University of Illinois
To further characterize the Glb1 gene, we have isolated cDNA and genomic clones corresponding to this locus. An embryo-specific cDNA library was constructed in the expression vector LambdaZAP (Stratagene). The RNA used for cDNA synthesis was polyA+ RNA from 27 DAP embryos of the maize inbred line VA26, which is homozygous for the Glb1-S allele. The primary library contained 500,000 clones from an estimated 0.1ug of cDNA. Screening of 250,000 clones with antiserum specific for GLB1 yielded 10 positive clones with insert sizes ranging from 700bp to 1800bp. The 1800bp clone was chosen for further characterization and was subjected to nucleotide sequence analysis. Because of the high (68%) G+C content it was necessary to use the dGTP analogs 7-deaza dGTP or dITP in the dideoxy termination reactions to obtain unambiguous sequence data. The amino acid sequence deduced from the cDNA sequence is in good agreement with the amino acid composition determined for GLB1-S. A 300bp restriction fragment from the 5' end of the 1800bp clone was used as a probe to rescreen the cDNA library for a full-length clone. The size of the longest clone (2200bp) obtained from this secondary screen corresponds to the size of Glb1-specific transcripts detected by Northern blot analysis.
The 1800bp clone was used as a probe in Southern blot analysis of maize DNA and found to hybridize with an EcoR1 fragment of 3.4kb in plants carrying either the L (Large), S (Small), or null Glb1 allele. We have obtained genomic clones for the S and null alleles by isolating sized EcoR1 fragments from an agarose gel and cloning into LambdaZAP. A genomic clone corresponding to the L allele was isolated by screening an EMBL3 phage library constructed from DNA of the inbred line W64A (this library was kindly provided by J.C. Wallace, Purdue University). Efforts are underway to determine the nucleotide sequence differences between the null and functional alleles.
The cDNA clones have also been used as probes to investigate Glb1
transcript levels in various tissues by Northern blot analysis. Embryos
homozygous for the null allele produce very low levels of Glb1-specific
transcripts but these are of a different size than those encoded by the
or S alleles. Glb1 transcripts have also been detected in
developing W64A endosperm, at much lower levels than in the embryo, but
not in unfertilized ears, immature tassels, or the leaves of 7-day-old
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