Cloning cDNA clone libraries

Expression vectors are engineered so that any cloned insert can be transcribed into RNA, and, in many instances, even translated into protein. cDNA expression libraries can be constructed in specially designed vectors derived from either plasmids or bacteriophage A. Proteins encoded by the various cDNA clones within such expression libraries can be synthesized in the host cells, and if suitable assays are available to identify a particular protein, its corresponding cDNA clone can be identified and isolated. Expression vectors designed for RNA expression or protein expression, or both, are available. 

At this time, more then thirty channel DNAs have been cloned and characterized from various sources, predominantly from Drosophila melanogaster, mouse, rat and human cDNA/genomic libraries [6-31]. Inspection of the derived primary K channel protein sequences indicates that voltage-gated channels belong to a... 

Yeast does not synthesise cycloartenol or triterpenes, and so approaches to clone plant OSCs by complementation in S. cerevisiae are not feasible because of the lack of appropriate mutants. However, LS-deficient yeast mutants accumulate high levels of 2,3-oxidosqualene, favouring the synthesis of novel cycfisation products generated by heterologous expression of OSCs. The absence of lano-sterol also facilitates analysis of the reaction products. Corey and co-workers isolated a cDNA encoding Arabidopsis thaliana CS by transforming a plant cDNA expression library into such a yeast mutant and screening protein preparations derived from pools of transformants for the ability to synthesise cycloartenol by TLC [39]. 

Indole alkaloids are derived from tr)y)tophan, which is formed in the shiki-mate pathway. In the case of the terpenoid indoles, tryptophan is usually first converted to tiyptamine by the enzyme tryptophan decarboxylase (TDC) (Fig. 2.9). This enz)une occurs in the cytosol and has been detected in all parts of the developing seedling and in cell cultures of C. roseus (De Luca, 1993). If appears to be a pyridoxoquinoprotein, as two molecules of pyridoxal phosphafe and two molecules of covalently bound pyrroloquinoline quinone were found per enz)une molecule (Pennings et al, 1989). A tdc cDNA clone has been isolafed by immunoscreening of a C. roseus cDNA expression library (De... 

EST Profiling. An EST is a short, single sequence run collecting data over about 200-400 bases from a clone selected from a cCNA library. Typically, cDNA clone libraries... 

Hetfman, D. M., Kcramisco, J. R., Fiddes, J- C-, TIujraas, G. I, and Hughes, S. H. (1983). Identification of clones that encode chicken ttopotnyosin by direct immunological screening of a cDNA expression library. Proc. Naii. Acad. Sci. U.S.A. 80, 31-35. 

Ho and Crapo (H14) cloned full-length Mn-SOD cDNAs from libraries of human liver and U937 cells and obtained a full primary sequence. The amino acid sequence predicted from the cDNA was almost the same as that obtained from the protein by chemical analysis (B5), except that one Gly and one Tyr were added and Glu replaced Gin residues at positions 42, 88, 109, and 131. The cDNA for human Mn-SOD encoded 222 amino acids. The first 24 amino acids were found to be a leader peptide essential for translocation into mitochondria. The homology between human and rat Mn-SOD was found to be 93% and the homology between human and mouse Mn-SOD was 94%. Moreover, the homology between rat and mouse enzymes was 96%. 

In a variation of genomic libraries, clone libraries of complementary DNA (cDNA) molecules, called cDNA libraries, are produced from mRNA molecules by reverse transcription. This technique can be used to evaluate the transcriptome of certain cell types under... 

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