Genome dynamics in aging mice

Abstract

Random spontaneous genome rearrangements are difficult to detect in vivo, especially in postmitotic tissues. Using a lacZ-plasmid reporter mouse model, we have previously presented evidence for the accumulation of large genome rearrangements in various tissues, including postmitotic tissues, during aging. These rearrangements, which were found to be organ-specific and to increase with age, have one breakpoint in the lacZ-reporter locus and the second elsewhere in the mouse genome. In this present work, we have used a mouse genome sequence database to physically characterize a total of 49 genome rearrangements in the brain, heart, and liver from young and old mice at two lacZ-plasmid reporter loci. Half of all breakpoints in the mouse genome occurred in chromosomes 3 and 4, each carrying a lacZ-reporter cluster, at distances varying from <100 kb to 66 Mb, indicating intrachromosomal deletions or inversions. The other half of the breakpoints in the mouse genome was found randomly on any of the other chromosomes, indicating translocations. Alternatively, part of the intra- and extrachromosomal events could involve transpositions. Regions of extended homology were not found at the breakpoints. These results lead us to postulate potential mechanisms for the origin of large genome rearrangements in mouse tissues and to predict their possible impact as a potential cause of aging.

Figure 1

Recovery of integrated LacZ-plasmids. Each integration site comprises a concatamer of ∼10 head-to-tail organized plasmid copies (only three plasmid copies are depicted). Individual plasmid copies are released from mouse genomic DNA (red curving lines) by HindIII digestion (H indicates HindIII site). After purification, self-ligation, and transformation into Escherichia coli host cells, the plasmids are recovered in the form of ampicillin-resistant colonies. The dashed arrows represent the occurrence and detection of a hypothetical genome rearrangement with one breakpoint in a lacZ-reporter gene (lightning-sign) and one in the mouse genome 5′ (①) or 3′ (②) of the integration site or on another chromosome (③). When not deleted, only the upstream truncated plasmid sequence will result in a mutant colony after HindIII digestion, as the origin of replication (yellow ellipse) and the ampicillin-resistance gene (A) are needed for selective propagation of the transformed E. coli host. The orientation of the cloned 5′ sequences of the integration sites (Seq. Int.; orange curve), indicated by the arrow, corresponds with the direction of the blue arrowheads in Fig. 3 and is arbitrary within this scheme. The physical location and orientation of the cloned 5′ integration site sequence and the recovered mouse genomic sequence in the mutant plasmid allow for characterization of the recovered genome rearrangement. See text for more details.

Figure 2

Chromosomal location of genomic mouse fragments recovered as parts of mutated plasmids from the brain, heart, and liver. The bars for chromosomes 3 and 4, containing the plasmid integration sites, are in orange.

Figure 3

Schematic representation of physical locations and orientations of the integrated transgene clusters and genomic mouse fragments, recovered as parts of mutant plasmids, on chromosomes 3 and 4. The red (proximal) and green (distal) arrowheads indicate the direction of the recovered mouse fragments, such that the base and tip of the arrowhead represent the breakpoint and the HindIII site, respectively (yellow downward arrow indicates direction unknown). The blue arrow indicates the location and orientation of the integrated transgene cluster (see also Fig. 1). The rounded physical distances from the integration site to the localized breakpoints in the mouse genome are shown. C indicates centromere; T, telomere.

Figure 4

Schematic depiction of proposed mechanisms for observed intrachromosomal rearrangements. The blue and red arrows indicate the orientation of the integrated plasmid loci and the recovered mouse sequences, respectively, on the original non-rearranged chromosome (left column). All four combinations are given for an arbitrarily orientated chromosome (green line). The middle column shows how two breakpoints (lightning signs) could lead to the inversion or deletion of the encompassed chromosomal sequence (yellow-orange dual tone line) and result in a recoverable mutation in the right column. The last row indicates the two options for a transposition, in which either the transgene locus or the recovered mouse sequence is copied or excised (as indicated by the pink and light blue arrows) and integrates in the breakpoint at the other location.