Cloning of an Epidermis-specific Cynops cDNA from Neurula Library.

A cDNA encoding one of the epidermis-specific proteins designated as the spot 6 was isolated from the Cynops embryo. Cynops neurula cDNA library was constructed with the plasmid vector containing the promoter sequence for SP6 RNA polymerase. After transcription and translation in vitro the final protein products were screened for the presence of spot 6 by two-dimensional gel electrophoresis. The total library was digested by 11 restriction endonucleases selected not to destroy the sequence in both the vector and the insert encoding spot 6 protein. The ATP-dependent DNase digestion eliminated the cDNA population sensitive to these endonucleases. These steps effectively enriched the sequence for spot 6 protein. The resultant sublibrary was repeatedly divided into smaller pools and was screened. The tryptic peptide analysis showed that the isolated clone produced the protein identical to the spot 6 protein originally defined in vivo. Northern analyses showed that the cloned gene was expressed as expected from the developmental behavior of the spot 6 in vivo.