De novo chromosome formations by large-scale amplification of the centromeric region of mouse chromosomes

Abstract

Chromosomes formed de novo which originated from the centromeric region of mouse chromosome 7, have been analysed. These new chromosomes were formed by apparently similar large-scale amplification processes, and are organized into amplicons of approximately 30 Mb. Centromeric satellite DNA was found to be the constant component of all amplicons. Satellite DNA sequences either bordered the large euchromatic amplicons (E-type amplification), or made up the bulk of the constitutive heterochromatic amplicons (H-type amplification). Detailed analysis of a heterochromatic megachromosome formed de novo by an H-type amplification revealed that it is composed of a tandem array of 10-12 large (approximately 30 Mb) amplicons each marked with integrated "foreign' DNA sequences at both ends. Each amplicon is a giant palindrome, consisting of two inverted doublets of approximately 7.5-Mb blocks of satellite DNA. Our results indicate that the building units of the pericentric heterochromatin of mouse chromosomes are approximately 7.5-Mb blocks of satellite DNA flanked by non-satellite sequences. We suggest that the formation de novo of various chromosome segments and chromosomes seen in different cell lines may be the result of large-scale E- and H-type amplification initiated in the pericentric region of chromosomes.

Alignment of biological microparticles by a polarized laser beam

“…Mammalian metaphase chromosomes were isolated from cultured Chinese hamster-mouse hybrid cell line H1D3 (Kereso et al 1996) by a standard method (Hadlaczky et al 1982). Cells were synchronized for mitosis with 0.1% colchicine and were burst in a glycine-hexylene glycol buffer supplemented with 0.1% Triton X-100 (GHT buffer).…”

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.…”

Chromosome engineering: prospects for gene therapy

“…Amplification of DNA at the integration site can result in chromosome breakage events, producing new large chromosomes typically in the 60-400 Mb size range. 18,19 Due to their size and high satellite DNA content, these can be purified in quantities sufficient for microinjection into mouse pronuclei, 20 or lipofection into a range of cell types. 21 The following sections will review our current understanding of the centromere and the state of progress and possible future directions in chromosome engineering technology.…”