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 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 ε-subunit of ATP synthase from bovine heart mitochondria. Complementary DNA sequence, expression in bovine tissues and evidence of homologous sequences in man and rat

1990 ◽  
Vol 265 (2) ◽  
pp. 321-326 ◽  
Author(s):  
O Viñas ◽  
S J Powell ◽  
M J Runswick ◽  
V Iacobazzi ◽  
J E Walker
Keyword(s):  
Dna Sequence ◽  
Atp Synthase ◽  
Protein Sequence ◽  
Nuclear Gene ◽  
Heart Mitochondria ◽  
Amino Acid Residues ◽  
Hybridization Probe ◽  
Related Sequence ◽  
Epsilon Subunit ◽  
Bovine Heart

The epsilon-subunit of ATP synthase from bovine heart mitochondria is assembled into the extrinsic membrane sector, F1-ATPase. The mature protein is 50 amino acid residues in length and its function is unknown. It is a nuclear gene product that is imported into the organelle. A mixture of 64 oligonucleotides 17 bases long, designed on the basis of the known protein sequence, was synthesized and used as a hybridization probe to isolate a cognate cDNA clone from a bovine library. The DNA sequence of this clone was determined, and the protein sequence of the epsilon-subunit deduced from it agrees exactly with that determined by direct sequence analysis of the protein isolated from bovine hearts. The bovine cDNA was used as a hybridization probe to examine the expression of the epsilon-subunit in various bovine tissues. mRNAs related to the cDNA are found in all of these tissues, and no evidence was obtained of the presence of mRNAs for the epsilon-subunit with similar coding sequences and dissimilar 3′ non-coding regions. By hybridization experiments with digests of DNA from cow, man and rat it has been shown that sequences related to the bovine cDNA are present in the genomes of all three species. More than one related sequence was detected in all cases, indicating the presence in all three genomes of more than one gene and/or pseudogenes.

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 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.

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

Abstract 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.

scholarly journals NADH:ubiquinone oxidoreductase from bovine heart mitochondria. cDNA sequences of the import precursors of the nuclear-encoded 39 kDa and 42 kDa subunits

1991 ◽  
Vol 278 (3) ◽  
pp. 821-829 ◽  
Author(s):  
I M Fearnley ◽  
M Finel ◽  
J M Skehel ◽  
J E Walker
Keyword(s):  
Amino Acid ◽  
Protein Sequence ◽  
Complex I ◽  
Mitochondrial Gene ◽  
Amino Acid Sequences ◽  
Heart Mitochondria ◽  
Blue Dye ◽  
Nadh:Ubiquinone Oxidoreductase ◽  
Cdna Clones ◽  
Bovine Heart

The 39 kDa and 42 kDa subunits of NADH:ubiquinone oxidoreductase from bovine heart mitochondria are nuclear-coded components of the hydrophobic protein fraction of the enzyme. Their amino acid sequences have been deduced from the sequences of overlapping cDNA clones. These clones were amplified from total bovine heart cDNA by means of the polymerase chain reaction, with the use of complex mixtures of oligonucleotide primers based upon fragments of protein sequence determined at the N-terminals of the proteins and at internal sites. The protein sequences of the 39 kDa and 42 kDa subunits are 345 and 320 amino acid residues long respectively, and their calculated molecular masses are 39,115 Da and 36,693 Da. Both proteins are predominantly hydrophilic, but each contains one or two hydrophobic segments that could possibly be folded into transmembrane alpha-helices. The bovine 39 kDa protein sequence is related to that of a 40 kDa subunit from complex I from Neurospora crassa mitochondria; otherwise, it is not related significantly to any known sequence, including redox proteins and two polypeptides involved in import of proteins into mitochondria, known as the mitochondrial processing peptidase and the processing-enhancing protein. Therefore the functions of the 39 kDa and 42 kDa subunits of complex I are unknown. The mitochondrial gene product, ND4, a hydrophobic component of complex I with an apparent molecular mass of about 39 kDa, has been identified in preparations of the enzyme. This subunit stains faintly with Coomassie Blue dye, and in many gel systems it is not resolved from the nuclearcoded 36 kDa subunit.

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