( HUCL was pooled from 29 diff...)Low Abundance cDNA Cloned Using Stratagenes Human Universal ,,, cDNA Library

HUCL was pooled from 29 different human tissues. This was advantageous since we and others^5,6 have reported that the ACF activity is widely but not ubiquitously expressed. Normalization of pooled cDNAs from multiple tissues therefore addresses the question of tissue-specific variations in expression levels of ACF. Finally, the average insert size of the clones is 1.7 kb, a range suitable for identifying a 1.75-kb sequence predicted to code for ACF, a protein of 65 kDa.

Fig.2

The cDNA insert from the 5' EST clone (AA678055) was labeled with ³²P-CTP and used as probe to screen the four HUCL master membranes.⁷ Hybridization was performed for an hour at 68C, and the blot was subjected to autoradiography. After adequate exposure, the first probe was stripped, and the HUCL membranes were reprobed with the 3' end (N77737) EST cDNA. Both nonoverlapping probes hybridized to the single cDNA pool at position F6 on membrane D (Figure 2). The coordinate position (F6) derived from the first screening was used to obtain the corresponding secondary membrane containing 96 individual clones from Stratagene. The secondary membrane was also screened sequentially using the two ESTs as probes. Both ESTs hybridized to a single clone at coordinate N8 of the secondary membrane. The purified cDNA clone corresponding to this position was obtained from Stratagene as a bacterial stab culture. Therefore, in two simple hybridization steps, we were able to identify a single cDNA that contained both EST sequences.

Analysis of the cDNA clone

The HUCL clone contained an in
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