Most of the maize genome (>90%) is made up of repetitive DNA, a large fraction of which is methylated. Genes comprise less than 3% of the genome and are found in contiguous stretches of less methylated DNA known as hypomethylated islands. Because of the repetitive DNA content in maize, sequencing the entire maize genome (2.5xl09 bp) is impractical. Some laboratories, mostly in industry, have adopted the strategy of large-scale sequencing of the RNA products transcribed from genes (cDNAs). An attractive alternative is to specifically sequence genes. One can take advantage of the tendency of the maize transposon Activator (Ac) to insert in hypomethylated DNA, the genomic component containing genes, to identify genes as sites into which Ac transposes (tac sites) and, then, to sequence the DNA adjacent to the transposon. An advantage of this approach is that, in addition to a sequence that can be compared to the existing database, it generates an insertion library. The collection of lines carrying Ac at many different locations in the maize genome will enable investigators to screen for subtle mutant phenotypes, particularly after obtaining information on where in the plant the genes are expressed. Many genes are expected to have minor effects and could be missed from a conventional transposon mutagenesis screen designed to identify gross changes in phenotype.
Taking advantage of the powerful endosperm genetics of maize, a simple and efficient Ac transposition assay based on the well-studied endosperm markers bz (bronze) and wx (waxy) has been developed. A collection of over 1200 independent Ac transposants has been generated and over one-third of these Ac sites have been mapped relative to the donor locus. In parallel, a panhandle PCR method, originally used in the human genome, has been adapted for the isolation of DNA adjacent to the insertion (tac sites). By sequencing tac sites, insertions have been identified, for example, in genes encoding a sulfur transporter, a MAPKK, and a sesquiterpene synthase.
This Ac mobilization scheme allows the isolation of tac sites that are either genetically linked or unlinked to the donor locus. However, because Ac has a strong tendency to transpose to closely linked sites, about one-half of the tac sites are linked to wx on chromosome 9. Clearly, it would be desirable to mobilize Ac from different starting sites in the genome. Toward that end, suitable maize lines that are readily transformable and regenerable are being developed. These lines will be transformed with a construct carrying an Ac element modified to facilitate the isolation of tac DNA. This construct should integrate at random sites in the genome, providing starting platforms for future Ac mobilization.
The expected outcomes of this proposal are: (a) The sequence of more than 1000 maize genes or gene fragments. (b) The elucidation of the function of a set of 50 genes based on the sequence of tac sites, the phenotype of Ac insertion mutations, and the pattern of gene expression. (c) The map location of those genes that correspond to unique sequences in the maize genome. (d) The development of transgenic maize lines that will facilitate the future isolation of genes (tac sites) from any location in the genome.
