Cloning of P8, A Transcription Factor Required of Skeletogenic Gene Expression

摘要

The transcription factor toward which this project was directed (formerly known as P8, now as Sp(G/C)F-1) interacts with regulatory regions of many sea urchin embryo genes, and probably acts as a general enhancer protein. During this year this factor was: (1) purified by affinity chromatography; (2) microsequence was obtained; (3) the factor was sequenced; (4) its DNA binding site was determined; (5) it was expressed in bacteria and an antibody generated; (6) its provenance was determined; and an unusual aspect of its structure was analyzed. The latter is that the factor appears in sea urchin embryo nuclear extract in five different forms of decreasing molecular weight. These forms are also presented in cell-free translation using synthetic mRNA from the cloned CDNA. We shoed that-unusually-there are five successive ATG start sites in this mRNA, all used more or less equally, thus accounting for five nested forms. The sequence reveals Sp(G/C)F-1 to be a novel DNA-binding protein, unlike any in the data banks. It is loaded into the egg in relatively large quantities during oogenesis and then appears in embryo nuclei.