Using restriction enzyme c/s-morphisms and direct sequencing analyses in SMS patients with a common deletion, the regions of strand exchange were mapped in 16 somatic-cell hybrids that retain the deletion chromosome which effectively isolates the recombinant SMS-REP from the copies on the non-deleted homolog. The crossovers were distributed throughout the region of homology between the proximal and distal SMS-REPs. However, despite approx 170 kb of more than 98% identity, 50% of the recombinant junctions occurred in a 12.0-kb region within the KER gene clusters in the C region (Fig. 1B). DNA sequencing of this recombination hotspot (27), or positional preference for strand exchange, in seven recombinant SMS-REPs narrowed the crossovers to an approx 8-kb interval. Four of them occurred in a 1655-bp region rich in polymorphic nucleotides that could potentially reflect frequent gene regions of homology between proximal and middle SMS-REPs. The proximal copy is the largest and is localized in the same orientations as the distal copy. The middle SMS-REP shows almost the same sequence and structure as the proximal copy except for two terminal deletions, an UPF3A gene interstitial deletion and a small (approx 2 kb) insertional duplication. However, it is inverted with respect to proximal and distal SMS-REPs. SMS-REP-specific CLP, TRE, and SRP cis-morphisms were confirmed by DNA sequencing. Fourteen genes/pseudogenes were found. The two additional KER copies in distal SMS-REP represent repeated fragments of the KER pseudogenes. Cross-hatched areas (NOS2A in the proximal and KER in the distal) denote two genes spanning the high homology and non-homology area between the distal and proximal SMS-REP copies, which suggest a three-step event for the hypothetical model of the evolution of the SMS-REPs. At the bottom, the chromosome 17 distribution of SMS-REP fragments, which constitute chromosome 17 LCR17s, is shown. The above data were obtained through BLAST analysis of sequence database. (B) Refining the regions of unequal crossover in somatic-cell hybrids. Top, the genomic structures of SMS-REPs, with the distal and proximal copies in direct orientation, telomere (tel) (left) and centromere (cen) (right). Bottom, regions of strand exchange within the recombinant SMS-REP of the deletion chromosome isolated in hybrids. Restriction cis-morphism markers enabling distinction between the distal and proximal SMS-REP copies are indicated (circles), with their positions corresponding to the proximal SMS-REP at the top of the figure. Some sites in the recombinant SMS-REP were derived from the distal SMS-REP (dottedcircles), and others were derived from the proximal copy (hatched circles). For each somatic-cell hybrid, the region of strand exchange within the recombinant SMS-REP and its size are indicated (blackened horizontal bar). The recombination event in hybrid 255-11D is centromeric to the CLP region (D) in the distal SMS-REP.
DNA Sequence Homology Comparison Among Different LCR17p Copies
Proximal Middle Distal LCR17pB LCR17pC LCR17pD CMT1A-REP SMS-REP SMS-REP
SMS, Smith-Magenis syndrome; CMT1A, Charcot-Marie-Tooth disease type 1A.
conversion. For further evaluation of the strand exchange frequency in patients with SMS, novel junction fragments from the recombinant SMS-REPs were identified (8).
As predicted by the reciprocal-recombination model, junction fragments were also identified from this hotspot region in patients with dup(17)(p11.2p11.2), documenting reciprocity of the positional preference for strand exchange. Several potential cis-acting recombination-promoting sequences were identified within the hotspot. Of note, a 2.1-kb AT-rich inverted repeat was found flanking the proximal and middle KER gene clusters but not the distal one (8).
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