Rapid cloning and characterization of new chromosome 10 DNA markers by Alu element-mediated PCR

Alu element-mediated polymerase chain reaction is a strategy for rapidly cloning and mapping human DNA markers from mixed DNA sources. A novel primer homologous to the 3' end of the human Alu repeat element provides the basis for preferential synthesis of human DNA fragments from human/rodent somatic cell hybrid DNA template. This approach has been used to isolate a series of new markers from chromosome 10. The Alu element-mediated PCR probes were regionally assigned on chromosome 10 by hybridization to Southern blots of Alu PCR-synthesized DNA derived from somatic cell hybrid template DNA. Alu element-mediated PCR is generally applicable and makes possible the analysis of complex genomes with a speed and sensitivity that has not been previously possible.     

Human repeat element-mediated PCR: cloning and mapping of chromosome 10 DNA markers.

Repeat element-mediated PCR can facilitate rapid cloning and mapping of human chromosomal region-specific DNA markers from somatic cell hybrid DNA. PCR primers directed to human repeat elements result in human-specific DNA synthesis; template DNA derived from a somatic cell hybrid containing the human chromosomal region of interest provides region specificity. We have generated a series of repeat element-mediated PCR clones from a reduced complexity somatic cell hybrid containing a portion of human chromosome 10. The cloning source retains the centromere and tightly linked flanking markers, plus additional chromosome 10 sequences. Twelve new inter-Alu, two inter-L1, and four inter-Alu/L1 repeat element-mediated PCR clones were mapped by hybridization to Southern blots of repeat element-mediated PCR products amplified from somatic cell hybrid DNA templates. Two inter-Alu clones mapped to the pericentromeric region. We propose that a scarcity of Alu elements in the pericentromeric region of chromosome 10 contributed to the low number of clones obtained from this region. One inter-Alu clone, pC11/A1S-6-c23, defines the D10S94 locus, which is tightly linked to MEN2A and D10Z1.     

Generation and fluorescent in situ hybridization mapping of yeast artificial chromosomes of 1p, 17p, 17q, and 19q from a hybrid cell line by high-density screening of an amplified library

A yeast artificial chromosome (YAC) library has been constructed from a somatic cell hybrid containing a t(1p;19q) chromosome and chromosome 17. After amplification, part of this library was analyzed by high-density colony filter screening with a repetitive human DNA probe (Alu). The human YACs distinguished by the screening were further analyzed by Alu fingerprinting and Alu PCR. Fluorescent in situ hybridization (FISH) was performed to localize the YACs to subchromosomal regions of chromosome 1p, 17, or 19q. We have obtained a panel of 123 individual YACs with a mean size of 160 kb, and 77 of these were regionally localized by FISH: 33 to 1p, 10 to 17p, 25 to 17q, and 9 to 19q. The YACs cover a total of 19.7 Mb or 9% of the 220 Mb of human DNA contained in the hybrid. No overlapping YACs have yet been detected. These YACs are available upon request and should be helpful in mapping studies of disease loci, e.g., Charcot-Marie-Tooth disease, Miller-Dieker syndrome, hereditary breast tumor, myotonic dystrophy, and malignant hyperthermia.     

Assignment of a human DNA double-strand break repair gene (XRCC5) to chromosome 2

The Chinese hamster ovary (CHO-K1) cell mutant XRS-6 is defective in rejoining of DNA double-strand breaks and is hypersensitive to X-rays, γ-rays, and bleomycin. Radiation resistance or sensitivity of somatic cell hybrids constructed from the fusion of XRS-6 cells with primary human fibroblasts strongly correlated with the retention of human chromosome 2 isozyme and molecular markers. Discordancies between some chromosome 2 markers and the radiation resistance phenotype in some of the hybrid cells suggested the location of the X-ray repair cross complementing 5 (XRCC5) gene on the p arm of chromosome 2. Introduction of human chromosome 2 by microcell-mediated chromosome transfer into the radiation-sensitive XRS-6 cells resulted in hybrid cells in which the radiation sensitivity was complemented. The chromosome 2p origin of the complementing human DNA in the microcell hybrids was supported by fluorescent in situ hybridization analysis of human metaphases using human DNA amplified from the hybrids by inter-Alu-PCR as chromosome-painting probes. XRCC5 is therefore provisionally assigned to human chromosome 2p.     

Generation of a panel of somatic cell hybrids containing fragments of human chromosome 12P by X-ray irradiation and cell fusion.

