Promoter activity of the two chicken 5-crystallin genes in a Hela cell extract

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Abstract
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 .apprx.2 mRNA (.apprx. 50 to 100) in the embryonic chicken lens.