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.

Cloning and expression of a complementary DNA encoding a bovine adrenal angiotensin II type-1 receptor

Abstract: Angiotensin II elicits different responses which affect cardiovascular, neuronal and electrolyte transport regulation. To understand the mechanisms responsible for its various actions, the receptor for angiotensin II has long been sought, but numerous attempts to purify the receptor have been unsuccessful owing to its instability and low concentration. We report here the expression cloning of a complementary DNA encoding a bovine angiotensin II receptor to overcome these difficulties. The receptor cDNA encodes a protein of 359 amino-acid residues with a transmembrane topology similar to that of other G protein-coupled receptors. COS-7 cells transfected with the cDNA expressed specific and high-affinity binding sites for angiotensin II, angiotensin II antagonist and a non-peptide specific antagonist for type-1 receptor. Dithiothreitol inhibited ligand binding. The concentration of intracellular Ca2+ and of inositol-1,4,5-trisphosphate increased in the transfected COS-7 cells in response to angiotensin II or angiotensin III, indicating that this receptor is the type-1 receptor for angiotensin II. Northern blot analysis revealed that the messenger RNA for this receptor is expressed in bovine adrenal medulla, cortex and kidney.

Antisense gene that inhibits synthesis of the hormone ethylene in transgenic plants

Abstract: ETHYLENE controls many physiological and developmental processes in higher plants, including ripening of fruit, abscission, senescence and responses to wounding1. Although the accumulation of messenger RNAs in ripening fruit and senescing leaves has been correlated with ethylene production and perception2–4, the regulatory mechanisms governing ethylene synthesis and the stimulation of gene expression by ethylene are not understood. We have previously shown that the complementary DNA, pTOM13, corresponds to an mRNA whose synthesis is correlated with that of ethylene in ripening fruit and wounded leaves5,6,8. The pTOM13 mRNA encodes a protein of relative molecular mass 35,0006. The cDNA and three related genomic clones have been sequenced, but the function of the protein is unknown7–9. We show here that antisense RNA, which has previously been used only to reduce the expression of genes of known function10–12, when applied to pTOM13, reduces ethylene synthesis in a gene dosage-dependent manner. Analysis of these novel mutants suggests that pTOM13 encodes a polypeptide involved in the conversion of 1-amino-cyclopropane-1-carboxylic acid to ethylene by the ethylene-forming enzyme (ACC-oxidase).

Use of eukaryotic expression technology in the functional analysis of cloned genes

Abstract: The purpose of this chapter is to describe ways in which eukaryotic expression technology can be used to identify and to analyze the function of cloned eukaryotic genes. The assumption is made that the clone of interest has been sequenced and an open reading frame has been identified. Although expression of genomic sequences will be briefly discussed, in general it is assumed that the sequence of interest is a cDNA. This chapter is divided into three sections. The first section describes several possible strategies for maximizing heterologous gene expression in the cells of higher eukaryotes. The second section deals with potential assays for gene expression based on function, and the third section describes some immunological approaches. Overall, the focus is on the use of techniques which yield information not obtainable from heterologous gene expression in bacteria or yeast.

An LFA-3 cDNA encodes a phospholipid-linked membrane protein homologous to its receptor CD2

Abstract: Recently the human T cell erythrocyte receptor CD2 has been shown to bind human erythrocytes through LFA-3, a heavily gly-cosylated surface protein of broad tissue distribution1,2. CD2–LFA-3 interactions are important for cytolytic conjugate formation3,4, for thymocyte adhesion5, and for T cell activation6. A complementary DNA clone encoding LFA-3 was isolated using a novel transient expression system of mouse cells11. The cDNA encodes a phospholipid-linked membrane protein whose extracellular domain shares significant homology with CD2. As CD2 is homologous with the neural cell adhesion molecule NCAM in immunoglobulin-like domains7, cellular adhesion molecules in both neural and lymphoid tissues could have a common ancestor.

Cloning and expression of murine interleukin-1 cDNA in Escherichia coli

Abstract: Interleukin-1 (IL-1), a peptide hormone produced by activated macrophages, possesses the ability to modulate the proliferation, maturation and functional activation of a broad spectrum of cell types1–7 and may play a major role in the initiation and amplification of immune and inflammatory responses through its action on these diverse cell populations8. IL-1 exhibits microheterogeneity in terms of its relative molecular mass (Mr, 13,000–19,000) and charge properties8, and although murine IL-1 has been purified9,10 and some of its basic structure–function relationships have been elucidated8, it has proved difficult to prepare sufficient amounts of IL-1 for direct and detailed sequence and structural studies. Here we report the cloning, sequence analysis and expression of murine IL-1 cDNA in Escherichia coli. The IL-1 cDNA codes for a polypeptide precursor of 270 amino acids. Biologically active IL-1 was produced in E. coli by expressing the carboxy-terminal 156 amino acids of the IL-1 precursor.