人体基因组随机单拷贝顺序的分离及其在染色体上的定位

本文从构建杂种细胞14-7-1的基因组文库出发,用种特异的探针分离出含有人体基因组顺序的重组子,并进一步分析了其中13个克隆,得到8个单拷贝顺序。通过与已建立的杂种细胞克隆分布板杂交以及染色体的原位杂交方法,将1个单拷贝顺序FD11-1定位在11p11-q11上。由于已经报道在11号染色体上具有3个连锁群,它们分别位于11p15、11p13和11q13上,因此,FD11-1有可能为11号染色体连锁基因图的建立提供1个有意义的座位。     

Isolation and mapping of 88 new RFLP markers on human chromosome 8.

To obtain new RFLP markers for construction of a high-resolution map of human chromosome 8, a cosmid library was constructed from a somatic hybrid cell that contained chromosome 8 as the only human component in mouse genomic background. Eighty-eight new RFLP markers were isolated and characterized, and 71 of them were sublocalized to chromosomal bands by fluorescent in situ hybridization (FISH). Of these, 36 were localized to the short arm, 34 to the long arm, and 1 to the centromeric region. Five markers defined VNTR loci. This work represents the first extensive isolation and physical mapping of RFLP markers on human chromosome 8. These new markers will serve as useful resources for linkage mapping of loci for inherited diseases and for efforts to identify a putative tumor suppressor gene(s) on chromosome 8.     

Isolation and mapping of cosmid markers on human chromosome 22, including one within the submicroscopically deleted region of DiGeorge syndrome

A genomic cosmid library was constructed from a Chinese hamster/human hybrid cell containing human intact chromosome 22 as its only human component. Of 1000 cosmids with inserts derived from human chromosome 22, 191 were tested for restriction fragment length polymorphisms (RFLPs). As a result, 64 clones detected RFLPs, including five variable number of tandem repeats systems. Of the remaining 127 cosmids, 111 detected a single copy sequence on human chromosome 22. Five somatic cell hybrids allowed us to assign all of the 64 polymorphic cosmids and 44 non-polymorphic cosmids to four different regions of human chromosome 22. In two patients with DiGeorge syndrome, one of the cosmids that had been sublocalized to 22pter-q11 detected hemizygosity. These 108 cosmid markers regionally assigned to human chromosome 22 should be useful for the construction of long-range physical maps and the identification of genetic alterations on the chromosome.     

Identification of 57 conventional RFLP and 6 VNTR systems with 32 DNA clones on chromosome 11p15

Fifty-four clones containing human inserts were selected from a cosmid library constructed from a somatic cell hybrid containing chromosome 11p15.3-p15.5 as its only human complement. In 32 of these clones, 63 polymorphic systems were identified with a panel of restriction enzymes: 57 conventional RFLP systems and 6 highly polymorphic VNTR systems. Although we examined the cosmid with only seven enzymes, 18 clones (including 6 VNTRs) were polymorphic with three or more enzymes. The results suggested that DNA sequences on the peritelomeric region of chromosome 11p tend to be highly variable. Because these markers are highly informative, they will be excellent resources for investigations of hereditary diseases and tumor suppressor genes in this region of chromosome 11.     

A highly polymorphic locus on chromosome 16q revealed by a probe from a chromosome-specific cosmid library

Summary A cosmid library was constructed from genomic DNA of a human-mouse somatic cell hybrid containing an 11q–16q translocation chromosome as the only human DNA. Cosmids with human inserts were prehybridized with total human DNA and were screened to find probes that revealed highly polymorphic loci. From one such cosmid, CF33-79, a single-copy subclone was isolated which revealed an insertion/deletion polymorphism with at least 11 alleles and a PIC of 0.77. Using a somatic cell hybrid mapping panel, the subclone was mapped to chromosome 16. By in situ hybridization with the entire cosmid used as a probe, chromosomal localization was shown at 16q2224.     

A high-resolution cytogenetic map of 168 cosmid DNA markers for human chromosome 11.

