Promoter activity of the two chicken delta-crystallin genes in a Hela cell extract.
The in vitro transcriptional activity of the two delta-crystallin genes (5'-delta 1-delta 2-3') of the chicken was studied in a whole Hela cell extract. Both the delta 1 and delta 2 promoters were recognized by RNA polymerase II in this heterologous system. The major RNA initiation site from the delta 1 promoter was the same in vitro as that which occurs in vivo, as judged by mapping with S1-nuclease, although other minor initiation sites upstream and downstream of the major initiation site were noted. A primer extension experiment showed that the longest RNA synthesized in vitro from a delta 2 template initiated near the beginning of the first exon. The delta 1 promoter was several-fold stronger than that of delta 2 under the present in vitro conditions. Transcription from the delta 1 promoter was abolished by a competitor fragment (c'-II; includes -328 to -63) purified from the delta 2 promoter, indicating that one or more common transcription factors binding upstream from the TATA box are required for in vitro function of the two delta-crystallin promoters. Thus, in the Hela cell extract both delta-crystallin genes contain a functional promoter. We consider the possibility that the single 5'CCAAT3' sequence present in the delta 1 promoter (but lacking in the delta 2 promoter) may contribute to its greater core activity under our conditions. The greater promoter activity of the delta 1-crystallin gene in the Hela cell extract was not sufficient to account for the large ratio of delta 1 to delta 2 mRNA (approximately 50 to 100) in the embryonic chicken lens.
Das
G C
GC
Laboratory of Molecular and Developmental Biology, National Eye Institute, Bethesda, MD 20892.
Piatigorsky
J
J
D000818:Animals; D002457:Cell Extracts / Q000502:physiology; D002645:Chickens / Q000235:genetics*; D002874:Chromosome Mapping; D003459:Crystallins / Q000235:genetics*; D005796:Genes; D006367:HeLa Cells / Q000166:cytology*; D006801:Humans; D011401:Promoter Regions, Genetic; D014158:Transcription, Genetic
D002457:Cell Extracts; D003459:Crystallins