Isolation and characterization of a vinculin cDNA from chick-embryo fibroblasts

A chick-embryo fibroblast lambda gt11 cDNA library was screened with affinity-purified antibodies to chick gizzard vinculin. One recombinant was purified to homogeneity and the fusion protein expressed in Escherichia coli strain C600. The fusion protein was unstable, but polypeptides that reacted with vinculin antibodies, but not non-immune immunoglobulin, were detected by Western blotting. The recombinant contained a single EcoRI fragment of 2891 bp with a single open reading frame. The deduced protein sequence could be aligned with that of six CNBr-cleavage peptides and two tryptic peptides derived from chicken gizzard vinculin. AUG-247 has tentatively been identified as the initiation codon, as it is contained within the consensus sequence for initiation sites of higher eukaryotes. The cDNA lacks 3′ sequence and encodes 74% of the vinculin sequence, presuming the molecular mass of vinculin to be 130,000 Da. Analysis of the deduced sequence showed no homologies with other protein sequences, but it does display a triple internal repeat of 112 amino acid residues covering residues 259-589. The sequences surrounding the seven tyrosine residues in the available sequence were aligned with the tyrosine autophosphorylation consensus sequence found in protein tyrosine kinases. Tyr-822 showed a good match to this consensus, and may represent one of the two major sites of tyrosine phosphorylation by pp60v-sre. Northern blots showed that the 2.89 kb vinculin cDNA hybridized to one size of mRNA (approx. 7 kb) in chick-embryo fibroblasts, chick smooth muscle and chick skeletal muscle. Southern blots revealed multiple hybridizing bands in genomic DNA.

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The isolation and characterization of the plasma membrane from chick embryo fibroblasts

Biochimica et Biophysica Acta (BBA) - Biomembranes ◽

10.1016/0005-2736(70)90001-5 ◽

1970 ◽

Vol 196 (2) ◽

pp. 125-140 ◽

Cited By ~ 75

Author(s):

James F. Perdue ◽

Judith Sneider

Keyword(s):

Plasma Membrane ◽

Chick Embryo ◽

Isolation And Characterization ◽

Embryo Fibroblasts

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Isolation and characterization of a gene encoding farnesyl diphosphate synthase from \(\textit{Panax vietnamensis}\) Ha et Grushv

ACADEMIA JOURNAL OF BIOLOGY ◽

10.15625/2615-9023/16356 ◽

2021 ◽

Vol 43 (4) ◽

pp. 119-128

Author(s):

Nguyen Van Giang ◽

Luu Han Ly ◽

Pham Le Bich Hang ◽

Le Thi Thu Hien

Keyword(s):

Farnesyl Diphosphate ◽

Farnesyl Diphosphate Synthase ◽

Amino Acid Residues ◽

Gene Encoding ◽

Reading Frame ◽

Ginsenoside Biosynthesis ◽

Isolation And Characterization ◽

Key Enzyme ◽

As Stress ◽

Root Tissues

Panax vietnamensis Ha et Grushv. is a species of the genus Panax native to Central Vietnam, containing a family of triterpene saponins named ginsenosides. This group of biomolecules possesses valuable therapeutic properties against cancer, hepatitis, diabetes, inflammation as well as stress and anxiety. Farnesyl diphosphate synthase (FPS) is a key enzyme participating in the ginsenoside biosynthesis pathway. In this study, a FPS gene from P. vietnamensis (PvFPS) was isolated and characterized. The PvFPS cDNA contained an open reading frame of 1032 bp, encoding a polypeptide chain of 342 amino acid residues. Nucleotide sequence comparison showed that FPS was highly conserved among most species, with two Aspartate-rich motifs responsible for product chain length determination strongly sustained. PvFPS was closely related to those of the same genera and order and differed from those from other kingdoms. PvFPS expression was detected at a greater level in root tissues than in leaves in all ages. Our findings provided information concerning the properties of a crucial gene in the ginsenoside biosynthesis, thus enhancing our understanding of this important pathway.

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Poly-3-Hydroxybutyrate Degradation in Rhizobium (Sinorhizobium) meliloti: Isolation and Characterization of a Gene Encoding 3-Hydroxybutyrate Dehydrogenase

Journal of Bacteriology ◽

10.1128/jb.181.3.849-857.1999 ◽

1999 ◽

Vol 181 (3) ◽

pp. 849-857 ◽

Cited By ~ 50

Author(s):

P. Aneja ◽

T. C. Charles

Keyword(s):

