No abstract

We have analyzed three classes of highly repetitive DNA sequences of Arabidopsis thaliana, composed of tamdemly repeated units of 180 bp, 500 bp, and 160 bp, respectively. The three families comprise approximately 2% of the Arabidopsis genome and are the major component of the highly repetitive DNA. The 500-bp element arose by duplication of one half of a 180-bp ancestor and insertion of a foreign segment between the two duplicated parts followed by amplification. The repeat elements contain occasionally palindromes and other motifs but none are significantly conserved. There is no significant similarity with previously published repetitive elements. Heterogeneity between monomers ranges from 6% to 17%. Monomers derived from different clusters in the genome are more diverged than monomers of the same array.

Genomic and cDNA clones for three inflorescence-specific genes from Arabidopsis thaliana were isolated and characterized. The genes are tandemly organized in the genome on a 10 kb fragment. The expression of these genes is coordinately regulated in a developmental and organ-specific pattern. They are expressed predominantly in anthers at the later stage of flower development. The primary structure of the encoded gene products exhibits comparable features consisting of a hydrophobic domain at the N-terminal region followed by repeated glycine-rich motifs. Little homology is observed either between the glycine-rich domain of the three genes or with previously described glycine-rich proteins from other plant species.

The transformation of mutant plants with a complete recombinant library derived from wild-type DNA followed by assay of transformed plants for complementation of the mutant phenotype is a promising method for the isolation of plant genes. The small genome of Arabidopsis thaliana is a good candidate for attempting this so-called shotgun transformation. We present the properties of an A. thaliana genomic library cloned in a binary vector, pC22. This vector, designed to introduce genomic libraries into plants, contains the oriV of the Ri plasmid pRiHR1 by which it replicates perfectly stably in Agrobacterium. Upon transfer of the library from E. coli to A. tumefaciens large differences in transfer efficiencies of individual recombinant clones were observed. There is a direct relation between transfer efficiency and stability of the recombinant clones both in E. coli and A. tumefaciens. The stability is independent of the insert size, but seems to be related to the nature of the insert DNA. The feasibility of shotgun transformation and problems of statistical sampling are discussed.

Two lines of transgenic Nicotiana tabacum transformed to kanamycin resistance by means of a binary Agrobacterium vector containing a nos-npt gene were investigated over three generations. Southern hybridization and crossing analyses revealed that a single copy of T-DNA had integrated in each line and that the kanamycin resistance was regularly transmitted to the progeny as a monogenic dominant trait. Homozygous transgenic plants were fully fertile, morphologically normal and did not significantly differ from wild-type plants in the quantitative characters examined (plant height, flowering time). The two lines showed very low, but significantly different levels of meiotic instability: kanamycin-sensitive plants occurred among backcross progeny from homozygous transgenic plants with frequencies of 6/45,000 and 25/45,000, respectively. The sensitive plants arose independently of each other and thus resulted from meiotic rather than mitotic events. These findings demonstrate for the first time that integrated foreign genes can be transmitted to progeny with the high degree of meiotic stability required for commercial varieties of crop plants. They emphasize the importance of non-homologous integration and of avoiding co-integration of inactive gene copies for achieving meiotically stable transformants.