cDNA cloning

Cold acclimation in plants is a polygenic phenomenon involving increased expression of several genes. The gene products participate either directly or indirectly towards increasing cold tolerance. Evidence of proteins having a direct effect on cold tolerance is emerging but limited. With isolated protoplasts from warm-grown kale (Brassica oleracea) as a model system, we tested protein fractions from winter bittersweet nightshade, Solanum dulcamara, stems for the presence of proteins that have a cryoprotective effect. Purification of one such fraction resulted in isolation of a 25 kDa protein. N-terminal Edman degradation amino acid sequence analysis showed that it has high homology to osmotin and osmotin-like proteins. When added to warm-grown protoplasts, it increased the cryosurvival of frozen-thawed protoplasts by 24% over untreated or BSA-treated controls at -8 degrees C. A cDNA library which was made in November from stems and leaves of S. dulcamara was successfully screened fo...

Both helper dependent expression systems, based on two components, and single genomes constructed by targeted recombination, or by using infectious cDNA clones, have been developed. The sequences that regulate transcription have been characterized mainly ...

The molecular pathology of many protein misfolding, toxic gain-of-function diseases, such as amyotrophic lateral sclerosis (ALS), is not well understood. Although protein misfolding and aggregation are common themes in these diseases, efforts to identify cellular factors that regulate this process in an unbiased fashion and on a global scale have been lacking. Using an adapted version of an extant β-gal-based protein solubility assay, an expression screen for cellular modulators of solubility of an ALS-causing mutant SOD1 was carried out in mammalian cells. Following fluorescence-activated cell sorting enrichment of a mouse spinal cord cDNA library for gene products that increased SOD1 solubility, high-throughput screening of the cDNA pools from this enriched fraction was employed to identify pools containing relevant modulators. Positive pools, containing approximately 10 cDNA clones each, were diluted and rescreened iteratively until individual clones that improved SOD1 folding/so...

Both helper dependent expression systems, based on two components, and single genomes constructed by targeted recombination, or by using infectious cDNA clones, have been developed. The sequences that regulate transcription have been characterized mainly ...

Many plant autocatalytic glycosyltransferases are implicated in plant polysaccharide biosynthesis. Cloning of cDNAs encoding potato (Solanum tuberosum L.) UDP-Glc:protein transglucosylase (UPTG, EC 2.4.1.112) and expression of the cDNA clone E11 in Escherichia coli have been previously reported. Here, we studied the functional expression of a second cDNA of the enzyme (E2 clone). Northern blots analysis, with specific cDNA probes for the two UPTG isoforms, showed a differential expression pattern of mRNA levels in different potato tissues. Moreover, both UPTG recombinant enzymes showed different kinetic parameters. The recombinant protein encoded by E2 clone has an apparent Imax for UDP-Xyl and UDP-Gal, significantly higher than for UDP-Glc. The Km values for UDP-Glc were 0.45-0.71 microM and the values for UDP-Xyl and UDP-Gal were slightly higher than that of the UDP-Glc (1.2-2.71 microM) for both UPTG recombinant enzymes. The present study revealed further evidence for the propose...

Aspartic proteinases (EC 3.4.23) are widely distributed in the plant kingdom, and a number of cDNAs have been isolated from different plants. Here we report the isolation an expression analysis of a cDNA from Solanum tuberosum L. (cv. Pampeana) named StAsp. The StAsp cDNA ...

A gene from Magnaporthe grisea was cloned using a cDNA clone of the Colletotrichum gloeosporioides cutinase gene as a heterologous probe; the nucleotide sequence of a 2 kb DNA segment containing the gene has been determined. DNA hybridization analysis shows that the M. grisea genome contains only one copy of this gene. The predicted polypeptide contains 228 amino acids and is homologous to the three previously characterized cutinases, showing 74% amino acid similarity to the cutinase of C. gloeosporioides. Comparison with previously determined cutinase sequences suggests that the gene contains two introns, 115 and 147 bp in length. The gene is expressed when cutin is the sole carbon source but not when the carbon source is cutin and glucose together or glucose alone. Levels of intracellular and extracellular cutinase activity increase in response to growth in the presence of cutin. The activity level is higher in a transformant containing multiple copies of the cloned gene than in t...

A cDNA clone encoding trypsin (AG-T) was isolated from the pyloric ceca of cold-adapted fish, arabesque greenling (Pleurogrammus azonus). The cDNA was composed of 892 bp with an open reading frame of 729 bp at nucleotide positions 25–753. Similar to all the known trypsin, the AG-T seemed to be synthesized as preproenzyme that contains a hydrophobic signal peptide, an activation pentapeptide and

Pathogenesis-related proteins (PR-proteins) are induced in plants in response to attack by microbial or insect pests. They have been classified into several groups (PR-1 through PR-14 at present) based on their amino acid sequences and biochemical functions. Many of these proteins that have been purified from infected plants or seed extracts possess antifungal or insecticidal activity. Genes and cDNA clones

We describe the use of a method called differential expression using customized amplification library (DECAL) to study the global changes in gene expression in iron-deficient versus iron-reconstituting cells of Synechocystis sp. strain PCC 6803. We identified a number of genes, such as isiA, idiA,psbA, cpcG, and slr0374, whose expression either increased or decreased in response to iron availability. Further analysis led to the identification of additional genes related to those identified by DECAL (e.g., psbC,psbO, psaA, apcABC,cpcBAC1C2D, and nblA) that were differentially regulated by iron availability. Expression of cpcG,psbC, psbO, psaA,apcABC, and cpcBAC1C2D increased, whereas that of isiA, idiA, nblA,psbA, and slr0374 decreased, in iron-reconstituting cells. S1 nuclease protection studies showed that increased transcript levels of psbA in iron-deficient cells was due to the increased expression of both psbA2 andpsbA3 genes, although the steady-state level ofpsbA2 remained hig...

A cDNA expression library of Entamoeba histolytica was screened with antiserum to native amoebic collagen binding proteins (CBPs), and two clones C13 and C7 which partially encode for the 30 kDa CBP were obtained. The sequenced clones were 90% homologous. C7 had a 69 ...

A rapidly growing area of genome research is the generation of expressed sequence tags (ESTs) in which large numbers of randomly selected cDNA clones are partially sequenced. The collection of ESTs reflects the level and complexity of gene expression in the sampled ...