We have employed an irradiation and fusion procedure to generate somatic cell hybrids containing various fragments of the short arm of human chromosome 12 using a 12p-only hybrid (M28) as starting material. For the initial identification of hybrids retaining human DNA, nonradioactive in situ hybridization was performed. Seventeen cell lines appeared to contain detectable amounts of human material. Detailed characterization of these hybrids by Southern blot analysis and chromosomal in situ suppression hybridization (chromosome painting), using hybrid DNAs as probes after Alu element-mediated PCR, resulted in a hybrid panel encompassing the entire chromosome 12p arm. This panel will provide a valuable resource for the rapid isolation of region-specific DNA markers. In addition, this panel may be useful for the characterization of chromosome 12 aberrations in, e.g., human germ cell tumors.     

Isolation of DNA fragments from a human chromosomal subregion by Alu PCR differential hybridization

The recent advent of Alu element-mediated PCR (Alu PCR) allows the rapid isolation of human-specific fragments from mixed DNA sources. This technique greatly facilitates the isolation of DNA fragments from specific regions of the human genome. We report a novel technique utilizing Alu PCR products as differential hybridization probes to isolate human DNA fragments from a chromosomal subregion. We used the Alu PCR products from a pair of somatic cell hybrids in which the human DNA content differs only in the 5q11.2-q13.3 region as differential hybridization probes. One hybrid (GM10114) retains an intact chromosome 5, while the other (HHW1064) contains a chromosome 5 deleted for the q11.2-q13.3 region. Phage from a flow-sorted chromosome 5 library were hybridized with the Alu PCR synthesis product from the chromosome 5 hybrid. Positively hybridizing phage were then screened with the Alu PCR product from the deletion 5 hybrid. Phage that hybridized to the Alu PCR product of the chromosome 5 hybrid but did not hybridize to the Alu PCR product of the deletion 5 hybrid were further characterized. We isolated five phage from 5q11.2-q13.3 using this differential hybridization procedure. Only one of these phage corresponded to a detectable difference between the ethidium bromide-stained Alu PCR products of the two somatic cell hybrids. This technique should be applicable to any somatic cell hybrid-deletion hybrid pair.     

A Sequence-Tagged Site Map of Human Chromosome 11

We report the construction of 370 sequence-tagged sites (STSs) that are detectable by PCR amplification under sets of standardized conditions and that have been regionally mapped to human chromosome 11. DNA sequences were determined by sequencing directly from cosmid templates using primers complementary to T3 and T7 promoters present in the cloning vector. Oligonucleotide PCR primers were predicted by computer and tested using a battery of genomic DNAs. Cosmids were regionally localized on chromosome 11 by using fluorescence in situ hybridization or by analyzing a somatic cell hybrid panel. Additional STSs corresponding to known genes and markers on chromosome 11 were also produced under the same series of standardized conditions. The resulting STSs provide uniform coverage of chromosome 11 with an average spacing of 340 kb. The DNA sequence determined for use in STS production corresponds to about 0.1% (116 kb) of chromosome 11 and has been analyzed for the presence of repetitive sequences, similarities to known genes and motifs, and possible exons. Computer analysis of this sequence has identified and therefore mapped at least eight new genes on chromosome 11.     

The Gene Encoding the Catalytic Subunit Cα of cAMP-Dependent Protein Kinase (Locus PRKACA) Localizes to Human Chromosome Region 19p13.1

We have determined the chromosomal localization of the gene for the catalytic subunit Cα of cAMP-dependent protein kinase (locus PRKACA) to human chromosome 19 using polymerase chain reaction (PCR) and Southern blot analysis of two different somatic cell hybrid mapping panels. In addition, PCR analysis of a chromosome 19 mapping panel revealed the presence of a human Cα-specific amplification product only in cell lines containing the region 19p13.1 to 19q12. Finally, two-color fluorescencein situhybridization to metaphase chromosomes using the human Cα cDNA and human chromosome 19 inter-Alu-PCR product as probes localized the human Cα gene to chromosome region 19p13.1.     

Alu-PCR Approach to Isolating NotI-Linking Clones from the 3p14-p21 Region Frequently Deleted in Renal Cell Carcinoma

In the mammalian genome CpG islands are associated with functional genes and cloning of these islands could be an alternative approach for cloning functional genes. Recently we have developed a new approach for cloning CpG islands and constructing NotI linking libraries. We have initiated the construction of a NotI restriction map for chromosome 3, especially focusing on the rearrangements in the 3p14-p21 region, which are associated with different malignancies. CpG islands from this region are useful for isolation of candidate tumor suppressor genes that map to this region and for isolating NotI-linking clones from 3p14-p21 for mapping purposes. Here we suggest a modification of Alu-PCR as an approach to isolating Not I sites (e.g., CpG islands) from defined regions of the chromosome. Instead of using whole chromosomal DNA for Alu-PCR, we have used representative NotI-linking libraries from hybrid cell lines containing either whole or deleted human chromosome 3 (MCH903.1 and MCH924.4, respectively). This decreases the complexity of the Alu-PCR products 10-100 times compared to the whole human genome. Using this modification, we can isolate NotI-linking clones, which are natural markers on the chromosome, rather than random genomic fragments. Among eight clones selected by this method, seven were from the region deleted in MCH924.4. The results clearly demonstrate the feasibility of Alu-PCR for isolating CpG islands from defined regions of the genome.     

