An STS content map of human chromosome 11: localization of 910 YAC clones and 109 islands.

Physical mapping of human chromosomes at a resolution of 100 kb to 1 Mb will provide important reagents for gene identification and framework templates for ultimately determining the complete DNA sequence. Sequence-tagged site (STS) content mapping, coupled with large fragment cloning in yeast artificial chromosomes, provides an efficient mechanism for producing first-generation, low-resolution maps of human chromosomes. Previously, we produced a set of standardized STSs for human chromosome 11 regionally localized by fluorescence in situ hybridization or somatic cell hybrid analysis. In this paper, we used these as well as other STS content, and identify 109 islands spanning an estimated 218 Mb on the 126-Mb chromosome. Since about 62% of the islands contain markers ordered on chromosome 11 by genetic or radiation hybrid analysis, this data set represents a first-order approximation of a physical map of human chromosome 11. This set of clones, contigs, and associated STSs will provide the material for the production of a continuous overlapping set of YACs as well for high-resolution physical mapping based upon sampled and complete DNA sequencing.

Large-scale screening of yeast artificial chromosome libraries using PCR.

The construction of physical maps of the human genome using sequence-tagged site content mapping requires that thousands of PCR amplifications be performed. On this scale, measures to reduce cost and to increase throughput become serious considerations. We describe relatively simple measures developed in our laboratory that increase the rate at which these reactions can be performed in a cost-effective manner. These measures have been extensively tested in our laboratory and are readily applicable in other laboratories including those performing library screening on a more modest scale.

Yeast artificial chromosome cloning of 3.2 megabases within chromosomal band 11q24 closely linking c-ets 1 and Fli-1 and encompassing the Ewing sarcoma breakpoint.

Human chromosome 11 harbors many genes of medical significance and cancer-related rearrangements. The availability of cloned DNA in cosmids and in yeast artificial chromosomes (YACs), combined with fluorescence in situ hybridization analysis, has led to the cloning of genes at sites of chromosomal breakpoints in acute leukemias in 11q23 and in Ewing tumors in 11q24. YAC cloning has facilitated the construction of contigs covering large portions of chromosomes for the detailed analysis of disease gene regions. Here we have cloned in YACs approximately 3.2 Mb of DNA within band 11q24, spanning the Ewing sarcoma breakpoint. Landmark cosmids 23.2 (D11S374) and 5.8 (D11S372), shown by FISH to flank the breakpoint within a 1.5- to 1.8-Mb segment, were used to seed two YAC "walks" both centromeric and telomeric to the breakpoint by YAC-end cloning and screening of two total genomic YAC libraries. The centromeric YAC contig, which consists of 23 overlapping YACs and orders 19 sequence-tagged sites (STSs), covers a minimum of 2.2 Mb and spans the Ewing sarcoma breakpoint. c-ets 1 and Fli-1, two members of the ets family, have been linked within 400 kb of intervening DNA within this contig, which also comprises a polymorphic microsatellite, D11S912 (CA)n, which we have localized within the Fli-1 gene. The telomeric YAC contig, which consists of 11 overlapping YACs, comprises 5 STSs and covers a minimum of 1 Mb distal to the breakpoint. Taken together, the two contigs, which consist of a total of 34 YACs and comprise 24 STSs, are separated by a maximum gap of 200-400 kb and cover as a whole 3.2 Mb of DNA. This represents about 70% of human chromosomal band 11q24, which extends over approximately 4.4 Mb of DNA.