Jingwei Yu

A region-specific microdissection library for human chromosome 2p23-->p21 and the analysis of an interstitial deletion of 2p21.

A region-specific library for human chromosome 2p23–p25 was constructed using microdissection and polymerase chain reaction (PCR)-mediated microcloning techniques. This library is large, comprising 300,000 recombinant microclones. The insert sizes range between 50–600 base pairs (bp) with a mean of 200 bp. About 50%–60% of the clones contain unique or very low copy number sequence inserts as determined by their weak or no hybridization to total human DNA. A subset of 48 microclones that did not hybridize to total human DNA after colony hybridization was analyzed, and 26 (54%) clones were shown to contain single-copy inserts and hybridize to human chromosome 2 DNAs, indicating that they are human chromosome 2 specific. The human genomic fragments identified by these clones after cleavage with HindIII have also been characterized. The single-copy microclones were used to analyze an interstitial deletion in the 2p23.3–p25.1 region — 46,XY, del(2) (pter→p25.1::p23.3→qter) — previously reported in a patient with severe growth and mental retardation and multiple anomalies. Of the 26 microclones analyzed, 14 clones were mapped to the deletion region. The availability of the 2p23–p25 region-specific library and the probes derived from the library should be valuable for fine structure physical mapping analysis and the cloning of disease-related genes localized to the region. These studies also demonstrate the efficiency with which useful probes can be quickly generated for genome studies and for positional cloning.

Three-region specific microdissection libraries for the long arm of human chromosome 2, regions q33-q35, q31-q32, and q23-q24

Three region-specific libraries have been constructed from the long arm of human chromosome 2, including regions 2q33-35 (2Q2 library), 2q31-32 (2Q3) and 2q23-24 (2Q4). Chromosome microdissection and the Mbol linker-adaptor microcloning techniques were used in constructing these libraries. The libraries comprised hundreds of thousands of microclones in each library. Approximately half of the microclones in the library contained unique or low-copy number sequence inserts. The insert sizes ranged between 50 and 800 bp, with a mean of 130–190 bp. Southern blot analysis of individual unique sequence microclones showed that 70–94% of the microclones were derived from the dissected region. 31 unique sequence microclones from the 2Q2 library, 31 from 2Q3, and 30 from 2Q4, were analyzed for insert sizes, the hybridizing genomic HindIII fragment sizes, and cross-hybridization to rodent species. These libraries and the short insert microclones derived from the libraries should be useful for high resolution physical mapping, sequence-ready reagents for large scale genomic sequencing, and positional cloning of disease-related genes assigned to these regions, e.g. the recessive familial amyotrophic lateral sclerosis assigned to 2q33-q35, and a type I diabetes susceptibility gene to 2q31-q33.

Region-specific microdissection library and single-copy microclones for human chromosome 2p11–p13

We report the construction and characterization of a region-specific microdissection library for human chromosome 2p11–p13. This library (designated 2P4 library) is large, comprising 600,000 recombinant microclones. Thirty to 40% of the clones contain unique sequences. The insert sizes range from 100 to 800 bp, with a mean of 380 bp. A subset of the microclones was selected, based on their weak or no hybridization to total human DNA, for further analysis. Of 50 single-copy microclones analyzed, 35 clones (70%) were derived from human and are chromosome 2-specific. The insert sizes and the hybridizing genomic HindIII fragments of these clones were also determined. The 2P4 microdissection library and the single-copy microclones from the library are useful in preparing STS (sequence-tagged site) to isolate corresponding YAC (yeast artificial chromosome) or other clones with large inserts and for isolating region-specific cDNA clones as candidate genes for cloning disease-related genes assigned to this region.

Complete set of eleven region-specific microdissection libraries for human chromosome 2.

The construction and characterization of 11 region-specific libraries for the entire human chromosome 2 have been completed, including four libraries for the short arm and six libraries for the long arm, plus a library for the centromere region. These libraries were constructed using the chromosome microdissection and microcloning technology. Eight libraries have been described previously. This paper presents the final three libraries: 2q21–q22 (designated 2Q5 library), 2q11–q14 (2Q6), and 2p11.1–q11.1 (2CEN). The sizes of the dissected regions ranged between 20 and 30 Mb, with the centromere region of about 4 Mb. All these libraries are large, potentially comprising hundreds of thousands of recombinant microclones. Between 77% and 97% of the microclones were shown to derive from respective dissected regions. From 26 to 66 unique sequence microclones were isolated and characterized in detail for each library. The microclones have short inserts, ranging between 50 and 600 bp, with a mean of about 200 bp. The short inserts can be conveniently sequenced as STSs to provide high density probes for the dissected region. A plasmid sub-library containing at least 20,000 microclones, and usually more, has been prepared from each library and deposited to ATCC for general distribution. The libraries have been used effectively in constructing high resolution physical maps and for contig assembly, as well as in positional cloning of disease genes assigned to the dissected region. Comparing to other chromosomes with detailed mapping information and densely populated probes, chromosome 2 remains largely under-exploited. The availability of a complete set of region-specific libraries and unique sequence microclones from the libraries should provide valuable resources for genome analysis, high resolution physical mapping, region-specific cDNA isolation, and positional cloning for chromosome 2.