Sixty-five radiation hybrids for the short arm of human chromosome 6: their value as a mapping panel and as a source for rapid isolation of new probes using repeat element-mediated PCR.

We have used an irradiation and fusion procedure to generate somatic cell hybrids that retain fragments of the short arm of human chromosome 6 (6p). To identify hybrids retaining human material, we performed repeat element-mediated PCR on crude lysates of cells from individual clones. Sixty-five hybrids were shown to contain human material and fifty of those contained one or more 6p-specific probes. Detailed characterization of these hybrids identified a subset that divides 6p into ten mapping intervals. Using repeat element-mediated PCR, we were able to isolate and map 61 new DNA fragments from specific regions of 6p. Fifteen of these fragments were used to screen for restriction fragment length polymorphisms (RFLPs), and nine identified RFLPs with one or more enzymes. The radiation hybrids described in this study provide a valuable resource for high-resolution mapping of 6p and for the rapid isolation of region-specific markers.     

Isolation and regional localisation of DNA sequences from a human chromosome 11-specific cosmid library

Summary A cosmid library has been prepared in the lorist-B vector from a mouse/human somatic cell hybrid containing region 11q23-11pter as the only human component. This chromosome region is stably maintained in the hybrid as a result of translocation onto one copy of mouse chromosome 13. Individual cosmids containing human DNA were isolated by their ability to hybridise with total human DNA, digested with either HindIII or EcoRI, and 33 individual unique sequences were identified. These fragments were then isolated and subcloned into the bluescribe plasmid vector. Regional localisation of these unique sequences was achieved using a panel of somatic cell hybrids containing different overlapping deletions of chromosome 11. The majority of the 33 mapped sequences derived from the long arm of chromosome 11. Two clones were located within the 11p13–p14 region, which is associated with a predisposition to Wilms' tumour. These probes supplement those already mapped to this chromosome and will assist in the generation of a detailed chromosome 11 linkage map.     

Five new microsatellite polymorphisms at the q21 region of human chromosome 21

Five clones, containing polymorphic CA-repeat sequences, have been isolated from a specific human chromosome 21 phage library and have been localised to band q21 of chromosome 21 using a somatic cell hybrid panel. These highly repetitive sequences (D21S1263, D21S1264, D21S1415, D21S1417 and D21S1420) have been characterised in the CEPH reference parents and have heterozygosities ranging from 0.30 to 0.81 and an average polymorphism information content (PIC) of 0.62. The relative order of these markers, based on the somatic cell hybrid panel, is cen-D21S1417, D21S1420-D21S1263, D21S1415-D21S1264-tel. The most polymorphic marker (D21S1264) has been included in the chromosome 21 genetic map. They have also been localised in the CEPH/ Généthon YAC panel, providing a refined localisation of these polymorphic sequences. These five CA-repeat markers should provide a better characterisation of the q21 region of chromosome 21.     

Alu-splice PCR: a simple method to isolate exon-containing fragments from cloned human genomic DNA

We have developed a simple, straightforward procedure to isolate exons from cloned human genomic DNA. The method is PCR based and relies upon the conservation of splice-site sequences and the frequency of Alu repeat elements in the genome to capture coding sequences. We designed two different sets of primers: a primer from each end of the Alu element and primers with the 5′ or 3′ splice-site consensus sequences. Putative exons were amplified by PCR using YAC DNA as starting material. We applied Alu-splice PCR to two overlapping YACs, 72H9 and 860G11, from human chromosome 21. Sequence and northern analysis of 37 initial clones resulted in the identification of five novel exons. Received: 17 July 1997 / Accepted: 28 August 1997     

Isolation of polymorphic DNA segments from human chromosome 21.

A somatic cell hybrid line containing only human chromosome 21 on a mouse background has been used as the source of DNA for construction of a recombinant phage library. Individual phages containing human inserts have been identified. Repeat-free human DNA subclones have been prepared and used to screen for restriction fragment length polymorphisms to provide genetic markers on chromosome 21. Nine independently isolated clones used as probes identified a total of 11 new RFLPs. Four of the DNA probes recovered from the library have been mapped unequivocally to chromosome 21 using a panel of somatic cell hybrid lines. A fifth probe detected an RFLP on chromosome 21 as well as sequences on other chromosomes. This set of RFLPs may now form the basis for construction of a genetic linkage map of human chromosome 21.     

