MEI1, AN ARABIDOPSIS GENE REQUIRED FOR MALE MEIOSIS - ISOLATION AND CHARACTERIZATION

The T-DNA tagged mutant gene of Arabidopsis thaliana, mei1, produces a fter meiosis an abnormal tetrad, consisting of five to eight microspor es of varying sizes and DNA contents. Plant DNA flanking the inserted T-DNA was isolated by inverse PCR. An approximately 16-kb DNA fragment spanning the T-DNA insertion site was isolated by screening a wild-ty pe genomic library, using the plant flanking DNA as a probe. Using RT- PCR and RNA isolated from very young flower buds, a cDNA fragment was obtained. Nucleotide sequence comparison of the cDNA and the genomic s equence in this region indicated a gene which contained two introns. T he 5' and 3' splice sites of neither intron comply with the :GU...AG: rule. In the mutant, the T-DNA had inserted into one of the introns. T he deduced sequence of the MEI1 wild-type gene, which contains 89 amin o acids, shows possible similarity with the human acrosin-trypsin inhi bitor, HUSI-II, and is about the same size. Two wildtype DNA fragments , both extending over the T-DNA insertion site, were introduced into m utant plants by Agrobacterium-mediated transformation and plants were selected for both hygromycin and kanamycin resistance. Several indepen dent male-fertile transformants were obtained with one of the DNA frag ments. The fragment showing complementation of the mutant phenotype in dicated that the sequence with similarity to the acrosin-trypsin inhib itor is MEI1. Within the 16-kb genomic fragment two other genes were i dentified; one showed no overall similarity to any protein sequence in the database and the other had almost complete identity with an Ambid opsis-transcribed sequence tag with similarity to ACC oxidase. Double mutants between mei1 and qrt1 were made, permitting better characteriz ation of the mei1 phenotype because the individual microspores continu ed to be held together after cellose dissolution.