YAC contig mapping of six expressed sequences encoded by human chromosome 21

Six cDNA clones from human chromosome 21 have been mapped in a set of complete YAC contig spanning the entire chromosome 21q. The mapping positions between two STSs on the YAC contig and the NotI coordinates starting from the telomere of 21q were determined for the cDNA clones. The YAC contig mapping positions agree well with those using a comprehensive somatic cell hybrid mapping panel.

Construction and characterization of region-specific microdissection libraries and single-copy microclones for short arm of human chromosome 2

The short arm of human chromosome 2, comprising approximately 93 million bp, has been divided into four regions to construct region-specific microdissection libraries to facilitate physical mapping and gene cloning. These four regions include 2p23-p25 (designated 2P1), 2p21-p23 (2P2), 2p14-p16 (2P3), and 2p11-p13 (2P4). Together with three previously constructed microdissection libraries of 2P1, 2P2 and 2P4, a fourth library for the region 2p14-p16 (2P3) has been constructed and characterized to complete all four region-specific libraries for the entire 2p. The 2P3 library is very large, potentially comprising 1,000,000 recombinant microclones with insert sizes ranging between 50 and 800 bp and a mean of 250 bp. Approximately 40% of the microclones contain unique sequences. Of the 77 single-copy microclones analyzed, 66 clones (86%) hybridized to both human and chromosome 2 DNAs, indicating that they were derived from human and are chromosome 2 specific. The hybridizing HindIII genomic fragments for the 66 microclones have also been determined.

Construction and characterization of three region-specific microdissection libraries for human chromosome 18

Three region-specific libraries for the entire human chromosome 18 were constructed using microdissection and MboI linker-adaptor microcloning techniques. The libraries included 18pter-p11.1 (designated 18P library), 18q 11.1-q12.3 (18Q1 library), and 18q21.1-qter (18Q2 library). Samples of the microclones from each library were analyzed in detail. The insert sizes ranged between 50–600 bp, with a mean of 180–220 bp for the three libraries. The libraries contained approximately 40–60% microclones with unique sequence inserts. More than 30 unique sequence microclones from each library were analyzed by Southern blot hybridization to demonstrate that they are human specific and were derived from chromosome 18. The human gemomic HindIII fragments hybridized to each microclone were determined and microclones crosshybridized to rodent species were identified. These region-specific libraries and the unique sequence microclones from the libraries are useful reagents for (1) isolating hughly polymorphic microsatellite markers for refined linkage analysis, (2) identifying corresponding YAC, BAC or other clones with large inserts for contig assembly and high resolution physical mapping, (3) isolating cDNA clones from the dissected region, and (4) convenient sequencing of the microclones to prepare high density markers and sequence-tagged sites (STSs). Such applications have been demonstrated in a series of similarly constructed microdissection libraries from other regions of the human genome.

Detailed analysis of a 17q21 microdissection library by sequence bioinformatics and isolation of region-specific clones

A region-specific microdissection library originating from human chromosome 17q21, was constructed using the MboI linker-adaptor microcloning techique. DNA sequencing of 241 microclones resulted in the identification of 74 novel coding sequences, paralogs of known genes, and known, but previously unmapped, genes or expressed sequence tags that were “virtually” mapped to chromosome 17q21. By pooling the microclones as multiplexed hybridizaion probes, and by virtue of their origin on 17q21, we were able to identify approximately 150 P1 clones from the human Reference Library Data Base P1 Library that potentially map to chromosome 17q21. Verification of the 17q21 location of 16 P1 clones was accomplished by PCR analysis, with STS primer pairs to known 17q21 genes or by FISH. Our results demonstrate the substantial advantage of combining the sequence analysis of microclones with multiplex hybridization strategies for gene discovery and mapping specific gene rich regions of the genome.

ASSIGNMENT OF THREE HUMAN MARKERS IN CHROMOSOME 21Q11 TO MOUSE CHROMOSOME 16

Three unique sequence microclones from human chromosome region 21q11 were assigned to mouse chromosome 16 using a mouse/Chinese hamster cell hybrid 96Az2 containing a single mouse chromosome 16. This comparative mapping provides further homology between human chromosome 21 and mouse chromosome 16 to include the very proximal portion of the long arm of human chromosome 21. Since this part of human chromosome 21 is associated with mental retardation in Down syndrome individuals, its homologous mouse region should also be included in the construction of mouse models for studying Down syndrome phenotypes including mental retardation.