DNA segments mapped by reciprocal use of microsatellite primers between mouse and rat

Rat microsatellite primers were used for detection of homologous DNA segments in the mouse species (Mus laboratorius, Mus musculus musculus, and Mus spretus). Twenty five (16.3%) of 153 rat primer pairs amplified specific DNA segments, when genomic DNA of mice was used as a template in the polymerase chain reaction (PCR). Size variation among inbred strains of mice was found for 13 DNA segments (8.5%). Eight out of the 13 polymorphic DNA segments were mapped to a particular chromosome with two sets of recombinant inbred strains, AKXL or BXD. Similarly, mouse microsatellite primers were used for detection of homologous DNA segments in rats (Rattus norvegicus). Twenty (12.0%) of 166 primer pairs amplified specific DNA segments from rat genome. Size variation among inbred strains of rats was found for seven DNA segments (4.2%). Eleven of these 20 DNA segments were mapped with a rat x mouse somatic cell hybrid clone panel and/or linkage analysis by use of backcross progeny. Our results suggest that the mapped DNA segments are really homologs between mouse and rat. These polymorphic DNA segments are useful genetic markers.     

A consensus Alu repeat probe for physical mapping

Physical mappinf of the human genome involves a variety of complex hybridization-based procedures, some of which rely upon the ability to seperate human clones derived from human-rodent hybrid cell lines from those that contain background rodent-derived DNA sequences. The ability to block the repititive element (Alu repeat) portion of inter-Alu PCR products derived from a variety of complex sources is also crucial for the isolation of unique DNA sequences. Here we report the construction and characterization of a new consensus Alu repeat probe (pPD39) designed for these purposes.     

Multicolor FISH Mapping with Alu-PCR-Amplified YAC Clone DNA Determines the Order of Markers in the BRCA1 Region on Chromosome 17q12-q21

A gene designated BRCA1, implicated in the susceptibility to early-onset familial breast cancer, has recently been localized to chromosome 17q12-q21. To date, the order of DNA markers mapped within this region has been based on genetic linkage analysis. We report the use of multicolor fluorescence in situ hybridization to establish a physically based map of five polymorphic DNA markers and 10 cloned genes spanning this region. Three cosmid clones and Alu-PCR-generated products derived from 12 yeast artificial chromosome clones representing each of these markers were used in two-color mapping experiments to determine an initial proximity of markers relative to each other on metaphase chromosomes. Interphase mapping was then employed to determine the order and orientation of closely spaced loci by direct visualization of fluorescent signals following hybridization of three probes, each detected in a different color. Statistical analysis of the combined data suggests that the order of markers in the BRCA1 region is cen-THRA1-TOP2-GAS-OF2-17HSI)-248yg9-RNU2-OF3-PPY/p131-EPB3-Mfd188-WNT3-HOX2-GP3A-tel. This map is consistent with that determined by radiation-reduced hybrid mapping and will facilitate positional cloning strategies in efforts to isolate and characterize the BRCA1 gene.     

The Evolutionarily Conserved CKAP2 Gene Is Located in Human Region 13q14.3 Often Rearranged in Various Tumors

The human CKAP2 gene, which is involved in diffuse large B-cell lymphomas, was localized by screening the GeneBridge 4 somatic cell radiation hybrid panel by means of the polymerase chain reaction (PCR). The CKAP2 gene was mapped between the WI-15460 and WI-3673 markers at the boundary between regions 13q14.3 and 13q21.1, at the distance of 14.39 cR (with 4.8 cR per cM) from the WI-5867 framework marker (lod score > 2.26). The human CKAP2 gene displayed high homology to mouse and rat expressed orthologs. A CKAP2-like sequence was found in human chromosome 14 and assumed to be a pseudogene resulting from duplication and subsequent mutations of the CKAP2 gene on chromosome 13. A possible role of the CKAP2 gene in oncogenesis associated with deletions and rearrangements of region 13q14.3–21.1 is discussed.     

Assignment of the human ST2 gene to chromosome 2 at q11.2

The humanSt2 locus has been assigned to chromosome 2, using a human ST2 cDNA clone, by a human/rodent somatic cell hybrid mapping panel. TheSt2 locus has also been mapped to chromosome 2811.2, using a human ST2 genomic DNA clone, by in situ hybridization. The locus is very tightly linked to theIl-1r1 locus. Together with the structural similarity of ST2 to IL-1RI, these data suggest functional relationships between these two genes.