( fying the correct complete cDNA sequenc...)Low Abundance cDNA Cloned Using Stratagenes Human Universal ,,, cDNA Library

fying the correct complete cDNA sequence is often hindered by the short sequences of the reported ESTs that are biased towards the 5 end. Since the EST cDNA inserts are generally not full-length and the clones are not always available, there is still a need to screen libraries to isolate a full-length cDNA. However, conventional library screening is tedious and time consuming, especially for a low-abundance protein.

To circumvent these problems, we used Stratagenes HUCL arrays to isolate a full-length clone that encodes Apobec-1 Complementation Factor (ACF), a novel RNA-binding protein that is involved in the post-transcriptional editing of apolipoprotein B (apo-B) mRNA in mammals. The HUCL array is a collection of more than 145,000 primary clones in 1536 pools of 96 clones each, which are arranged on four master membranes. Because of the librarys large representation of high-quality cDNA clones, human genes can be quickly identified and subsequently isolated.

Purification of ACF

Editing apo-B mRNA involves a site-specific deamination reaction that converts cytidine⁶⁶⁶⁶ to uridine and generates a premature translational stop codon.² Although the catalytic subunit of the editing enzyme was discovered in 1993, the identity of the RNA-binding subunit has been elusive, despite intensive efforts by several groups. We previously characterized and purified a 65-kDa protein from baboon kidney that had all the properties expected for the RNA-binding subunit. ^3,4 The protein was purified over 140,000-fold, and silver staining of the final purified fraction showed the presence of a single protein of 65
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