To further characterize the Glb1 gene, we have isolated genomic clones corresponding to the Large (L) and null alleles. The Glb1-L clone was obtained from a partial lambda library of sequences from the inbred line W64A, and the clone for the null allele was obtained by digesting DNA from Glb1-0/0 plants with EcoRI, fractionating the reaction on an agarose gel, and eluting fragments of about 3.4 kb which were subsequently cloned into LambdaZAP. We have determined the entire nucleotide sequence of the 3.4 kb EcoRI clone corresponding to the L allele. Both strands were completely sequenced. This sequence includes 380 nucleotides 5', and 767 nucleotides 3', to the sequence of the cDNA clone pcGlb1S. Primer extension analysis indicates that the 5' end of the Glb1-L transcript maps to a position 45 nucleotides upstream of the initiator methionine codon. A TATA sequence element is found 33 nucleotides upstream of the transcription start site. Sequences with homology to the ABA response elements from the Em gene of wheat (Marcotte et al., Plant Cell 1:969, 1989) are present 116 and 75 nucleotides 5' to the start of transcription, consistent with the observation that Glb1 expression is positively regulated by ABA (Kriz et al., Plant Physiol., in press). Glb1-L contains four introns ranging in size from 82 to 113 nucleotides. The size difference observed in the proteins encoded by the L and S alleles appears to be largely attributable to a 36-nucleotide duplication in the 3' portion of the coding region.
Nucleotide sequence analysis of the
null allele is almost complete. From sequence comparisons and Southern
blot analysis of maize DNA it appears the null allele is more closely related
to the L allele than to the S allele. The null allele possesses
the small 3' duplication present in the L allele, and there are
no nucleotide differences between the two alleles in the 410 nucleotides
upstream of the initiator methionine codon. The null allele appears to
possess the four introns, and the nucleotide sequences immediately surrounding
the splice sites are no different from those in the L allele. There
are, however, differences from the L sequence within the introns.
There are also several differences in the 3' regions of the two alleles,
including a small deletion and insertion. Experiments are in progress to
determine the nature of the defect in the null allele, which may very well
be due to post-transcriptional processing errors.
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