽  
pp. 749-754 ◽  
Author(s):  
G McAllister ◽  
P Whiting ◽  
E A Hammond ◽  
M R Knowles ◽  
J R Atack ◽  
...  
Keyword(s):  
Rat Brain ◽  
Cell Signalling ◽  
Biochemical Properties ◽  
Recombinant Enzyme ◽  
Cdna Clones ◽  
Coding Region ◽  
Inositol Monophosphatase ◽  
Human Enzyme ◽  
Key Enzyme

Inositol monophosphatase (EC 3.1.3.25) is a key enzyme in the phosphoinositide cell-signalling system. Its role is to provide inositol required for the resynthesis of phosphatidylinositol and polyphosphoinositides. It is the probable pharmacological target for lithium action in brain. Using probes derived from the bovine inositol monophosphatase cDNA we have isolated cDNA clones encoding the human and rat brain enzymes. The enzyme is highly conserved in all three species (79% identical). The coding region of the human cDNA was inserted into a bacterial expression vector. The expressed recombinant enzyme was purified and its biochemical properties examined. The human enzyme is very similar to the bovine enzyme.

Association of genetic variants of the prolactin gene with milk production traits in Russian Red Pied cattle

2007 ◽  
Vol 2007 ◽  
pp. 156-156 ◽  
Author(s):  
Masoud Alipanah ◽  
Lobov Kalashnikova ◽  
Genadi Rodionov
Keyword(s):  
Genetic Marker ◽  
Regulatory Function ◽  
Cdna Clones ◽  
Production Traits ◽  
Coding Region ◽  
Nucleotide Substitutions ◽  
Prolactin Gene ◽  
Pcr Rflp ◽  
Rsai Polymorphism

Prolactin plays an important regulatory function in mammary gland development, milk secretion, and expression of milk protein genes. Hence the PRL gene is a potential genetic marker of production traits in dairy cattle. The gene was mapped on chromosome 23 by Hallerman et al. (1988). It consists of 5 exons and four introns (Camper et al. 1984) encoding the 199-amino-acid mature protein (Wallis 1974). On the basis of sequence analysis of four different cDNA clones, seven possible nucleotide substitutions were described by Sasavage et al. (1982). One of them, recognized by RsaI endonuclease, has become a popular genetic marker used for genetic characterization of cattle populations by means of PCR-RFLP (Mitra et al., 1995). Two allelic variants (B and b) have been distinguished at the DNA level, based on RsaI polymorphism in the third exon of the coding region. It has been suggested that prolactin alleles correlate with milk yield (Lewin et al., 1992).

Molecular Cloning and Characterization Analysis of Pyruvate Phosphate Dikinase Gene from Dunaliella parva

2011 ◽  
Vol 347-353 ◽  
pp. 2438-2442
Author(s):  
Chang Hua Shang ◽  
Shun Ni Zhu ◽  
Zhen Hong Yuan ◽  
Zhong Ming Wang
Keyword(s):  
Inorganic Phosphate ◽  
Function Analysis ◽  
Untranslated Regions ◽  
Coding Region ◽  
Volvox Carteri ◽  
Pyruvate Phosphate Dikinase ◽  
Dunaliella Parva ◽  
Significant Homology ◽  
Key Enzyme

Pyruvate phosphate dikinase (PPDK) catalyzes the reversible conversion of AMP, phosphoenolpyruvate (PEP) and pyrophosphate (PPi) to ATP, pyruvate and inorganic phosphate (Pi). It is a key enzyme in gluconeogensis and photosynthesis that is responsible for reversing the reaction performed by pyruvate kinase in Embden-Meyerhof-Parnas glycolysis. A cDNA clone for the Dunaliella parva PPDK was isolated by sequencing. Then the 3'-RACE and 5'-cDNA amplification were conducted based on the obtained sequence. The molecular characterization of the PPDK gene was described.The Dunaliella parva PPDK gene cDNA sequence was 3249 bp, which contained 2595 bp coding region and 654 bp 3'-untranslated regions. The deduced amino acid sequence of Dunaliella parva PPDK showed significant homology to the known PPDK from Volvox carteri and Chlamydomonas reinhardtii. This study provided foundation for further research on the function analysis and overexpression of PPDK genes. To our knowledge this is the first reported.

