Cloning, cDNA

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. 

Inouye, S., Watanabe, K., Nakamura, H., and Shimomura, O. (2000). Secretional luciferase of the luminous shrimp Oplophorus gracilirostris cDNA cloning of a novel imidazopyrazinone luciferase. FEBS Lett. 481 19-25. 

Nakajima, Y., etal. (2004). cDNA cloning and characterization of a secreted luciferase from the luminous Japanese ostracod, Cypridina noctiluca. Biosci. Biotechnol. Biochem. 68 565-570. 

Tatsumi, H., Kajiyama, N., and Nakano, E. (1992). Molecular cloning and expression in Escherichia coli of a cDNA clone encoding luciferase of a firefly, Luciola lateralis. Biochim. Biophys. Acta 1131 161-165. 

Expressed Sequence Tag. A short DNA sequence usually representing the most terminal regions of a cDNA clone. 

Haas, LG. Meo, T. (1988). cDNA cloning of the immunoglobulin heavy chain binding protein. Proc. Natl, Acad. Sci. USA 85,2250-2254. 

Xu, C. (1993). cDNA cloning of a mouse factor that activates transcription from a metal response element of the mouse metallothionein-1 gene in yeast. DNA Cell Biol. 12, 517-525. 

Gerlach, W.L., Pryor, A.J., Dennis, E.S., Ferl, R.J., Sachs, M.M. Peacock, W.J. (1982). cDNA cloning and induction of the alcohol dehydrogenase gene (Adhl) of maize. Proceedings of the National Academy of Sciences, USA, 79, 2981-5. 

Hooft van Huijsduijnen, R.A.M., Van Loon, L.C. Bol, J.F. (1986). cDNA cloning of six mRNAs induced by TMV infection of tobacco and a characterization of their translation products. EMBO Journal, 5, 2057-61. 

Natter S. Seiberler S, Hufnagl P, Binder BR, Hirschl AM. Ring J, Abeck D. Schmidt T, Valent P, Valenta R Isolation of cDNA clones coding for IgE autoantigens with serum IgE from atopic dermatitis patients. FASEB J 1998 12 1559-1569. 

Feng Y, Broder CC, Kennedy PE, Berger EA (1996) HIV-1 entry cofactor functional cDNA cloning of a seven-transmembrane, G protein-coupled receptor. Science 272 872-877 Fernandez EJ, LoUs E (2002) Structure, function, and inhibition of chemokines. Annu Rev Pharmacol Toxicol 42 469-499... 

Harrison JK, Barber CM, Lynch KR (1994) cDNA cloning of a G-protein-coupled receptor expressed in rat spinal cord and brain related to chemokine receptors. Neurosci Lett 169 85-89 Harrison JK, Jiang Y, Chen S et al (1998) Role for neuronally derived fractalkine in mediating interactions between neurons and CX3CR1-expressing microglia. Proc Natl Acad Sci USA 95 10896-10901... 

A solution to the problem of introns is to isolate mRNA extracted from the human pancreas cells that make insulin. These cells are rich in insulin mRNA from which introns have already been spliced out. Using the enzyme reverse transcriptase it is possible to convert this spliced mRNA into a DNA copy. This copy DNA (cDNA), which carries the uninterrupted genetic information for insulin can be cloned. Although yeast cells (Saccharomyces) can splice out introns it is normal practice to eliminate them anyway by cDNA cloning. 

Finding the amino-acid sequence of a receptor protein has been approached in three main ways. The final aim of all three methods is to obtain a cDNA clone coding for the protein since the base sequence of this DNA allows the amino-acid sequence of the protein to be predicted ... 

Goyer, A. et al.. Folate biosynthesis in higher plants. cDNA cloning heterologous expression and characterization of dihydroneopterin aldolases, Plant. Physiol, 135, 103, 2004. 

Figure 4. RNA Blot Analysis of pSubunit, PG, and D21 Expression during Fruit Development and Ripening. Total RNA (25 pg) isolated from the indicated tomato tissues was probed with eiAer a psubunit cDNA clone, a cDNA for the catalytic PG polypeptide, or a cDNA for the constitutively expressed mRNA D21. Identical specific activities were used in each hybridization and all blots were exposed for 8 hr.

Grierson, D., Tucker, G.A., Keen, J., Ray, J., Bird, C.R. Schuch, W. (1986) Sequencing and identification of a cDNA clone for tomato polygalacturonase. Nucleic Acid Research. 14 8595-8603. 

Hall, L. N., Bird, C. R., Picton,S. P., Tucker, G. A., Seymour, G. B and Grierson, D (1994) Molecular characterisation of cDNA clones representing pectin esterase isozymes from tomato. Plant Molecular Biology. 25. 313-318. 

Ray,J.,Knapp,J.,Grierson,D.,Bird,C and Schuch,W. (1988) Identification and sequence determination of a cDNA clone for tomato pectinesterase. European Journal of Biochemistry. 174. 119-124. 

Figure 2. Comparison of Phaseolus vulgaris pectin methylesterase cDNA clones PE3M (cv. Masai), PE3V PE2V (cv. Verona) and genomic clone PEIV (cv. Verona). Homology boxes are printed in bold [6]. Numbers refer to the aa sequence of the full length cDNA clone PE3M.

With the use of oligonucleotide probes based on the amino acid sequences of these protease V8-obtained peptides and of cyanogen bromide fragments of the porcine H,K-ATPase P subunit, cDNA clones for the rat [12,25] and rabbit [74] H,K-ATPase P subunit were then isolated. 

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