We have constructed a high-resolution cytogenetic map with 168 DNA markers, including 90 RFLP markers for human chromosome 11. The cosmid clones were mapped by fluorescence in situ suppression hybridization, in which discrete fluorescent signals can be detected directly on prometaphase R-banded chromosomes. Although these cosmid clones were distributed throughout the chromosome, they had some tendency to localize in the regions of R-positive band, such as 11p15, 11p11.2, 11q13, 11q23, and 11q25. Since these regions of chromosome 11 are considered to contain genes responsible for certain genetic diseases, cancer breakpoints involved in chromosome rearrangements, and tumor-suppressor genes, this high-resolution cytogenetic map will contribute to the molecular characterization of such genes. This map will also provide many landmarks essential for construction of the complete physical map with contigs of cosmid and YAC clones.     

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.     

Construction and Characterization of a Human Chromosome 2-Specific BAC Library

We have constructed a human chromosome 2-specific bacterial artificial chromosome (BAC) library using DNA from the somatic cell hybrid GM10826. The average size of the clones is about 63 kb. The coverage and distribution of the library were estimated by screening with known polymorphic genetic markers and fluorescence in situ hybridization (FISH). Twentyone markers tested positive when DNA pools prepared from approximately one-sixth of the library were screened with 33 known markers. This is consistent with the theoretical calculation of 63% coverage at one genomic equivalent. This suggested that the coverage of the library is approximately 5-6×. FISH analysis with 54 BACs revealed single site hybridization to chromosome 2, and the clones were distributed randomly on the chromosome. We have also performed direct sequencing of the BAC insert ends to generate sequence-tagged sites suitable for mapping and chromosome walking. This is the first reported human chromosome 2-specific BAC library and should provide a resource for physical mapping and disease searching for this chromosome.     

cDNA surveying of specific tissue expression of human chromosome 19 sequences.

cDNA surveying is a straightforward approach for identifying sequences in genomic clones expressed in specific tissues. It has been applied to a subchromosomal region of human chromosome 19 (19q13.2-q13.4), a region that contains several known expressed sequences including the locus for myotonic dystrophy (DM). Genomic clones were selected from this region by probing a human placental cosmid library with a chromosome 19q-specific minisatellite sequence, or human genomic clones were isolated from a cosmid library constructed from a human chromosome 19q13.2-q13.3 hamster hybrid cell line using human repetitive DNA as probe. Pooled cDNAs synthesized from RNA of specific tissues characteristically affected in DM were depleted in repetitive sequences and used as hybridization probes against gridded cosmid arrays. DNA from the cDNA-positive cosmid clones was transferred to nylon filters and reprobed with cDNAs to identify restriction fragments that were expressed in these tissues. Hybridizing restriction fragments were subcloned, sequenced, and demonstrated to be nonrepetitive. Primer pairs complementary to subcloned sequences were constructed and used for PCR amplification of cDNA synthesized from RNA of tissues affected in myotonic dystrophy. PCR products were sequenced to verify the identity of expressed genomic DNA and its corresponding cDNA.     

Isolation and analysis of DNA markers specific to human chromosome 15.

Chromosome-specific DNA markers provide a powerful approach for studying complex problems in human genetics and offer an opportunity to begin understanding the human genome at the molecular level. The approach described here for isolating and characterizing DNA markers specific to human chromosome 15 involved construction of a partial chromosome-15 phage library from a human/Chinese hamster cell hybrid with a single human chromosome 15. Restriction fragments that identified unique- and low-copy loci on chromosome 15 were isolated from the phage inserts. These fragments were regionally mapped to the chromosome by three methods, including Southern analysis with a mapping panel of cell hybrids, in situ hybridization to metaphase chromosomes, and quantitative hybridization or dosage analysis. A total of 42 restriction fragments of unique- and low-copy sequences were identified in 14 phage. The majority of the fragments that have been characterized so far exhibited the hybridization pattern of a unique locus on chromosome 15. Regional mapping assigned these markers to specific locations on chromosome 15, including q24-25, q21-23, q13-14, q11-12, and q11. RFLP analysis revealed that several markers displayed polymorphisms at frequencies useful for genetic linkage analysis. The markers mapped to the proximal long arm of chromosome 15 are particularly valuable for the molecular analysis of Prader-Willi syndrome, which maps to this region. Polymorphic markers in this region may also be useful for definitively establishing linkage with one form of dyslexia. DNA probes in this chromosomal region should facilitate molecular structural analysis for elucidation of the nature of instability in this region, which is frequently associated with chromosomal aberrations.     

