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Home / Science
High-Resolution Physical Maps of Chromosomes 16 and 19 Completed
By:Aaron Hall
Chromosome 16.
The LANL integrated chromosome 16 map announced at the Santa Fe workshop is composed of a cytogenetic breakpoint map, a low-resolution physical map in mega-YACs, and a high-resolution physical map in cosmids and mini-YACs. Over 600 genes, DNA markers, and microsatellite repeats generated by investigators worldwide have been integrated into the new map. The magnitude of this accomplishment is underscored by comparisons with physical maps already available: chromosome 16 is twice the size of chromosome 21 and 3 times the size of the Y euchromatic region, and resolution is over 100 times greater than previously achieved for those chromosomes.
Cytogenetic map.
The backbone of the new map is a cytogenetic breakpoint map developed with DOE funding by David Callen's group at Adelaide Women and Children's Hospital (AWCH), Australia. The breakpoint map is derived from 78 mouse-human somatic cell hybrids representing 90 independently ascertained chromosome breakpoints. With the 4 fragile sites, these divide the 85 Mb of euchromatin into 91 intervals (average resolution about 1 Mb).
YAC map.
At the second level is a low-resolution YAC contig map composed of about 600 CEPH mega-YACs and 220 flow-sorted chromosome 16-specific YACs that are smaller but of higher quality, with a very low chimeric rate and deletion frequency; these YACs are localized to and ordered within the breakpoint intervals with 350 STSs; average interval between markers is about 260 kb. The chromosome 16 mega-YAC map provides clonal coverage of 98% of the 90-Mb euchromatin regions, with 2 gaps on the p arm and 4 on the q arm.
Cosmid map.
A high-resolution "sequence-ready" cosmid contig map comprises the next resolution level (average resolution, 5 kb) and consists of 4000 fingerprinted cosmids assembled into contigs covering 60% of the chromosome. The cosmids are anchored to the YAC and cytogenetic breakpoint maps via STSs developed from cosmid contigs and by hybridizations between YACs and cosmids. About 2000 cosmids in 320 contigs have been localized to the cytogenetic map. The group has 90% coverage for the chromosome in fingerprinted cosmids, but not all have been placed on the integrated map.
Genetic map integration.
Correlated with the physical map is a highly informative microsatellite-based genetic map developed at AWCH; it consists of 78 PCR-typable markers at 2.6-cM median intervals (3.2 average). Almost 200 genetic markers provide a resolution of <1 cm on the genetic map.
In 1988 the LANL group chose to map chromosome 16 for several reasons. Among them is the chromosome's 98-Mb size, which is compatible with a cosmid-fingerprinting mapping approach such as that used for the 100-Mb Caenorhabditis elegans genome. Resources critical to the project include a monochromosomal hybrid containing 16 as a single human chromosome; flow-cytometry instrumentation for constructing specific cosmid and YAC libraries; and robotics for making high-density gridded arrays from the libraries, pooling YAC libraries, screening by PCR, and setting up reactions in other mapping steps. Other essential resources are a mapping panel containing 78 hybrid cell lines, 90 independently ascertained breakpoints on the chromosome, and the Genethon YAC map. LANL collaborated closely with AWCH investigators in integrating the physical, genetic, and cytogenetic maps.
Many other LANL physical-mapping resources provide powerful tools for using mapping data, including a mega-YAC contig arrayed on gridded membranes, with YACs ordered as they occur on the chromosome; two flow-sorted chromosome-specific Cla I and Sac II YAC libraries arrayed on gridded membranes and pooled for PCR-based screening; 10x-coverage cosmid libraries arrayed on gridded membranes and 90% coverage in EcoR I and Hind III restriction-mapped clones; and a high-resolution somatic hybrid cell mapping panel that provides a standard for localizing new clones to the map.
All chromosome 16 mapping data are available worldwide on Internet (http://www-ls.lanl.gov) or through Norman Doggett (505/665-3024, doggett@gnome.lanl.gov).
Chromosome 19
The LLNL high-resolution chromosome 19 cosmid-based physical map spans 90% of the euchromatin in 63 contiguous islands consisting of BACs, PACs, YACs, and cosmids where size, order, and distance are known. Only 50 Mb of the 60-Mb chromosome contain unique sequence, with the remainder consisting of centromeric and telomeric repeats.
Metric map construction.
The chromosome 19-specific cosmids were clustered into contigs using automated restriction-fragment fingerprinting analysis. At the next mapping level, contigs were ordered relative to each other, and physical distances between certain clones within contigs were measured by FISH to metaphase chromosomes (resolution, 400 to 500 kb), somatic interphases, and sperm pronuclear interphases (resolution, 50 kb). LLNL's goal was to obtain sequentially ordered contigs spanning telomere to telomere, with known distances separating cosmid pairs and the resulting þmetricþ map serving as a backbone for further map building and other efforts. The average distance separating pairs of cosmids is 250 kb for 80 cosmids from the p arm (range, 50 to 700 kb) and 270 kb for 126 cosmids on the q arm (range, 50 to 840 kb). [Genomics 23(3), 582-91 (October 1994).]
Closing gaps.
Gaps between ordered cosmid contigs were spanned using the larger insert libraries with a variety of hybridization and STS technologies; simple cosmid walking achieved continuity at the cosmid level. Over 38 Mb of EcoR I restriction maps were used to close gaps in some regions. Remaining gaps will be closed by those with a particular interest in a region.
Integrating genes and genetic markers.
Significant strides have been made toward integrating genetic and physical maps. Over 400 genes and genetic markers have been localized on cosmids, and almost 300 have been incor-porated into the metric map. More than 340 STSs, ESTs, and clone markers have been mapped and ordered.
Chromosome 19 may be far richer in genes than the previous estimates of about 2000. Researchers are now finding a gene every 25 kb, and at least 20 to 25 gene families with a minimum of 3 to 5 members are represented on the chromosome. These biologically interesting genes will be targeted for study as the high-resolution EcoR I map is being completed. Genomic sequencing is now being done in selected regions of interest, especially on three DNA repair genes. Software has been designed to integrate and display cosmid, YAC, FISH, and restriction maps, as well as sequence, hybridization, and screening data.
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