Inverse Single-Strand RACE: An Adapter-Independent Method of 5′RACE

Eyal, Yoram; Neumann, H.; Or, Etti; Frydman, Ahuva

doi:10.2144/99274bm04

Cited by 22 publications

(23 citation statements)

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“…A 1.2-kb fragment was obtained, subcloned to pGEM-T vector, and sequenced. In the second step we cloned the 5Ј end of the gene by an inverse single-strand 5Ј-RACE PCR method, developed by Eyal et al (39), and using three specific primers, all of which were based on sequences from the 1.2-kb fragment downstream of the TGA. The primers used in RACE were FP10A to synthesize cDNA, a forward primer FP10, and a reverse primer FP11A for PCR.…”

Section: Reinhardtii Glutathione Peroxidase Cdna Contains Anmentioning

confidence: 99%

A Selenoprotein in the Plant Kingdom

Fu¹,

Wang²,

Eyal³

et al. 2002

Journal of Biological Chemistry

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154

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Selenoproteins that contain the rare amino acid selenocysteine in their primary structure have been identified in diverse organisms such as viruses, bacteria, archea, and mammals, but so far not in yeast or plants. Among the most thoroughly investigated families of selenoenzymes are the animal glutathione peroxidases (GPXs). In the last few years, genes encoding GPX-like homologues from Chlamydomonas and higher plants have been isolated, but, unlike the animal ones, all of them have cysteine (rather than selenocysteine) residues in their catalytic site. In all organisms investigated that contain selenoproteins, selenocysteine is encoded by a UGA opal codon, which is usually a stop codon. We report here that, in Chlamydomonas reinhardtii, the cDNA-cloned sequence of a GPX homologue contains an internal TGA codon in frame to the ATG. Specific mRNA expression, protein production, and enzyme activity are selenium-dependent. Sequence analysis of the peptides produced by proteolytic digestion, performed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), confirmed the presence of a selenocysteine residue at the predicted site and suggest its location in the mitochondria. Thus, our data present the first direct proof that a UGA opal codon is decoded in the plant kingdom to incorporate selenocysteine.

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Section: Reinhardtii Glutathione Peroxidase Cdna Contains Anmentioning

confidence: 99%

A Selenoprotein in the Plant Kingdom

Fu¹,

Wang²,

Eyal³

et al. 2002

Journal of Biological Chemistry

Self Cite

154

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“…Further extension of the 5Ј sequence was performed using a SMART RACE cDNA amplification kit (Clontech), according to the manufacturer's instructions, using human heart poly(A) ϩ RNA and brain (cerebellum) poly(A) ϩ RNA (Clontech). Inverse RACE on AT6.1 RNA was performed as described (11), using ThermoScript (Invitrogen), 10% GC-Melt (Clontech), and 5% Me 2 SO, reverse transcribing with primer R1 (5Ј-GCCCCGCACGCCGCAACAGTTCG-3Ј). Circularized DNA was amplified by PCR using nested reverse primer R2 (5Ј-CAGCGCCGCCACTCGGCCATGCAAG-3Ј) and forward primer F1 (5Ј-GCGAACTGTTGCGGCGTGCG-3Ј).…”

Section: Methodsmentioning

confidence: 99%

Type XXIII Collagen, a New Transmembrane Collagen Identified in Metastatic Tumor Cells

Banyard

Bao²,

Zetter

2003

Journal of Biological Chemistry

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We have identified a transmembrane collagen, collagen XXIII, in rat prostate carcinoma cells. Differential display of mRNA expression in prostate carcinoma sublines with varying metastatic potential revealed overexpression of this transcript in the metastatic AT6.1 subline. cDNA cloning identified a 2733-bp transcript from AT6.1 RNA, encoding a protein of 532 amino acids, together with a 3067-bp human homologue, resulting in a 540-amino acid protein. Collagen XXIII is predicted to be a type II membrane protein consisting of an aminoterminal cytoplasmic domain, a transmembrane region, and three collagenous domains flanked by short noncollagenous domains. Collagen XXIII is a new member of the transmembrane collagen family, showing structural homology with the transmembrane collagens XIII and XXV. We present evidence that collagen XXIII is expressed as a ϳ75-kDa protein at the cell surface and that it can be cleaved by furin protease activity. Cleavage results in a ϳ60-kDa soluble protein that forms a multimeric complex and exhibits a low affinity interaction with heparin.The extracellular environment consists of a complex mix of matrix macromolecules together with sequestered growth factors. Each of these components can interact with cells to influence their function and behavior. Proteolytic cleavage of these molecules can release soluble growth factors and matrix fragments that also possess biological activity. The collagen superfamily represents the most abundant group of extracellular matrix macromolecules, and it is becoming clear that this family encompasses a structurally and functionally diverse group of proteins (1). Collagens can be divided into two major groups: fibrillar and nonfibrillar, with a key characteristic of both being the presence of a repeating Gly-Xaa-Yaa, where Xaa and Yaa are frequently proline and hydroxyproline, respectively. A subfamily of the nonfibrillar collagens is the transmembrane collagens. This group currently consists of three members, collagens XIII and XVII and the recently identified collagen XXV, each containing a single-pass hydrophobic transmembrane domain. Both collagens XIII and XVII show widespread distribution in epithelia (2, 3), whereas collagen XXV is specifically overexpressed in neurons (4). Functionally both collagen XIII and XVII have been co-localized in cell adhesions, collagen XVII has been co-localized in hemidesmosomes (5), collagen XIII has been co-localized in focal contacts (2), and their extracellular domains have been shown to support cell adhesion (6 -8). We report here the cloning of a new transmembrane collagen overexpressed in rat prostate adenocarcinoma cells and characterize its cellular localization, its cleavage, and the properties of its soluble ectodomain.

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“…5Ј-RACE analysis of the hexA transcript was performed using a modification of the adapter-independent method of Eyal et al (23). First, the oligonucleotide APFAS2T71R was 5Ј-end phosphorylated using T4 polynucleotide kinase (New England Biolabs).…”

Section: Methodsmentioning

confidence: 99%

Hexanoate Synthase, a Specialized Type I Fatty Acid Synthase in Aflatoxin B1 Biosynthesis

Hitchman

2001

Bioorganic Chemistry

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Inverse Single-Strand RACE: An Adapter-Independent Method of 5′RACE

Cited by 22 publications

References 0 publications

A Selenoprotein in the Plant Kingdom

A Selenoprotein in the Plant Kingdom

Type XXIII Collagen, a New Transmembrane Collagen Identified in Metastatic Tumor Cells

Hexanoate Synthase, a Specialized Type I Fatty Acid Synthase in Aflatoxin B1 Biosynthesis

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