Four restriction fragment length polymorphisms revealed by probes from a single cosmid map to human chromosome 12q

Summary Human gene mapping would be greatly facilitated if marker loci with sufficient polymorphism information content were generally available. As a source of such markers, we have used cosmids from a human genomic library. We have used a rapid method for screening random cosmids to identify those homologous to genomic regions especially rich in restriction fragment length polymophisms (Litt and White 1985). This method allows whole cosmids to be used as probes against Southern transfers of genomic DNA; regions of cosmid probes homologous to repeated genomic sequences are rendered unable to anneal with Southern transfers by prerendered of the probes with a vast excess of non-radioactive genomic DNA. From one cosmid (C1-11) identified by this procedure, we have isolated four single-copy probes, each of which identifies a polymorphic locus. Despite the existence of some linkage disequilibrium in this system, the polymorthism information content was computed as 0.73. Using a somatic cell hybrid mapping panel, we have mapped probes from cosmid 1–11 to human chromosome 12q. Additionally, in situ hybridization of the whole cosmid to metaphase spreads allowed more precise assignment of the locus to the region 12cenq13. The locus revealed by probes from cosmid 1–11 has been designated D12S6.     

Construction and characterization of a yeast artificial chromosome library containing 1.5 equivalents of human chromosome 21.

A library of yeast artificial chromosomes (YACs) was constructed from a human/hamster somatic cell hybrid containing human chromosome 21 (q11-qter). Cells were embedded in agarose, and the DNA was partially digested with EcoRI, released into solution by agarase treatment of the agarose plugs, ligated into pYAC4, and transferred into yeast. Double screening of the yeast transformants with human and hamster genomic DNA allowed the selection of clones hybridizing only with human DNA. The library consists of 321 clones, amounting to 1.5 equivalents (61 Mb) of chromosome 21. The mean YAC size calculated from 178 clones is 190 +/- 100 kb. Screening of the library with eight sequence-tagged sites gave six positives. Among 21 YACs tested by in situ hybridization, 17 mapped to chromosome 21.     

Human chromosome 11 complements ataxia-telangiectasia cells but does not complement the defect in AT-like Chinese hamster cell mutants

It has been shown that the X-ray-sensitive Chinese hamster V79 mutants (V-E5, V-C4 and V-G8) are similar to ataxia-telangiectasia (A-T) cells. To determine whether the AT-like rodent cell mutants are defective in the gene homologous to A-T (group A, C or D), human chromosome 11 was introduced to the V-E5 and V-G8 mutant cells by microcell-mediated chromosome transfer. Forty independent hybrid clones were obtained in which the presence of chromosome 11 was determined by in situ hybridization. The presence of the region of chromosome 11q22–23 was shown by molecular analysis using polymorphic DNA markers specific for the ATA, ATC and ATD loci. Seventeen of the obtained monochromosomal Chinese hamster hybrids contained a cytogenetically normal human chromosome 11, but only twelve hybrid cell lines were shown to contain an intact 11q22–23 region. Despite the complementation of the X-ray sensitivity by a normal chromosome 11 introduced to A-T cells (complementation group D), these twelve Chinese hamster hybrid clones showed lack of complementation of X-ray and streptonigrin hypersensitivity. The observed lack of complementation does not seem to be attributable to hypermethylation of the human chromosome 11 in the rodent cell background, since 5-azacytidine treatment had no effect on the streptonigrin hypersensitivity of the hybrid cell lines. These results indicate that the gene defective in the AT-like rodent cell mutants is not homologous to the ATA, ATC or ATD genes and that the human gene complementing the defect in the AT-like mutants seems not to be located on human chromosome 11.     