scholarly journals Cloning and Characterization of the Zymobacter palmae Pyruvate Decarboxylase Gene (pdc) and Comparison to Bacterial Homologues

2002 ◽  
Vol 68 (6) ◽  
pp. 2869-2876 ◽  
Author(s):  
Krishnan Chandra Raj ◽  
Lee A. Talarico ◽  
Lonnie O. Ingram ◽  
Julie A. Maupin-Furlow
Keyword(s):  
Codon Usage ◽  
Specific Activity ◽  
Pyruvate Decarboxylase ◽  
Biochemical Properties ◽  
Kinetic Properties ◽  
E Coli ◽  
Genes Encoding ◽  
Key Enzyme ◽  
Zymobacter Palmae

ABSTRACT Pyruvate decarboxylase (PDC) is the key enzyme in all homo-ethanol fermentations. Although widely distributed among plants, yeasts, and fungi, PDC is absent in animals and rare in bacteria (established for only three organisms). Genes encoding the three known bacterial pdc genes have been previously described and expressed as active recombinant proteins. The pdc gene from Zymomonas mobilis has been used to engineer ethanol-producing biocatalysts for use in industry. In this paper, we describe a new bacterial pdc gene from Zymobacter palmae. The pattern of codon usage for this gene appears quite similar to that for Escherichia coli genes. In E. coli recombinants, the Z. palmae PDC represented approximately 1/3 of the soluble protein. Biochemical and kinetic properties of the Z. palmae enzyme were compared to purified PDCs from three other bacteria. Of the four bacterial PDCs, the Z. palmae enzyme exhibited the highest specific activity (130 U mg of protein−1) and the lowest Km for pyruvate (0.24 mM). Differences in biochemical properties, thermal stability, and codon usage may offer unique advantages for the development of new biocatalysts for fuel ethanol production.

scholarly journals Cystathionine γ-synthase from Arabidopsis thaliana: purification and biochemical characterization of the recombinant enzyme overexpressed in Escherichia coli

1998 ◽  
Vol 331 (2) ◽  
pp. 639-648 ◽  
Author(s):  
Stéphane RAVANEL ◽  
Bertrand GAKIÈRE ◽  
Dominique JOB ◽  
Roland DOUCE
Keyword(s):  
Biochemical Characterization ◽  
Biochemical Properties ◽  
Recombinant Enzyme ◽  
Enzyme Inactivation ◽  
Replacement Reaction ◽  
Protein Precursor ◽  
Long Wavelength ◽  
Ping Pong ◽  
Km Value

Cystathionine γ-synthase catalyses the first reaction specific for methionine biosynthesis in plants, the γ-replacement of the phosphoryl substituent of O-phosphohomoserine by cysteine. A cDNA encoding cystathionine γ-synthase from Arabidopsis thalianahas been cloned and used to overexpress the enzyme in Escherichia coli.The native recombinant enzyme is a homotetramer composed of 53 kDa subunits, each being tightly associated with one molecule of pyridoxal 5´-phosphate that binds at lysine-379 of the protein precursor. The replacement reaction follows a Ping Pong mechanism with a Vmax of 33.6 units/mg and Km values of 2.5 mM and 460 µM for O-phosphohomoserine and cysteine respectively. The protective effect of O-phosphohomoserine against enzyme inactivation by propargylglycine indicated that the Kd for the substrate is approx. 1/2500 of its Km value. Thus most of these biochemical properties are similar to those previously reported for plant and bacterial cystathionine γ-synthases. However, the plant enzyme differs markedly from its enterobacterial counterparts because it catalyses a very faint γ-elimination of O-phosphohomoserine in the absence of cysteine, this process being about 1/2700 as fast as the γ-replacement reaction and approx. 1/1500 as fast as the γ-elimination catalysed by the E. colienzyme. This huge difference could be attributed to the inability of the A. thalianacystathionine γ-synthase to accumulate a long-wavelength-absorbing species that is characteristic for the efficient γ-elimination reaction catalysed by the enterobacterial enzyme.

