( sert of 1.94 kb and encoded an open rea...)Low Abundance cDNA Cloned Using Stratagenes Human Universal ,,, cDNA Library

sert of 1.94 kb and encoded an open reading frame of 586 amino acids.⁷ The predicted amino acid sequence contained 20 of the 23 peptides that were obtained by mass spectrometry, which was strong evidence that we had isolated the correct sequence. Based on DNA sequencing, the ACF cDNA encoded a novel protein with a predicted molecular mass of 64,274 Da.⁷ The N-terminal region of ACF showed the presence of RNA recognition motif (RRM)-type RNA binding domain, which is consistent with its function as the RNA-binding subunit of the editing enzyme. The C-terminal region of ACF (residues 304-586) was unique.

Once we had the clone in hand, we were able to quickly establish that ACF is required for editing apo-B mRNA both in vitro and in vivo. Taken together, our results support a model of the editing enzyme in which ACF is the RNA binding subunit that docks the catalytic subunit to edit the cytidine⁶⁶⁶⁶ of apo-B mRNA.

Conclusions

We determined that screening of Stratagenes HUCL was a straightforward and highly effective alternative to conventional library plating and screening. Using the HUCLs normalized cDNAspooled from multiple human tissuesgreatly facilitated our cloning of ACF, a very low-abundance protein. The single positive ACF clone was readily detected against a very low background after hybridization. The final purified clone was available as a bacterial stab for further characterization after only two simple screenings.

REFERENCES

1. Lelias, J-M., et al. (1998) Strategies 11: 29-32.
2. Chan, L., et al. (1998) Strategies 11: 29-32.
3. Mehta, A
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