( ...)Low Abundance cDNA Cloned Using Stratagenes Human Universal ,,, cDNA Library

A time-saving alternative to conventional library plating and screening

Anuradha Mehta Donna Driscoll

Department of Cell Biology, Cleveland Clinic Foundation, Cleveland, OH

We chose Stratagenes Human Universal cDNA Library ¹ (HUCL) to isolate a full-length cDNA clone encoding a novel low-abundance protein called Apobec-1 Complementation Factor (ACF) for several important reasons: The simple two-step hybridization protocol allowed us to quickly screen a large number of clones, normalization increased the chance of finding low-abundance cDNAs, the librarys varied tissue base aided in isolating a protein that was widely expressed but only in small amounts, and the average size of the library clones ranged within ACFs expected sequence size. We successfully isolated ACF cDNA, which was crucial in allowing us to define the minimal protein requirements for the C to U editing of apolipoprotein-B mRNA.

Purification of proteins and identification of their cognate cDNAs are powerful tools for studying the molecular mechanisms underlying biological systems. Highly sensitive mass spectrometric techniques provide valuable peptide sequence information from very limited amounts of protein, which can be used to screen the wealth of information available in the NCBI databasesincluding both nonredundant and Expressed Sequence Tag (EST) databases. In the case of a novel protein, identi
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