Carbon Source ◽

Sole Carbon Source ◽

Sinorhizobium Meliloti ◽

Transcriptional Start Site ◽

Consensus Sequence ◽

Start Codon ◽

Start Site ◽

Gene Encoding ◽

Reading Frame ◽

Isolation And Characterization

ABSTRACT We have cloned and sequenced the 3-hydroxybutyrate dehydrogenase-encoding gene (bdhA) from Rhizobium (Sinorhizobium) meliloti. The gene has an open reading frame of 777 bp that encodes a polypeptide of 258 amino acid residues (molecular weight 27,177, pI 6.07). The R. meliloti Bdh protein exhibits features common to members of the short-chain alcohol dehydrogenase superfamily. bdhA is the first gene transcribed in an operon that also includes xdhA, encoding xanthine oxidase/dehydrogenase. Transcriptional start site analysis by primer extension identified two transcription starts. S1, a minor start site, was located 46 to 47 nucleotides upstream of the predicted ATG start codon, while S2, the major start site, was mapped 148 nucleotides from the start codon. Analysis of the sequence immediately upstream of either S1 or S2 failed to reveal the presence of any known consensus promoter sequences. Although a ς54 consensus sequence was identified in the region between S1 and S2, a corresponding transcript was not detected, and a rpoN mutant of R. meliloti was able to utilize 3-hydroxybutyrate as a sole carbon source. The R. meliloti bdhA gene is able to confer uponEscherichia coli the ability to utilize 3-hydroxybutyrate as a sole carbon source. An R. meliloti bdhA mutant accumulates poly-3-hydroxybutyrate to the same extent as the wild type and shows no symbiotic defects. Studies with a strain carrying alacZ transcriptional fusion to bdhAdemonstrated that gene expression is growth phase associated.

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Molecular analysis of FMRFamide- and FMRFamide-related peptides (FaRPS) in the cuttlefish Sepia officinalis.

Journal of Experimental Biology ◽

10.1242/jeb.200.10.1483 ◽

1997 ◽

Vol 200 (10) ◽

pp. 1483-1489 ◽

Cited By ~ 4

Author(s):

P K Loi ◽

N Tublitz

Keyword(s):

Basic Amino Acid ◽

Sepia Officinalis ◽

Cleavage Sites ◽

Amino Acid Residues ◽

Base Pairs ◽

Reading Frame ◽

Isolation And Characterization ◽

The Brain

The display of complex color patterns of the cuttlefish Sepia officinalis is under the regulation of the FMRFamide-related peptide (FaRP) family, but their exact identities are unknown. We report the isolation and characterization of a full-length FaRP cDNA from the brain of S. officinalis. This cDNA is 1850 base pairs long, including an open reading frame of 996 base pairs. The cDNA encodes a precursor protein containing four FaRPs: ALSGDAFLRF, FIRF, FLRF and FMRF. Each propeptide has a C-terminal glycine residue that is presumably converted post-translationally to an amide. Every FaRP propeptide is also flanked by basic amino acid residues at the amino and carboxy termini, indicative of putative cleavage sites during post-translational processing. Each of the four FaRPs encoded by this cDNA causes chromatophore expansion when assayed in an in vitro chromatophore bioassay. Thus, it is likely that one or more of the FaRPs identified in this study are involved in controlling chromatophore activity in cuttlefish.

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Isolation and characterization of a vinculin cDNA from chick embryo fibroblasts

Biochemical Society Transactions ◽

10.1042/bst0150794 ◽

1987 ◽

Vol 15 (5) ◽

pp. 794-796

Author(s):

PETER JONES ◽

GLYN J. PRICE ◽

MATTHEW D. DAVISON ◽

DAVID R. CRITCHLEY

Keyword(s):

Chick Embryo ◽

Isolation And Characterization ◽

Embryo Fibroblasts

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Isolation and characterization of cDNA for human 120 kDa mitochondrial 2,4-dienoyl-coenzyme A reductase

Biochemical Journal ◽

10.1042/bj3040787 ◽

1994 ◽

Vol 304 (3) ◽

pp. 787-792 ◽

Cited By ~ 26

Author(s):

K T Koivuranta ◽

E H Hakkola ◽

J K Hiltunen

Keyword(s):

Amino Acid ◽

Amino Acid Sequences ◽

Cdna Clones ◽

Human Tissues ◽

Amino Acid Residues ◽

Beta Oxidation ◽

Reading Frame ◽

Isolation And Characterization ◽

The Difference ◽

Coa Reductase

2,4-Dienoyl-CoA reductase (EC 1.3.1.34) participates in beta-oxidation of (poly)unsaturated enoyl-CoAs and it appears in mammalian mitochondria as two isoforms with molecular masses of 120 and 60 kDa [Hakkola and Hiltunen (1993) Eur. J. Biochem. 215, 199-204]. The 120 kDa isomer is a homotetrameric enzyme, and here we report cDNA cloning of its subunit from human. cDNA clones were isolated by reverse transcriptase-PCR from a fibrosarcoma cell line and by screening from a human liver lambda gt11 cDNA library. The 1128 bp clone contained an open reading frame of 1008 bp encoding a polypeptide of 335 amino acid residues with a predicted molecular mass of 36066 Da. This polypeptide represents the immature monomer of the 120 kDa enzyme, and it contains a predicted N-terminal mitochondrial targeting signal. The amino acid (nucleotide) sequence of human 2,4-dienoyl-CoA reductase shows 82.7% (81.7%) similarity (identity) to the corresponding sequence from the rat. Northern-blot analysis gave a single mRNA species of 1.2 kb in several human tissues, the amounts present in the tissues tested ranking as follows: heart approximately liver approximately pancreas > kidney >> skeletal muscle approximately lung. Immunoblotting of human and rat liver samples with an antibody to the subunit of the rat 120 kDa isoform indicates that the mature human enzyme is larger than its counterpart in the rat. The comparison of amino acid sequences for rat and human enzymes proposes that the difference in the size is 10 amino acid residues. The results show that the rat and human reductases are similar in many characteristics and that the reductase is expressed in human tissues capable of beta-oxidation of fatty acids.

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