We have further analyzed our genomic sucrose synthase clone by S1-mapping experiments and partial DNA sequencing. A partial restriction map is shown in the figure.
By using terminally labeled DNA subfragments, the direction of transcription was determined from the left to the right with respect to the above restriction map. The extension of the gene is at least 4.5 kb, exceeding the size of mature mRNA of 2.8 kb. The difference is due to the fact of many small introns. By S1-digestion of RNA protected DNA, electrophoretic separation and subsequent hybridization to radioactive subfragments, the number of introns was determined to be 14, if all the reproducibly found fragments correspond to exons and are not the product of incorrect splicing.
The position of two introns near the central BglII site on the above map was verified by sequencing of genomic and cDNA. The leftmost S1-resistant DNA hybridizes to the DNA fragment between the leftmost BglII and the SphI site. It may correspond to the transcription start. Otherwise, a very small leader sequence may be located further upstream and have escaped detection.
W. Werr, W.-B. Frommer and P. Starlinger
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