We have continued the study of our genomic clone of the Shrunken gene by both S1 mapping experiments with RNA-DNA hybrids and partial sequencing of the 5'- and 3'-ends of the gene. The structure of the gene is shown in the figure. Two introns are found at the 3'-end by comparing the genomic DNA sequence with the cDNA sequence. The rest of the gene structure was examined by digestion of RNA-DNA hybrids with endonuclease S1. Another 13 introns were detected by this method and positioned within genomic restriction fragments. The first exon is approximately 50 bp long and has been located between the left-most HindIII site and an XbaI site further 5'. Because the third 95 bp exon contains a PvuII site, it was possible to locate its 5'-intron-exon junction within our sequence. By using synthetic oligonucleotides complementary to a sequence derived from the third exon for priming a reverse transcriptase reaction in the presence of dideoxynucleotides, we have obtained some of the sequence of the 5'-end of the mRNA. This allowed us to position the first two exons within our genomic sequence data and tells us that we are looking at the 5'-end of the mRNA. The transcription start was precisely mapped by S1 endonuclease digestion of RNA-DNA hybrids. There is a TATA box-like sequence (TCTATTTAT) 25-30 bp in front of the multiple start sites, and also a sequence resembling a CCAAT box (CCATCT) around position -90. The first ATG is located within the second exon, the following open reading frame is continued in exon 3 and exon 4 as far as the latter is sequenced.
All introns so far sequenced start with GT and stop with AG, as in nearly all animal and plant genes examined up to now. The transcribed region extends 5.5 kb at the genomic level, while the length of the mRNA is 2.7 kb. The gene is interspersed with 15 introns which are larger at the 5'-end than at the 3'-end.
W. Werr, W. B. Frommer and P. Starlinger
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