Complementary DNA

About: Complementary DNA is a research topic. Over the lifetime, 55301 publications have been published within this topic receiving 2752650 citations. The topic is also known as: cDNA & DNA, Complementary.

Solution-phase library screening for the identification of rare clones: isolation of an alpha 1D-adrenergic receptor cDNA.

Abstract: alpha 1-Adrenergic receptor (alpha 1-AR) subtypes (alpha 1A and alpha 1B) play a critical role in vascular smooth muscle contraction and circulatory homeostasis. Transcripts for these guanine nucleotide-binding protein-coupled receptors are extremely low in abundance, however, and isolation of their cDNAs is difficult. We have developed a novel technique for identifying rare clones in a cDNA library, which has been used successfully to isolate a cDNA clone encoding an alpha 1D-AR. A 564-bp polymerase chain reaction product encoding a region between the third and sixth transmembrane domains of the alpha 1D-AR was first generated using rat brain mRNA as template and highly degenerate primers. The primers corresponded to those domains but contained mismatches to the alpha 1B-AR sequences. A 3-kb transcript was identified with this polymerase chain reaction probe, by Northern analysis of rat hippocampus. However, traditional plaque hybridization failed to identify a cDNA in a rat hippocampus lambda gt10 library. By solution-phase screening of virtually the entire library, a cDNA containing a 3-kb insert was identified, amplified, and purified. This insert encodes a 560-amino acid protein corresponding to the topology of guanine nucleotide-binding protein-coupled receptors. This receptor has approximately 71% amino acid identity, in the transmembrane regions, to the hamster and rat alpha 1B-ARs. Characterization of the receptor expressed in COS-7 cells, by ligand binding and photoaffinity labeling, revealed some of the characteristics of an alpha 1A-AR. However, unlike alpha 1A-ARs characterized previously in membrane preparations or in solubilized partially purified preparations, the expressed receptor could be extensively inactivated by chlorethylclonidine. In addition, it displays ligand-binding properties that are not consistent with an alpha 1A-AR. This indicates that the cDNA clone that we have isolated encodes a novel alpha 1-AR subtype, which we classify as the alpha 1D-AR.

The plant hormone abscisic acid mediates the drought-induced expression but not the seed-specific expression of rd22, a gene responsive to dehydration stress in Arabidopsis thaliana

Abstract: Nine cDNA clones, corresponding to genes that are responsive to dehydration (named RD), have been isolated from Arabidopsis thaliana. The sequence of a putative protein encoded by one of the RD cDNA clones, RD22, exhibits considerable homology to an unidentified seed protein (USP) of Vicia faba. Northern analysis showed that RD22 mRNA is induced by salt stress as well as by water deficit but not by cold or heat stress. RD22 mRNA appeared after the application of abscisic acid (ABA), an indication that transcription of RD22 mRNA is induced by endogenous ABA, the production of which is triggered by drought and salt stress. The induction of RD22 mRNA by ABA was inhibited by cycloheximide. Thus, it appears that protein synthesis is required for the induction of this mRNA by ABA. By contrast, protein synthesis was not required for the ABA-responsive induction RD29 mRNA, which corresponds to another dehydration-responsive gene of A. thaliana. These results suggest that there are at least two mechansisms for the induction of dehydration-responsive genes by ABA. RD22 mRNA was also expressed during the early and middle stages of seed development, showing a pattern of expression similar to that of USP. The seed-specific expression of RD22 seems not to be regulated by ABA. Structural analysis of the RD22 genomic clone revealed that the structural gene (designated rd22) contains three introns, and only a single copy of the gene is present in the A. thaliana genome, while the gene for USP from V. faba is actually a family of genes with 10 to 20 members. The site of initiation of transcription was determined by primer extension. Possible cis-acting elements involved in the expression of rd22 are discussed.

Structural analysis of the sequence coding for an inducible 26-kDa protein in human fibroblasts.

Abstract: When human fibroblast cells were stimulated with poly(I) X poly(C) in the presence of cycloheximide for the production of interferon-beta (IFN-beta), a 26-kDa protein could be immunoprecipitated by antiserum raised against partially purified human IFN-beta [Content, J., De Wit, L., Pierard, D., Derynck, R., De Clercq, E. & Fiers, W. (1982) Proc. Natl Acad. Sci. USA 79, 2768-2772]. In our hands this 26-kDa protein showed no antiviral activity. Other investigators have, however, reported the presence in the same conditions of a second type of IFN, a so-called beta 2 species [Weissenbach, J., Chernajovsky, Y., Zeevi, M., Shulman, L., Soreq, H., Nir, U., Wallach, D., Perricaudet, M., Tiollais, P. & Revel, M. (1980) Proc. Natl Acad. Sci. USA 77, 7152-7156] of which the mRNA structure and protein characteristics strongly suggests identity with the 26-kDa product. In this paper we describe the nucleotide sequence of the 26-kDa cDNA and part of the corresponding genomic clone. The cDNA clones were isolated from a library made with mRNA from induced human fibroblasts. As, however, the information thus obtained was still incomplete, genomic clones were isolated from a total human DNA library. In this way, the entire region coding for the 26-kDa protein was established, as well as the neighbouring sequences including the inducible promoter area. From the deduced polypeptide sequence a number of characteristics of the 26-kDa protein can be explained. It turns out that the 26-kDa protein gene and the so-called 'IFN-beta 2' gene are identical. However, extensive homology searches indicate that the 26-kDa protein does not show statistically significant sequence homology with any known interferon species. Hence, the question of whether the 26-kDa product represents a novel IFN species remains open.

Guar Seed ß-Mannan Synthase Is a Member of the Cellulose Synthase Super Gene Family

Abstract: Genes for the enzymes that make plant cell wall hemicellulosic polysaccharides remain to be identified. We report here the isolation of a complementary DNA (cDNA) clone encoding one such enzyme, mannan synthase (ManS), that makes the beta-1, 4-mannan backbone of galactomannan, a hemicellulosic storage polysaccharide in guar seed endosperm walls. The soybean somatic embryos expressing ManS cDNA contained high levels of ManS activities that localized to Golgi. Phylogenetically, ManS is closest to group A of the cellulose synthase-like (Csl) sequences from Arabidopsis and rice. Our results provide the biochemical proof for the involvement of the Csl genes in beta-glycan formation in plants.