scholarly journals Sheep 6-phosphogluconate dehydrogenase. Revised protein sequence based upon the sequences of cDNA clones obtained with the polymerase chain reaction

1992 ◽  
Vol 288 (3) ◽  
pp. 1061-1067 ◽  
Author(s):  
D O Somers ◽  
S M Medd ◽  
J E Walker ◽  
M J Adams
Keyword(s):  
Polymerase Chain Reaction ◽  
Protein Sequence ◽  
Direct Analysis ◽  
Pentose Phosphate ◽  
Cdna Clones ◽  
Mature Protein ◽  
Chain Reaction ◽  
Phosphogluconate Dehydrogenase ◽  
Sheep Liver ◽  
Polymerase Chain

Sheep liver 6-phosphogluconate dehydrogenase (6-PGDH) is an enzyme of the pentose phosphate pathway. Evidence has appeared which suggests that the 6-PGDH protein sequence determined previously by direct analysis of the protein isolated from ovine liver is incorrect. Determining the enzyme's DNA sequence was considered to be the best way of solving the problem. In the first instance, a degenerate forward and a degenerate reverse primer were designed on the basis of the known protein sequence, and a partial-length cDNA clone was isolated from total sheep liver cDNA using the polymerase chain reaction. The clone encoded the expected part of the protein sequence. The clone was unsuccessfully used as a prime-cut probe to screen a sheep liver library and a bovine heart library. As a result, the polymerase chain reaction was utilized again to successfully generate a family of overlapping cDNA clones encoding a mature protein of 482 amino acids. The mature protein sequence encoded by the cDNA differs significantly from the sequence derived by direct analysis of the protein, but on closer examination the fundamental difference is caused by the incorrect placement of three enzyme fragments obtained by cyanogen bromide cleavage during the direct sequence analysis of the protein. Placing the fragments in the correct order results in the two sequences being virtually identical except for some minor amino acid changes between the amide and acid forms, and a small number of deletions and insertions.

scholarly journals The δ-subunit of ATP synthase from bovine heart mitochondria. Complementary DNA sequence of its import precursor cloned with the aid of the polymerase chain reaction

1990 ◽  
Vol 266 (2) ◽  
pp. 421-426 ◽  
Author(s):  
M J Runswick ◽  
S M Medd ◽  
J E Walker
Keyword(s):  
Polymerase Chain Reaction ◽  
Atp Synthase ◽  
Protein Sequence ◽  
Heart Mitochondria ◽  
Cdna Clones ◽  
Mature Protein ◽  
Chain Reaction ◽  
Bovine Heart ◽  
Polymerase Chain ◽  
Delta Subunit

The delta-subunit of ATP synthase from bovine heart mitochondria is part of the extrinsic membrane domain, F1-ATPase. The mature protein is 146 amino acids in length and its function is obscure. It is encoded by a nuclear gene and is imported into the organelle. Two mixtures of oligonucleotides 17 bases long, designed on the basis of the known protein sequence, have been synthesized and employed as primers on bovine cDNA in the polymerase chain reaction. By this means a segment of bovine cDNA encoding part of the delta-subunit has been amplified, and this DNA segment has been employed to identify related cDNA clones in a library. These clones encode the mitochondrial import precursor of the delta-subunit; the protein sequence of the mature protein deduced from it is exactly the same as that determined earlier by direct sequence analysis. The clones have also been used to show that both the bovine and human genomes seem to contain a single gene for the delta-subunit.

scholarly journals Identification of novel protein tyrosine phosphatases of hematopoietic cells by polymerase chain reaction amplification

Blood ◽  
1991 ◽  
Vol 78 (9) ◽  
pp. 2222-2228 ◽  
Author(s):  
T Yi ◽  
JL Cleveland ◽  
JN Ihle
Keyword(s):  
Polymerase Chain Reaction ◽  
Polymerase Chain Reaction Amplification ◽  
Hematopoietic Cells ◽  
Protein Tyrosine Phosphatases ◽  
Cdna Clones ◽  
Tyrosine Phosphatases ◽  
Chain Reaction ◽  
Protein Tyrosine ◽  
Cdna Sequences ◽  
Polymerase Chain

Polymerase chain reaction (PCR) conditions were used to amplify cDNAs that encode putative protein tyrosine phosphatases (PTPs) from a murine interleukin-3-dependent myeloid cell line. Primers for the reactions were based on conserved sequences of the catalytic domain that are shared among all known PTPs. Sequencing of 100 PCR-amplified cDNA clones identified seven different cDNA sequences. Two of these sequences were identical to the murine PTP genes Ly5/CD45/LCA and LRP/R- PTP-alpha. Two of the cDNA sequences were 95% identical to human PTP epsilon (HPTP epsilon) and rat brain PTP (PTP1B), respectively, and are likely to represent their murine homologs. Three of the cDNA sequences encoded novel potential PTPs. One of the putative PTPs was ubiquitously expressed while a second was predominantly expressed in brain, kidney, and liver and at much lower levels in a variety of other cell tkpes and tissues. The third novel putative phosphatase was expressed primarily in hematopoietic cells and tissues in a pattern that was comparable with Ly5/CD45/LCA. Further characterization of these novel PTPs will provide insights into the growth regulation of hematopoietic cells.

