Identification of transposon‐tagged genes by the random sequencing of Mutator‐tagged DNA fragments from Zea mays

Abstract

SummaryWe have used a universal adaptor ampli®cation procedure to isolate random Mutator-tagged fragments from Mutator-active maize plants. Direct sequence characterization of 761 Mutator-tagged fragments indicated that a signi®cant number were homologous to sequences within the public databases. The ability of Mutator-tagged fragments to detect homology was not related to the length of the sequence within the range 100±400 bp. However, fragments above this size did show an increased chance of detecting homology to either expressed sequence tags or genes. Characterization of the insertion sites of the Mutator elements suggested that while it does target transcribed regions, Mutator does not appear to have any site preference within the transcription unit. Hybridization of previously unidenti®ed Mutator-tagged fragments to arrayed cDNA libraries con®rmed that many of these also showed homology to transcribed regions of the genome. Examination of back-crossed progeny con®rmed that all the insertions examined were germinal; however, in all but one case, sel®ng ®ve individual Mutatortagged lines failed to reveal an obvious phenotype. This study suggests that the random sequencing of Mutator-tagged fragments is capable of producing both a signi®cant number of interesting transposon tagged genes and mutant plant lines, all of which could be extremely valuable in future gene discovery and functional genomics programmes.

Two Cytosolic Glutamine Synthetase Isoforms of Maize Are Specifically Involved in the Control of Grain Production

“…The Mu insertion event within Gln1-4 was identified by the random sequencing of Mu-tagged fragments as described by Hanley et al (2000). Sequence analysis of both events indicated that in the case of gln1-3, Mu1 (mutant a) had inserted within exon 8, and in the case of gln1-4, Mu5 had inserted within the intron separating exons 7 and 8 ( Figure 1).…”

“…been isolated that have insertions in three of the five GS1 genes (Hanley et al, 2000;S. Haines and K.J.…”

A Unique Set of 11,008 Onion Expressed Sequence Tags Reveals Expressed Sequence and Genomic Differences between the Monocot Orders Asparagales and Poales[W]

“…Mutator -like homologs may be functional transposases; however, they also may have evolved other functions, such as transcriptional regulation (Hudson et al, 2003). The presence of potentially mutagenic class-I TEs in the onion genome raises the possibility of developing gene-tagging systems (Hanley et al, 2000). Previous surveys also revealed relatively high frequencies of retrotransposon-like ESTs in monocot EST collections (Vicient et al, 2001;Echenique et al, 2002).…”

Differential methylation of genes and repeats in land plants

“…Although useful for gene annotation, EST sequencing is limited to genes that are detectably expressed, and misses non-coding sequences such as introns and promoters (Palmer et al 2003). Another approach has exploited the tendency of some DNA transposons to insert preferentially in genes (Hanley et al 2000;Raizada et al 2001). However, transposon insertion is not random (Greenblatt 1984;May et al 2003) and yields a partial representation of genes when attempted on a large scale (Palmer et al 2003;Fernandes et al 2004).…”