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. 2001 Jan 2;98(1):171-5.
doi: 10.1073/pnas.98.1.171.

Pericentromeric organization at the fusion point of mouse Robertsonian translocation chromosomes

S Garagna  1 , N Marziliano, M Zuccotti, J B Searle, E Capanna, C A Redi
Affiliations

Pericentromeric organization at the fusion point of mouse Robertsonian translocation chromosomes

S Garagna et al. Proc Natl Acad Sci U S A. .

Abstract

In mammals, Robertsonian (Rb) translocation (the joining of two telo/acrocentric chromosomes at their centromere to form a metacentric) is the most effective process in chromosomal evolution leading to speciation; its occurrence also affects human health (through the induction of trisomies) and the fertility of farm animals. To understand the mechanism of Rb translocation, we used the house mouse as a model system and studied the organization of pericentromeric satellite DNAs (satDNA) of telocentrics and Rb chromosomes, both minor and major satDNA. The chromosome-orientation fluorescence in situ hybridization (CO-FISH) technique was used to analyze the major satDNA. To detect the very small amount of minor satDNA, a procedure was developed that combines CO-FISH with primed in situ labeling and conventional FISH and is five times more sensitive than the CO-FISH procedure alone. It was found that both the major and the minor satDNA tandem repeats are oriented head-to-tail in telocentric and Rb chromosomes, and their polarity is always the same relative to the centromere. We suggest that all tandemly repetitive satDNAs in a species probably are locked into such a symmetry constraint as a universal consequence of chromosomal evolution. Rb translocation breakpoints were found localized within the minor satDNA of telocentrics, and these sequences contributed symmetrically to the formation of the centromeric region of the Rb chromosomes. These results are important for an understanding of the geometry of Rb translocations and suggest the study of DNA orientation as a new tool for investigating these rearrangements.

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Figures

Figure 1
Figure 1
Schematic representation of the CO-FISH/PRINS/FISH method as applied to mouse chromosomes. Single-stranded (ss) chromatids (A) were hybridized with a ss minor satDNA oligo (thick dash) (B). Sat-2 (thick dash) and sat-3 (thin dash) oligos then were used as primers for PRINS amplification (C) followed by conventional FISH with the R198 probe (stars) (D). Although the amplification steps involved both chromatids at a hybridization site, the efficiency of amplification was higher for the double-stranded chromatid reconstituted by ss oligo hybridization, so that a bright signal was detected only for that chromatid. For telocentric chromosomes, hybridization signals were always present on only one chromatid (I), whereas for Rb chromosomes hybridization signals were present either on both chromatids each side of the centromere (i.e., in contra-lateral disposition: G) or on only one of the two chromatids (H). A single signal on telocentrics (E) and two contra-lateral signals on Rb chromosomes (F) also were detected after CO-FISH with the major satDNA probe. These patterns of hybridization indicate that the tandem repeats of both major and minor satDNAs are oriented head-to-tail along the DNA strand. The contra-lateral disposition relative to the centromere of the hybridization signals in Rb chromosomes shows that the DNA polarity is maintained through the centromere. Both telocentrics contributed minor satDNA to the newly formed centromeric regions of Rb chromosomes. The centromeres are represented by black dots.
Figure 2
Figure 2
(A) CO-FISH staining of a chromosome spread from a POS mouse using a major satellite oligonucleotide (sat-1). A contra-lateral signal was obtained for Rb chromosomes (examples: arrows) and a single lateral signal on telocentrics (examples: arrowheads). (B and E) Conventional FISH with the minor satDNA probe (R 198) on a telocentric (B) and an Rb chromosome (E): two bright spots are visible in the centromeric regions of both chromosomes. (D, G, and I) CO-FISH/PRINS/FISH staining of the minor satDNA in the centromeric regions of telocentric and Rb chromosomes. In telocentric chromosomes, hybridization signals were present on one chromatid only (D); in Rb chromosomes, hybridization signals were present either on one (G) or on both chromatids (I). (C, F, and H) DAPI (4′-6-diamidino-2-phenylindole) staining of chromosomes after the CO-FISH/PRINS/FISH procedure. The AT-rich heterochromatin located in the pericentromeric region of mouse chromosomes fluoresces brightly (arrow).
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