Use of YAC fragmentation to delimit a duplicated region on human chromosome 21

One advantage of using yeast artificial chromosomes (YACs) to study complex genomes is the ability to clone large DNA fragments. In addition, since yeasts are able to integrate DNA by homologous recombination, YACs can be modified by targeted transformation, sequences being added or replaced by cloned DNA fragments. Similarly, deletion derivatives of YACs can be obtained after transformation with chromosome fragmentation vectors. CFVs contain a yeast telomere, a selectable marker, and a targeting segment homologous to a sequence or sequences present in the YAC. Centric CFVs contain a centromere, whereas acentric CFVs do not. Recombination of the CFV targeting sequence with a homologous YAC segment introduces the yeast telomere, deleting all YAC sequences proximal (centric vector) or distal (acentric vector) to the recombination site. Human Alu or LINE elements can be used as targets to create sets of nested deletion derivatives that facilitate restriction mapping, localization of genes on YACs, and elimination of chimeric portions of a YAC insert. Cloning and sequencing extremities of fragmented YACs will provide new markers along the original YAC. Here, this technique is used to delimit a duplicated region on the long arm of normal human Chromosome (Chr) 21. We have previously identified this duplicated region, and cloned it in YACs, showing that it is 135-500 kb long and that the proximal and the distal copies are at least 12 Mb apart, in 21q11.1 and 21q22.1 respectively. We also showed that YAC 2D7y21 contains the complete distal copy of the duplication, and we constructed a high-resolution restriction map of this YAC that allowed identification of CpG islands. This duplicated region could be the origin of chromosomal rearrangements, such as the one described in a patient carrying an interstitial deletion on the long arm of Chr 21 which starts and ends in each of the two copies of the duplication. In addition, lack of the distal portion of this duplication in cases of partial monosomy 21 could be responsible for some clinical features observed in these patients: transverse palmar crease, arthrogryposis, hypertonia, and mental retardation. Finally, sequences homologous to this duplicated region have been detected only in chimpanzee and baboon. Although it is still unclear whether this duplication exists in nonhuman primates, these data indicate that it probably appeared recently in evolution, leaving putative sequence motifs important for the duplication