An integrated YAC-overlap and ‘cosmid-pocket’ map of the human chromosome 21

Abstract

We describe here the construction of an ordered clone map of human chromosome 21, based on the identification of ordered sets of YAC clones covering > 90% of the chromosome, and their use to identify groups of cosmid clones (cosmid pockets) localised to subregions defined by the YAC clone map. This is to our knowledge the highest resolution map of one human chromosome to date, localising 530 YAC clones covering both arms of the chromosome, spanning > 36 Mbp, and localising more than 6300 cosmids to 145 intervals on both arms of the chromosome. The YAC contigs have been formed by hybridising a 6.1 equivalents chromosome 21 enriched YAC collection displayed on arrayed nylon membranes to a series of 115 DNA markers and Alu-PCR products from YACs. Forty eight mega-YACs from the previously published CEPH-Genethon map of sequence tagged sites (STS) have also been included in the contig building experiments. A YAC tiling path was then size-measured and confirmed by gel-fingerprinting. A minimal tiling path of 70 YACs were then used as probes against the 7.5 genome equivalents flow sorted chromosome 21 cosmid library in order to identify the lists of cosmids mapping to alternating shared--non-shared intervals between overlapping YACs ('cosmid pockets'). For approximately 1/5 of the minimal tiling path of YACs, locations and non-chimaerism have been confirmed by fluorescence in situ hybridisation (FISH), and approximately 1/5 of all cosmid pocket assignments have independent, confirmatory marker hybridizations in the ICRF cosmid reference library system. We also demonstrate that 'pockets' contain overlapping sets of cosmids (cosmid contigs). In addition to being an important logical intermediate step between the YAC maps published so far and a future map of completely ordered cosmids, this map provides immediately available low-complexity cosmid material for high resolution FISH mapping of chromosomal aberrations on interphase nuclei, and for rapid positional isolation of transcripts in the highly resolved regions of genetic interest.

A High-Resolution Physical Map of Human Chromosome 21p Using Yeast Artificial Chromosomes

“…The locations of YACs 831B6, 781G5, and 52H5 in our contig map are compatible with these maps. In the hybridization-based physical map of chromosome 21 reported by Nizetic et al (1994), D21S5, D21S187, D21S1276, D21S188, D21S191, D21S1277, and D21S1278 were localized in the 21p region. The order of these markers and YACs are the same as in our map; however, the orientation is reversed.…”

“…Xba21 did not amplify the somatic cell hybrid DNA, and pA 2 was positive on mouse and hamster DNA; therefore, it could not be mapped unambiguously. (Chumakov et al 1992;Nizetic et al 1994). PCR amplification of the selected YACs identified 42 YACs that were positive for the markers with which they were selected.…”

“…The highly repetitive nature of chromosome 21p and its extensive homology with other acrocentric chromosomes have hampered the search for chromosome 21p-specific markers and the construction of genetic and physical maps of this region. Most maps of chromosome 21 span the entire long arm (21q) (Chumakov et al 1992;Nizetic et al 1994;Gardiner et al 1995;Korenberg et al 1995), whereas only a few of them extend a short distance into the p-arm (Nizetic et al 1994;Doering et al 1995).…”

Localization of 102 exons to a 2.5 Mb region involved in Down syndrome

“…Therefore, the cosmids are annotated with letters to reflect those clones that have also been reported (12). Estimates of fragment sizes between various markers based on pulsed field gel electrophoresis and restriction fingerprinting of YACs (15,19,25) are given below the bottom scale along with the estimated size of the same fragment from this map in parentheses. The cosmid location of cDNA clones reported by other groups (11,13) is indicated by a square if the cosmid was also present in this collection of clones.…”

“…This cDNA detects a predominant 8 kb mRNA species that appears to be expressed at varying levels in all adult tissues assessed. The extensive amount of physical and genetic information and resources available for chromosome 21 was instrumental in allowing the construction of the next phase of higher resolution physical and transcript maps by us and others (11)(12)(13)(18)(19)(20). Our approach was to: (i) use the YAC map to identify a subset of cosmids for a 2.5 Mb region; (ii) bin the cosmids according to regions of YAC overlap; (iii) assay the cosmids for the presence of known markers; (iv) isolate exons from the cosmid bins; and (v) use the isolated exons as markers to further order cosmids within and between bins.…”