Evidence for a megareplicon covering megabases of centromeric chromosome segments

Abstract

We have analysed the replication of the heterochromatic megachromosome that was formed de novo by a large-scale amplification process initiated in the centromeric region of mouse chromosome 7. The megachromosome is organized into amplicons approximately 30 Mb in size, and each amplicon consists of two large inverted repeats delimited by a primary replication initiation site. Our results suggest that these segments represent a higher order replication unit (megareplicon) of the centromeric region of mouse chromosomes. Analysis of the replication of the megareplicons indicates that the pericentric heterochromatin and the centromere of mouse chromosomes begin to replicate early, and that their replication continues through approximately three-quarters of the S-phase. We suggest that a replication-directed mechanism may account for the initiation of large-scale amplification in the centromeric regions of mouse chromosomes, and may also explain the formation of new, stable chromosome segments and chromosomes.

Transfer and Stable Transgene Expression of a Mammalian Artificial Chromosome into Bone Marrow‐Derived Human Mesenchymal Stem Cells

“…For reprints contact: Reprints@AlphaMedPress.com formed upon the integration of exogenous DNA sequences into the specific regions of acrocentric chromosomes-those containing both pericentromeric heterochromatin and the tandemly repeated ribosomal genes (rDNA). Ensuing breakage during mitosis generated new chromosomes ranging in size from 10 to 360 megabases [10][11][12]. Considering that these ACEs, termed satellite DNA-based artificial chromosomes (SATACs), can be manipulated or engineered to contain a variety of exogenous sequences, they present a unique opportunity for a variety of applications, including gene therapy [13,14].…”

De novo formed satellite DNA-based mammalian artificial chromosomes and their possible applications

“…Basically, any cultured mammalian cell line that contains rDNA region is suitable for the production of de novo formed SATACs. (Csonka et al 2000;Hadlaczky et al 1991;Hadlaczky 2001;Holló et al 1996;Keresö et al 1996;Lindenbaum et al 2004;Praznovszky et al 1991). There is no evidence in humans that amplification of these pericentromeric sequences is deleterious to an individual, as polymorphisms in the short arms of acrocentric chromosomes have been shown to consist of amplified pericentromeric heterochromatin and/or rDNA (Conte et al 1997;Friedrich et al 1996;Sands 1969;Sofuni et al 1980;Stergianou et al 1993;Trask et al 1989A;Trask et al 1989B;Verma et al 1977;Warburton et al, 1976) and have been inherited with no adverse effects (Bernstein et al 1981;Cooper and Hirschhorn 1962;Schmid et al 1994;Therkelsen 1964;Yoder et al 1974).…”

“…Ren et al 2006;Kouprina et al 2013;Kouprina et al 2014). Four different strategies have been developed for the construction of artificial chromosomes (Irvine et al 2005): (i) in the synthetic approach the artificial chromosome is assembled from chromosomal components (Basu et al 2005a;Basu et al 2005b; Harrington et al 1997;Henning et al 1999;Ikeno et al 1998;Ikeno et al 2009;Kaname et al 2005;Kouprina et al 2003;Nakashima et al 2005;Suzuki et al 2006), (ii) the 'top down' method applies the in vivo telomere-associated fragmentation of existing chromosomes (Au et al 1999;Auriche et al 2001;Carine et al 1986;Choo et al 2001;Farr et al 1995;Heller et al 1996;Ishida et al 2000;Katoh et al 2004;Mills et al 1999;Shen et al 2000;Voet et al 2001;Wong et al 2002) , (iii) naturally occurring minichromosomes may be engineered to construct an artificial chromosome (Raimondi 2011), and (iv) de novo chromosome generation can be induced via targeted amplification of specific chromosomal segments (Carl 2004;Csonka et al 2000;Holló et al 1996;Keresö et al 1996;Lindenbaum et al 2004;Praznovszky et al 1991;Tubak et al 1991). To date, two technologies, the 'top down' and the induced de novo chromosome generation approaches have advanced to the point suitable for biotechnological applications.…”

Generation of stable engineered chromosomes in soybean

“…Satellite artificial chromosome (SATAC) technology (Hadlaczky 2001;Holló et al 1996;Keresõ et al 1996) or artificial chromosome engineering (ACE) technology (Lindenbaum et al 2004), makes use of a universal property of pericentric heterochromatic regions associated with ribosomal DNA (rDNA) gene loci to undergo large scale amplification upon integration of foreign DNA . Insertion of foreign DNA into rDNA regions has been potentiated by cotransformation with a large excess of homologous targeting DNA (such as a portion of the 45S rDNA gene) (Csonka et al 2000;Lindenbaum et al 2004).…”