Molecualr analysis of In-D; a semi-dominant allele of the anthocyanin regulatory gene Intensifier
--Scheffler, B1, Rojek, R, Pusch, I, Wienand, U
1University, MS, USDA-ARS-NPURU, PO Box 8048, 38677

The C2 and White-pollen (Whp) loci both encode for chalcone synthase (CHS). In the aleurone tissue, the Intensifier locus (In) regulates the production of CHS coming from the Whp locus. Kernels that are in/in allow CHS to be produced from Whp locus, while CHS production from Whp is inhibited in In/(In or in) kernels. Franken, P, U Niesbach-Klosgen, U Weydemann, L Marechal-Drouard, H Saedler, U Wienand (1991, EMBO J. 10:2605-2612) investigated the expression of the Whp locus and the results from their study indicated that In might regulate, directly or indirectly, Whp at the post-transcriptional level. Burr, FA, B Burr, BE Scheffler, M Blewitt, U Wienand, EC Matz (1996, The Plant Cell 8:1249-1259) have cloned and characterized In. The results from this study show that In encodes for a myc-related protein, thus indicating In may act as a suppressor of transcription. In order to elicit the regulatory function of In, we began an investigation of In-D. In-D is a semi-dominant mutation of In that inhibits overall production of anthocyanins in the aleurone tissue.

Genomic Southern analysis of In-D shows that there are minor differences within the coding region of In and In-D. Sequence analysis of a lambda In-D genomic clone uncovered significant differences between the two loci in introns 2 and 6. Intron 6 of In-D is1000 bp smaller than In's intron.

A large number of cDNAs were isolated from a lambda Zap cDNA library, constructed from mRNA isolated from the aleurone of 30 DAP In-D kernels. Sequence analysis of these clones shows, as in the case of In, that In-D exhibits missplicing. The missplicing patterns between the two alleles are very similar except for intron 2. In-D also exhibits premature polyadenylation in intron 2. One half of the cDNA clones showed high homology to the In-D genomic clone, but there were significant differences to indicate that they did not originate from this copy of In-D. Upon further genomic DNA analysis of In-D and segregating populations from In-D/In and In-D/in, it appears that In-D consists of two complete, but structurally different, copies of the wild-type allele. Upon sequence analysis of this second copy major differences between the duplicated copies in introns 3, 5, 6 and 7 were detected.

Northern and Western analysis demonstrate that In-D is expressed at significantly higher levels when compared to In, and that total CHS production is inhibited or delayed.

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