11p15.5-specific libraries for identification of potential gene sequences involved in Beckwith-Wiedemann syndrome and tumorigenesis.

Constitutional and somatic chromosomal abnormalities of the chromosome 11p15 region are involved in an overgrowth malformation syndrome, the Beckwith-Wiedemann syndrome (BWS), and in several types of associated tumors. The bias in parental origin for the different etiologic forms of this syndrome and for loss of heterozygosity in the tumors suggests that a gene (or genes) mapping to this region undergoes genomic imprinting. However, the precise localization of the locus (or loci) for the BWS and associated tumors is still unknown and more markers are required. We therefore isolated 11p15 markers from two libraries: the first one obtained by microdissection of the chromosome 11p15.5 region and the second one, a phage library, constructed from a hybrid cell line containing this region as its sole human DNA. Of 19 microclones isolated from the microdissection library, 11 were evolutionarily conserved. Four phage clones were isolated; one (D11S774) detected a highly informative variable number of tandem repeats (VNTR) and another (D11S773) a biallelic polymorphism. These clones were sublocalized using a panel of somatic cell hybrids that defines eight physical intervals in 11p15.5. Twenty-one clones map to the distal interval that harbors the BWS locus.     

Isolation of probes specific to human chromosomal region 6p21 from immunoselected irradiation-fusion gene transfer hybrids

A hybrid cell line (R21/B1) containing a truncated human chromosome 6 (6pter-6q21) and a human Y chromosome on a hamster background was irradiated and fused to A23 (TK-) or W3GH (HPRT-) hamster cells. Clones containing expressed HLA class I genes (4/40) were selected using monoclonal antibodies. These clones were recloned and analyzed with a panel of probes from the HLA region. One hybrid (4G6) contained the entire HLA complex. Two other hybrids (4J4 and 4H2) contained only the HLA class I region, while the fourth hybrid (5P9) contained HLA class I and III genes in addition to other genes located in the 6p21 chromosomal region. In situ hybridization showed that the hybrid cells contained more than one fragment of human DNA. Alu and LINE PCR products were derived from these cells and compared to each other as well as to products from two somatic cell hybrids having the 6p21 region in common. The PCR fragments were then screened on conventional Southern blots of the somatic cell hybrids to select a panel of novel probes encompassing the 6p21 region. In addition, the origin of the human DNA fragments in hybrid 4J4 was determined by regional mapping of PCR products.     

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.     

Preparation of monosomal lines containing individual human 15, 21, and X chromosomes]

Sorting of human--mouse or human--hamster hybrid cells with particular human chromosomes was performed by in situ hybridization. Total human genomic DNA was heavily labelled with. H and hybridized to metaphase spreads from hybrid clone cells. The method allowed us to not only identify human chromosomes in hybrid cells but also to detect terminal translocations and insertions from 1-2 bands in length to large ones. Biochemical markers of some human chromosomes were analysed using electrophoretic technique in the clones selected. Cytogenetic analysis (G staining) of these clones was made to visualize human chromosomes. Total 99 initial hybrid human--hamster and 26 human--mouse clones were obtained. 53 clones were analysed by in situ hybridization, only one of them being monochromosomal; the latter contained human X chromosome on the background of Chinese hamster chromosomes. Two other monochromosomal clones containing particular 15 and 21 chromosomes, respectively, were obtained by more complicated way from human--mouse hybrid clones using back selection, repeated hybridization and passing through a number of subsequent subclonings.     

A radiation hybrid map of the proximal short arm of the human X chromosome spanning incontinentia pigmenti 1 (IP1) translocation breakpoints.

