Analysis of Barbara McClintock's Tiny Fragment/X Component

About This Grant

Nobel laureate Barbara McClintock discovered a “tiny fragment” chromosome, which she postulated contained an “X component” that could rapidly reorganize the genome. This fragment chromosome rearranges itself as well as other regions of the genome. The regions that would participate in these rearrangements are now known to contain highly repetitive sequences. They present a challenge for the DNA copying mechanism that is often stalled in repetitive sequences and needs to be reinitiated. If this reinitiation is defective, DNA breaks occur at these sites and could form the basis of the rearrangements produced by the X component if the breaks in various places in the genome occur simultaneously and are eventually repaired with swapped partners. The types of rearrangements produced are similar to those that commonly occur during karyotype evolution, so an understanding of the X component will reveal a novel genomic activity that can have a profound effect on chromosomal changes. An educational program is proposed to digitize microscope slides of chromosomal aberrations and their behavior from an historical collection and an instructional video on the technique of whole chromosome painting in maize will be produced and uploaded to the laboratory Youtube channel. The proposed experiments will test the hypothesis that dramatic changes in transmission frequency of the tiny fragment chromosome are the result of genomic rearrangements catalyzed by the X component carried on the minichromosome and define their structure with whole chromosome paints and DNA sequencing. Experiments will test the hypothesis that silencing is operating versus fragment loss by comparing mRNA to genomic DNA via droplet digital PCR from total nucleic acid isolations from different sectors on mosaic plants with or without Bronze1 expression. RNAseq experiments will assess the silencing versus activity of all the genes on tiny fragment in these sectors. Based on previous examples, cases of mosaic patterns of Bronze1 will be tested for heritability. The gene WSS1, a DNA-dependent metalloprotease, is present on tiny fragment and its normal function is to facilitate the reinitiation of replication of highly repetitive sequences. With under or overexpression of this gene in other species, DNA double strand breaks occur, making this gene a strong candidate as the X component. CRISPR-Cas9 editing of this gene in the endogenous chromosome will test if chromosomal rearrangements analogous to those produced by the X component result. Transformation of this gene to produce overexpression will test the same. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.