Functional analysis of theArabidopsiscentromere by T-DNA insertion-induced centromere breakage

Abstract

Two minichromosomes (␣ and ␦) in addition to two other aberrant chromosomes (␤ and ␥) were found in a transgenic Arabidopsis plant produced by an in planta vacuum infiltration technique. The minichromosomes were successfully separated by successive crossing and selfing and added to wild-type Columbia (Col-0) as a supernumerary chromosome. FISH indicated that both of the two minichromosomes originated from the short arm of chromosome 2. The mini ␣ chromosome contained the whole short-arm 2S and a truncated centromere (180-bp repeat cluster), whereas mini ␦ lacked the terminal region including telomere repeats. Pachytene FISH clearly revealed that mini ␦ comprised a ring chromosome carrying two copies of the region from the 180-bp repeat cluster to BAC-F3C11. Both of the 180-bp clusters (each Ϸ500 kb in length) were thought to possess normal centromere functions because the centromere-specific histone H3 variant (HTR12) was detected on both clusters. Notwithstanding this dicentric and ring form, mini ␦ was stably transmitted to the next generations, perhaps because of its compact size (<4 Mb). Chromosome ␤ also comprised a dicentric-like structure, with one of the two 180-bp repeat sites derived from chromosome 1 and the other from chromosome 2. However, the latter was quite small and failed to bind HTR12. The data obtained in this study indicated that 500 kb of the 180-bp array of the chromosome 2 centromere, from the edge of the 180-bp array on the short-arm side, is sufficient to form a functional domain.minichromosomes ͉ ring chromosome

Structure and Evolution of Plant Centromeres

“…This suggests that the minichromosome stability is controlled genetically, as shown in S. pombe . Recently, two other minichromosomes (mini-α and mini-δ ) were found in a transgenic Arabidopsis plant (Murata et al 2008) . Both minichromosomes originated from a short arm of chromosome 2.…”

Engineered plant minichromosomes

“…The dicentric ring consisted of approximately 570 kb of centromeric repeats, 270 kb of mitochondrial DNA, and parts of the short arm of chromosome 2, all in duplicate. The initial ring chromosome (Murata et al, 2008;Yokota et al, 2010) and its spontaneous derivatives (Yokota et al, 2011) could be meiotically transmitted, albeit at a much lower frequency than wild-type chromosomes.…”

“…Alternative routes to generate artificial minichromosomes have been developed (Murata et al, 2006(Murata et al, , 2007(Murata et al, , 2008(Murata et al, , 2013Yokota et al, 2011). Originally, among the progeny of a transgenic A. thaliana plant, individuals were found with different chromosome rearrangements, involving the transgene locus.…”

Artificial chromosome formation in maize (Zea mays L.)

“…The size of CENH3-binding domains appears to be similar across species [in Drosophila, about 400 kb (Sun et al 2003); in the human Y chromosome, about 500 kb (Tyler-Smith et al 1993); in rice chromosome 8, about 750 kb ; in Arabidopsis, about 500 kb (Murata et al 2008); and in maize, 300-700 kb (Jin et al 2004)] suggesting a possible size requirement for functional centromeres. Analysis of minimal centromere size has been performed on human minichromosomes (Yang et al 2000) demonstrating that functional centromeres require a minimum of about 100 kb of alphoid DNA.…”

“…In plants, naturally occurring minichromosomes have been observed in various species (Gindullis et al 2001;Schubert 2001;Murata et al 2006Murata et al , 2008. A minimal size of approximately 5% of the haploid genome has been suggested for chromosome stability and transmission through meiosis (Schubert 2001).…”