Molecular mechanism in the formation of a human ring chromosome 21.

Abstract

We have characterized the structural rearrangements of a chromosome 21 that led to the de novo formation of a human ring chromosome 21 [r(21)]. Molecular cloning and chromosomal localization of the DNA regions flanking the ring junction provide evidence for a long arm to long arm fusion in formation of the r(21). In addition, the centromere and proximal long arm region of a maternal chromosome 21 are duplicated in the r(21). Therefore, the mechanism in formation of the r(21) was complex involving two sequential chromosomal rearrangements. (i) Duplication of the centromere and long arm of one maternal chromosome 21 occurred forming a rearranged intermediate. (ii) Chromosomal breaks in both the proximal and telomeric long arm regions on opposite arms of this rearranged chromosome occurred with subsequent reunion producing the r(21).Ring chromosomes in humans represent a class of aberrant chromosomes observed frequently in congenital anomalies and occasionally with normal phenotypes (1). Most ring chromosomes arise de novo, yet occasional familial transmission has been reported (2-5). The frequency of ring chromosomes is 1 in 25,000 recognized conceptions (6), and all human chromosomes have been observed as rings. Almost 50% of ring autosomes are derived from the acrocentric chromosomes (7). The proposed mechanism of ring formation, breakage of both short and long arms of a chromosome with subsequent end to end fusion (8), remains unproven. In several reports of acrocentric ring chromosomes, a Robertsonian translocation produced by short arm fusion has been observed in the patient, a parent, or a sibling (9)(10)(11)(12)(13)(14)(15)(16) (19). The R2-10 line was also back selected for loss of the r(21) (cell line R2-1OW-S5) by growth in nonselective (purine supplemented) medium, followed by selective medium with the addition of bromodeoxyuridine and light (22). For mapping, a panel of somatic cell hybrids, 72532X-6, 153E7bx, and 2FuR1 (23), with characterized rearrangements of human chromosome 21 was used (19).Cloning the Chromosomal Break Points. DNA from the patient's blood lymphocytes was digested with EcoRI and a size-selected library (7-9 kb) was made in Agtwes B. About 300,000 recombinant phage were screened (24) with pPW231C and a single clone, A21BP (Fig. 1B), was purified. DNA from the region on the 3' side of the break point was cloned (A2lpq) by using probe CW21A from a flow-sorted, HindIII-digested chromosome 21 library in Charon 21A (ID code, LL21NS02; American Type Culture Collection number, 57713).Immunofluorescence Studies. For quantification of DNA, photographic negatives of 4',6-diamidino-2-phenylindole (DAPI)-stained (25) images were scanned using a Loats (Westminster, MD) video densitometer equipped with a twodimensional gel-scanning software package. Indirect immunofluorescence was performed on mitotic spreads (26). Chromosomes were first stained with DAPI and then with a monoclonal anti-centromere antibody prepared against the cloned 80-kDa human autoantigen

Dysfunction of chromosomal loop attachment sites: illegitimate recombination linked to matrix association regions and topoisomerase II.

“…Significantly, we demonstrate here that the in vivo breakpoint of a previously described chromosomal translocation (31) corresponds to a "hotspot" of in vitro DNA cleavage by Topo II within the MAR of the mouse immunoglobulin K-chain gene. Furthermore, we found that a MAR has been deleted during rabbit immunoglobulin K-chain gene evolution (32) and that MARs reside at the recombination junction of a human ring chromosome 21 adjacent to a long interspersed repetitive element (LINE) (33). These results provide evidence for a dysfunction of MARs in illegitimate recombination, since these sequences are sometimes found at points of DNA insertion, deletion, and translocation.…”

“…4C). These MARs are separated by a 750-bp region that contains the 3' end of a LINE (33), which resides on a 0.8-kb Nco I fragment that does not exhibit significant binding (Fig. 4 B and C).…”

“…Rabbit K1-and K2-chain gene segments correspond to Sst I fragments of 5.4 and 3.1 kb, respectively, from the joiningconstant intron region of the b4 allotype (32) inserted into a pBR322 derivative. Plasmid pR21BP consists of a 3.8-kb EcoRI-HindflI fragment encompassing the recombination junction ofa human ring chromosome 21 inserted into pBR322 (33). RESULTS Sequences That Specifically Bind Topo H Include MARs.…”

Transcriptional Regulation of BRCA1

“…Topoisomerase II sites also occur near rearrangement breakpoints in the Dystrophin gene (Hu et al, 1991) and ring chromosome 21 (Wong et al, 1989). In vitro and in vivo evidence for the role of topoisomerase II in nonhomologous recombination is well established in prokaryotes (O'Connor et al, 1985;Ikeda, 1986) and a role for vertebrate topoisomerase II in nonhomologous recombination has been demonstrated in vitro (Bae et al, 1988).…”

Isolation and characterization of a DNA fragment containing various kinds of repetitive sequences located on human chromosome 21

“…The human and rodents cell lines, as well as human/ rodent somatic cell hybrids, WALT, 2Fur, ACEM, R2-10, and 21q + cells were used and their properties have been described previously (Oates and Patterson, 1977;Kozak et al, t977;Van Keuren et al, 1986;Patterson et al, 1983;Wong et al, 1989;Drabkin et al, 1895).…”