Topic
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.
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TL;DR: Lisitsyn et al. as discussed by the authors adapted representational difference analysis (RDA) for use with cDNA, which was found to be extremely sensitive, reproducible, and predominantly lacked false positives.
Abstract: Detection of differentially regulated genes has been severely hampered by technical limitations. In an effort to overcome these problems, the PCR-coupled subtractive process of representational difference analysis (RDA) [Lisitsyn, N. et al. (1993) Science 259, 946-951] has been adapted for use with cDNA. In a model system, RAG-1 and RAG-2, the genes responsible for activating V(D)J recombination, were identified in a genomic transfectant by cDNA RDA in a small fraction of the time taken by conventional means. The system was also modified to eliminate expected difference products to facilitate the identification of novel genes. Additional alterations to the conditions allowed isolation of differentially expressed fragments. Several caffeine up-regulated clones were obtained from the pre-B cell line 1-8, including IGF-1B, and a predicted homologue of the natural killer cell antigen, NKR-P1. The approach was found to be fast, extremely sensitive, reproducible, and predominantly lacked false positives. cDNA RDA has the capacity and adaptability to be applied to a wide range of biological problems, including the study of single gene disorders, characterization of mutant and complemented cell types, developmental or post-event expression time courses, and examination of pathogen-host interactions.
947 citations
TL;DR: A plasmid vector for cloning cDNAs in Escherichia coli is described; the same vector also promotes expression of the cDNA segment in mammalian cells, and it is confirmed that the alpha-globin transcript produced in transfected cells is initiated correctly, spliced at either of the two introns, and polyadenylated either at the site coded in the c DNA segment or at the distal SV40polyadenylation signal.
Abstract: This paper describes a plasmid vector for cloning cDNAs in Escherichia coli; the same vector also promotes expression of the cDNA segment in mammalian cells. Simian virus 40 (SV40)-derived DNA segments are arrayed in the pcD vector to permit transcription, splicing, and polyadenylation of the cloned cDNA segment. A DNA fragment containing both the SV40 early region promoter and two introns normally used to splice the virus 16S and 19S late mRNAs is placed upstream of the cDNA cloning site to ensure transcription and splicing of the cDNA transcripts. An SV40 late region polyadenylation sequence occurs downstream of the cDNA cloning site, so that the cDNA transcript acquires a polyadenylated 3' end. By using pcD-alpha-globin cDNA as a model, we confirmed that the alpha-globin transcript produced in transfected cells is initiated correctly, spliced at either of the two introns, and polyadenylated either at the site coded in the cDNA segment or at the distal SV40 polyadenylation signal. A cDNA clone library constructed with mRNA from SV40-transformed human fibroblasts and this vector (about 1.4 X 10(6) clones) yielded full-length cDNA clones that express hypoxanthine-guanine phosphoribosyltransferase (Jolly et al., Proc. Natl. Acad. Sci. U.S.A., in press).
944 citations
TL;DR: A cDNA clone encoding CD28 is isolated by a simple and highly efficient cloning strategy based on transient expression in COS cells based on the use of an efficient method to prepare large plasmid cDNA libraries.
Abstract: CD28 (Tp44) is a human T-cell-specific homodimer surface protein that may participate in T-cell activation. We have isolated a cDNA clone encoding CD28 by a simple and highly efficient cloning strategy based on transient expression in COS cells. Central to this strategy is the use of an efficient method to prepare large plasmid cDNA libraries. The libraries are introduced into COS cells, where transient expression of surface antigen allows the isolation of cDNAs by way of monoclonal antibody binding. The CD28 cDNA encodes a highly glycosylated membrane protein with homology to the immunoglobulin superfamily and directs the production of a homodimer in transfected COS cells.
934 citations
TL;DR: These findings demonstrate that a mechanism of de novo methylation of genes might exist that can be induced and targeted in a sequence-specific manner by their own mRNA.
932 citations
TL;DR: The results demonstrate that while ER beta shares many of the functional characteristics of ER alpha, the molecular mechanisms regulating the transcriptional activity of mER beta may be distinct from those of ERalpha.
Abstract: Estrogen receptor β (ERβ) is a novel steroid receptor that is expressed in rat prostate and ovary We have cloned the mouse homolog of ERβ and mapped the gene, designated Estrb, to the central region of chromosome 12 The cDNA encodes a protein of 485 amino acids that shares, respectively, 97% and 60% identity with the DNA- and ligand-binding domains of mouse (m) ERα Mouse ERβ binds to an inverted repeat spaced by three nucleotides in a gel mobility shift assay and transactivates promoters containing synthetic or natural estrogen response elements in an estradiol (E2)-dependent manner Scatchard analysis indicates that mERβ has slightly lower affinity for E2 [dissociation constant (Kd) = 05 nm] when compared with mERα (Kd = 02 nm) Antiestrogens, including 4-hydroxytamoxifen (OHT), ICI 182,780, and a novel compound, EM-800, inhibit E2-dependent transactivation efficiently However, while OHT displays partial agonistic activity with ERα on a basal promoter linked to estrogen response elements in Cos-1 c
929 citations