Characterization of the Original Christmas Disease Mutation (Cysteine 206 → Serine): From Clinical Recognition to Molecular Pathogenesis

1992 ◽  
Vol 67 (01) ◽  
pp. 063-065 ◽  
Author(s):  
Sherryl A M Taylor ◽  
Jacalyn Duffin ◽  
Cherie Cameron ◽  
Jerome Teitel ◽  
Bernadette Garvey ◽  
...  
Keyword(s):  
Nucleotide Substitution ◽  
Novel Mutation ◽  
Factor Ix ◽  
Causative Mutation ◽  
Coding Region ◽  
Body Of Knowledge ◽  
Polymerase Chain ◽  
Splice Junctions

SummaryChristmas disease was first reported as a distinct clinical entity in two manuscripts published in 1952 (1, 2). The eponym associated with this disorder, is the surname of the first patient examined in detail and reported by Biggs and colleagues in a paper describing the clinical and laboratory features of seven affected individuals (3). This patient has severe factor IX coagulant deficiency (less than 0.01 units/ml) and no detectable circulating factor IX antigen (less than 0.01 units/ml). Coding sequence and splice junctions of the factor IX gene from this patient have been amplified in vitro through the polymerase chain reaction (PCR). One nucleotide substitution was identified at nucleotide 30,070 where a guanine was replaced by a cytosine. This mutation alters the amino acid encoded at position 206 in the factor IX protein from cysteine to serine. The non conservative nature of this substitution, the absence of this change in more than 200 previously sequenced factor IX genes and the fact that the remainder of the coding region of this gene was normal, all provide strong circumstantial evidence in favour of this change being the causative mutation in this patient. The molecular characterization of this novel mutation in the index case of Christmas disease, contributes to the rapidly expanding body of knowledge pertaining to Christmas disease pathogenesis.

Application of commercial test-systems to identify gram-negative facultatively anaerobic bacteria

2016 ◽  
Vol 3 (1) ◽  
pp. 43-48 ◽  
Author(s):  
V. Patyka ◽  
L. Butsenko ◽  
L. Pasichnyk
Keyword(s):  
Erwinia Amylovora ◽  
Anaerobic Bacteria ◽  
Biochemical Properties ◽  
Test System ◽  
Phytopathogenic Bacteria ◽  
Gram Negative ◽  
Test Systems ◽  
Facultatively Anaerobic ◽  
Microbiological Methods

Aim. To validate the suitability of commercial API 20E test-system (bioMerieux) for the identifi cation and characterization of facultative gram-negative phytopathogenic bacterial isolates. Methods. Conventional mi- crobiological methods, API 20E test-system (bioMerieux) according to the manufacturer’s instructions. Re- sults. The identifi cation results for Erwinia amylovora, Pectobacterium carotovorum and Pantoea agglome- rans isolates were derived from the conventional and API 20E test systems, which, were in line with the literature data for these species. The API 20E test-system showed high suitability for P. agglomerans isolates identifi cation. Although not all the species of facultatively anaerobic phytopathogenic bacteria may be identi- fi ed using API 20E test-system, its application will surely allow obtaining reliable data about their physiologi- cal and biochemical properties, valuable for identifi cation of bacteria, in the course of 24 h. Conclusions. The results of tests, obtained for investigated species while using API 20E test-system, and those of conventional microbiological methods coincided. The application of API 20E test-system (bioMerieux) ensures fast obtain- ing of important data, which may be used to identify phytopathogenic bacteria of Erwinia, Pectobacterium, Pantoea genera.

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