The radius of gyration of an apomyoglobin folding intermediate.

Sph site of pSL301 (Invitrogen). Colonies were screened for inserts by PCR with T7 and T3 sequences located outside the cloning site as primers. 10. Selected clones were manually sequenced as described [G. (1989)] with 5'-GACGTCGAC-CTGAGGTAATTATAACC-3' as primer. Sequence files were analyzed by means of the SAGE software group (7), which identifies the anchoring enzyme site with the proper spacing and extracts the two intervening tags and records them in a database. The 1000 tags were derived from 413 unique ditags and 87 repeated ditags. The latter were counted only once to eliminate potential PCR bias of the quantita-tion, as described in the text. 12. Pancreatic mRNA from the same preparation was used for SAGE and to construct a cDNA library in the ZAP Express vector. The ZAP Express cDNA Synthesis kit (Stratagene) was used according to the manufacturer's protocol. Analysis of 15 randomly selected clones indicated that 100% contained cDNA inserts. Plates containing 250 to 500 plaques were hybridized as described [ cDNA probes for trypsinogen 1, trypsinogen 2, procarboxypeptidase Al, chymotrypsinogen, and elastase IIIB were derived by RT-PCR from pancre-as RNA. The sequences of primers are available from the authors upon request. The trypsinogen 1 and 2 probes were 93% identical and hybridized to the same plaques under the conditions used. Likewise , the elastase IIIB probe was >95% identical to protease E. 13. Plates containing 250 to 2000 plaques were hybrid-ized to oligonucleotide probes with the same conditions previously described for standard probes ex