Mammalian cDNA library

Fig. 2 Yeast-based functional screen for receptor-independent activators of G-protein signaling. The yeast strain indicated in A was generated as described previously (Cismowski et al. 1999, 2002) and transformed with mammalian cDNA libraries in the galactose-inducible yeast expression vector pYES2. The strategy for identifying mammalian cDNAs that promoted growth (i.e., activated G-protein signaling pathway) is indicated in B. GALlp GALl promoter)...

Genomic DNA is much more complex than cDNA. Since cDNAs are synthesized in the laboratory from mRNAs using reverse transcriptase, they are no more diverse than the number of mRNAs present at the time of isolation. However, only about 2% of the mammalian genome codes for the synthesis of proteins and their mRNAs. Thus genomic DNA libraries are at least 50 times more diverse than cDNA libraries. cDNA libraries are not easier to make, however, both because of the inherent instability of mRNA compared to DNA and because reverse transcriptase prematurely terminates when copying long mRNAs. 

TES-32 is the most abundant single protein product secreted by the parasite. It is also heavily labelled by surface iodination of live larvae (Maizels et al., 1984, 1987), and is known by monoclonal antibody reactivity to be expressed in the cuticular matrix of the larval parasite (Page et al, 1992a). TES-32 was cloned by matching peptide sequence derived from gel-purified protein to an expressed sequence tag (EST) dataset of randomly selected clones from a larval cDNA library (Loukas et al., 1999). Because of the high level of expression of TES-32 mRNA, clones encoding this protein were repeatedly sequenced and deposited in the dataset (Tetteh et al., 1999). Full sequence determination showed a major domain with similarity to mammalian C-type (calcium-dependent) lectins (C-TLs), together with shorter N-terminal tracts rich in cysteine and threonine residues. Native TES-32 was then shown to bind to immobilized monosaccharides in a calcium-dependent manner (Loukas et al., 1999). 

The DNA or cDNA library is then introduced into a preparation of bacterial host cells. Usually, the first host selected is a laboratory strain of E. coli which has been grown and pretreated with inorganic salts to make uptake of DNA easier. The ability to take up foreign DNA is called competence, cells which have been specially prepared for the purpose are called competent cells. Other methods to transfer DNA into cells include electroporation (application of an external electric field to permeabUize the cell wall), transfection (where a recombinant bacterial virus is used to transfer the DNA to the target cell) or ballistic methods (by using DNA-coated particle projectiles). The last method has been used to introduce foreign DNA into plant cells and mammalian cells. 

This pathway, which is driven by the presence of free Gfly, was modified to allow for screening of mammalian cDNA expression libraries to isolate those cDNAs that activated transcription of a pheromone-responsive promoter downstream of activated G-protein. Modifications included eliminating the pheromone receptor, replacing the endogenous yeast Ga... 

Very recendy a new, physiologic inhibitor of calcineurin has been described by Liu and colleagues at MIT and OriGene (Sun et al., 1998). This protein, called cabin-1 (calcineurin binding protein) is a 2220-residue nuclear protein, which was isolated from a murine T-cell cDNA library in a yeast two-hybrid system with a truncated, catalydcally inac-dve calcineurin used as the bait. Cabin-1 was shown to be a phospho-protein capable of binding to and inhibiting the phosphatase activity of calcineurin, and the interaction between cabin-1 and calcineurin was shown to be sensitive to disruption by FK506 or CsA. In a mammalian... 

Takahashi et al.61s further identified the primary structure by preparing a cDNA library from A. fumigatus induced with fructosylpropylamine and isolated a clone using a polyclonal Amadoriase II antibody. The structure comprised 438 amino acid residues, corresponding to 48.798 kDa. The identity of the Amadoriase n cDNA was further confirmed by expression in Escherichia coli cells with an inducible expression system. Northern-blotting analysis showed that Amadoriase II was induced by fructosylpropylamine in a dose-dependent manner. The sequence determined showed the enzyme to represent a new family of mammalian enzymes. The sequence exhibited 82 and 36% identity and 92 and 65% similarity, respectively, with the two sequences determined by Yoshida et al.616 Amadori products have been implicated in the formation of H202, but the in vivo mechanism needs to be elucidated further. 

Recombinant DNA methods have been extensively used to isolate and analyze the sequence of numerous receptors from various mammalian complimentary DNA (cDNA) libraries using the polymerase chain reaction (PCR) technique (98) to clone receptors that can then be expressed in various pro- and eukaryotic cell lines and Xenopus oocyte. Conserved regions in receptors that are involved in ligand binding, coupling to transductional systems and ion channel formation, have thus been identi-... 

For higher eukaryotes, the most common approach to cloning is PCR amplification from cDNA libraries in order to avoid the intron problem. Another challenge when expressing eukaryotic proteins is that eukaryotic proteins frequently do not fold properly in E. coli and will not be processed with the correct post-translational modifications, and therefore often other eukaryotic expression hosts must be used. Options include using yeast cells, insect cell lines or mammalian cell lines. 

Manufacture of high-density protein arrays presents a greater challenge due to the inherent heterogeneity in the physico-chemical properties and stability of proteins. However, arrays of recombinant proteins representing all yeast open reading frames (ORFs) have been manufactured and used for identification of novel binding activities [6], Escherichia coli transformed with cDNA libraries to express mammalian proteins have been arrayed to identify autoantibodies in serum from the mouse model of systemic lupus erythematosus [7]. 

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