Radiation hybrid mapping was used in combination with physical mapping techniques to order and estimate distances between 14 loci in the proximal region of the short arm of the human X chromosome. A panel of radiation hybrids containing human X-chromosomal fragments was generated from a Chinese hamster-human cell hybrid containing an X chromosome as its only human DNA. Sixty-seven radiation hybrids were screened by Southern hybridization with sets of probes that mapped to the region Xp11.4-Xcen to generate a radiation hybrid map of the area. A physical map of 14 loci was constructed based on the segregation of the loci in the hybrid clones. Using pulsed-field gel electrophoresis (PFGE) analyses and a somatic cell hybrid mapping panel containing naturally occurring X; autosome translocations, the order of the 14 loci was verified and the loci nearest to the X-chromosomal translocation breakpoints associated with the disease incontinentia pigmenti 1 (IP1) were identified. The radiation hybrid panel will be useful as a mapping resource for determining the location, order, and distances between other genes and polymorphic loci in this region as well as for generating additional region-specific DNA markers.     

A panel of irradiation-reduced hybrids selectively retaining human chromosome 11p13: their structure and use to purify the WAGR gene complex

The irradiation-fusion technique offers a means to isolate intact subchromosomal fragments of one mammalian species in the genetic background of another. Irradiation-reduced somatic cell hybrids can be used to construct detailed genetic and physical maps of individual chromosome bands and to systematically clone genes responsible for hereditary diseases on the basis of their chromosomal position. To assess this strategy, we constructed a panel of hybrids that selectively retain the portion of human chromosome band 11p13 that includes genes responsible for Wilms tumor, aniridia, genitourinary anomalies, and mental retardation (constituting the WAGR syndrome). A hamster-human hybrid containing the short arm of chromosome 11 as its only human DNA (J1-11) was gamma-irradiated and fused to a Chinese hamster cell line (CHO-K1). We selected secondary hybrid clones that express MIC1 but not MER2, cell-surface antigens encoded by bands 11p13 and 11p15, respectively. These clones were characterized cytogenetically by in situ hybridization with human repetitive DNA and were tested for their retention of 56 DNA, isozyme, and antigen markers whose order on chromosome 11p is known. These cell lines appear to carry single, coherent segments of 11p spanning MIC1, which range in size from 3000 kb to more than 50,000 kb and which are generally stable in the absence of selection. In addition to the selected region of 11p13, two cell lines carry extra fragments of the human centromere and two harbor small, unstable segments of 11p15. As a first step to determine the size and molecular organization of the WAGR gene complex, we analyzed a subset of reduced hybrids by pulsed-field gel electrophoresis. A small group of NotI restriction fragments comprising the WAGR complex was detected in Southern blots with a cloned Alu repetitive probe. One of the cell lines (GH3A) was found to carry a stable approximately 3000-kb segment of 11p13 as its only human DNA. The segment encompasses MIC1, a recurrent translocation breakpoint in acute T-cell leukemia (TCL2), and most or all of the WAGR gene complex, but does not include the close flanking markers D11S16 and delta J. This hybrid forms an ideal source of molecular clones for the developmentally fascinating genes underlying the WAGR syndrome.     

Isolation and mapping of polymorphic cosmid clones used for sublocalization of the multiple endocrine neoplasia type 1 (MEN1) locus

Summary Multiple endocrine neoplasia type 1 (MEN1) is characterized by neoplasia of the parathyroids, the pancreas, and the pituitary. Tumorigenesis involves unmasking of a recessive mutation at the MEN1 locus, which has been mapped to the centromeric part of chromosomal region 11q. In order to localize the MEN1 gene further and to make its isolation possible, a number of new markers were isolated. Two radiation-reduced somatic cell hybrids were identified that only contained markers close to and flanking the MEN1 region. DNA from these hybrids was used for the construction of a cosmid library, and clones containing human inserts were isolated. In addition, cosmid clones were isolated for locus expansion of 7 other markers that were mapped to the 11q12–13.2 region. The 33 newly isolated clones together with 25 previously published markers from this region were analyzed in a panel of radiation-reduced somatic cell hybrids. From the hybridization pattern, the region was divided into 11 parts. New restriction fragment length polymorphisms were identified in 7 of the newly isolated cosmid clones and in one plasmid. These were then used to sublocalize meiotic cross-overs more precisely in two MEN1 families, thus refining the mapping of the disease gene.