Cloning and sequencing of a cDNA encoding an ovine oestrus-associated oviducal protein

1996 ◽  
Vol 8 (2) ◽  
pp. 305 ◽  
Author(s):  
JT Marshall ◽  
CD Nancarrow ◽  
AG Brownlee
Keyword(s):  
Amino Acids ◽  
Polymerase Chain Reaction ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Northern Blot ◽  
Mature Protein ◽  
Chain Reaction ◽  
Cloning And Sequencing ◽  
Glycosylation Sites ◽  
Polymerase Chain

Ovine oestrus-associated oviducal glycoprotein (oEGP) is synthesized and secreted specifically by the ampullary region of the ovine oviduct during the peri-ovulatory stages of the oestrous cycle. A cDNA that encodes oEGP was isolated and sequenced. Isolation of oEGP was achieved using the polymerase chain reaction (PCR) with primers based on a bovine oestrus-associated oviducal glycoprotein cDNA (bOGP) sequence. A 1599-bp cDNA encodes, in part, a deduced 519-amino acid sequence of mature protein which carries two potential N-linked glycosylation sites. The deduced amino acid sequence is more than 95% identical to that of bOGP and more than 74% identical to the first 491 amino acids of human oestrogen-dependent oviducal glycoprotein (hOGP). Northern blot hybridizations of RNA from several sheep tissues detected mRNA (2.4 kb) only in an ampulla oviduct sample.

scholarly journals A Colorimetric Assay to Quantify Dehydrogenase Activity in Crude Cell Lysates

2002 ◽  
Vol 7 (2) ◽  
pp. 135-140 ◽  
Author(s):  
Kimberly M. Mayer ◽  
Frances H. Arnold
Keyword(s):  
Polymerase Chain Reaction ◽  
Dehydrogenase Activity ◽  
Colorimetric Assay ◽  
Bacterial Cells ◽  
Phenazine Methosulfate ◽  
Chain Reaction ◽  
Phosphogluconate Dehydrogenase ◽  
Polymerase Chain ◽  
Kinetics Of ◽  
Purple Formazan

Nitroblue tetrazolium (NBT) in the presence of phenazine methosulfate (PMS) reacts with the NADPH produced by dehydrogenases to produce an insoluble blue-purple formazan. Endpoint assays taking advantage of this reaction have been successfully used to detect the activity of several dehydrogenases. Here we present a version of this assay suitable for determining the kinetics of 6-phosphogluconate dehydrogenase catalysis in crude lysates of bacterial cells prepared in 96-well plates. Using the assay to screen a small library of variant 6-phosphogluconate dehydrogenases generated by error-prone polymerase chain reaction, we were able to identify three variants with improved activity and thermostability over the parent enzyme. These enzymes were partially purified and shown to be expressed at higher levels than the parent (leading to the increase in activity), and all three variants were indeed more thermostable than the parent (temperature midpoints 4-7°C higher) after purification. Thus the NBT-PMS assay appears suitable for screening libraries of variant dehydrogenases.

scholarly journals Induction of a Grapevine Germin-Like Protein (VvGLP3) Gene Is Closely Linked to the Site of Erysiphe necator Infection: A Possible Role in Defense?

2007 ◽  
Vol 20 (9) ◽  
pp. 1112-1125 ◽  
Author(s):  
Dale Godfrey ◽  
Amanda J. Able ◽  
Ian B. Dry
Keyword(s):  
Polymerase Chain Reaction ◽  
Cell Wall ◽  
Recombinant Protein ◽  
Plasmopara Viticola ◽  
Cdna Clones ◽  
Chain Reaction ◽  
Erysiphe Necator ◽  
Fusion Construct ◽  
Polymerase Chain ◽  
Insight Into

Germin-like proteins (GLP) have various proposed roles in plant development and defense. Seven novel GLP cDNA clones were isolated from grapevine (Vitis vinifera cv. Chardonnay). Reverse transcriptase-polymerase chain reaction expression analysis revealed that the VvGLP genes exhibit diverse and highly specific patterns of expression in response to a variety of abiotic and biotic treatments, including challenge by Erysiphe necator, Plasmopara viticola, and Botrytis cinerea, suggesting a diversity of roles for each of the GLP family members. Significantly, one of the grapevine GLP genes, VvGLP3, is induced specifically by E. necator infection and expression is closely linked to the site of infection. Subcellular localization of VvGLP3 determined by transient expression of a VvGLP3:GFP fusion construct in onion cells indicated that the recombinant protein was targeted to the cell wall. Recombinant VvGLP3 was successfully expressed in Arabidopsis thaliana and the partially purified recombinant protein was demonstrated to have superoxide dismutase activity. This data has provided an insight into the diverse nature of the GLP family in grapevine and suggests that VvGLP3 may be involved in the defense response against E. necator.

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