Saturation of human chromosome 3 with unique sequence hybridization probes

We have generated chromosome 3-specific recombinant libraries in both lambda and cosmid cloning vectors starting with somatic cell hybrids (hamster/human) containing either an intact chromosome 3 or a chromosome 3 with an interstitial deletion removing 75% of long-arm sequences. The libraries contained between 2 X 10(5) and 5 X 10(6) independent recombinants. Approximately 2% of the recombinants in these libraries contain inserts of human DNA. These were identified by hybridizing the recombinants to radioactively labeled total human DNA. Over 2500 recombinants containing human DNA were isolated from these various libraries and DNA was prepared from each of them. This represents 80,000 kb of cloned chromosome 3 sequences. One-third of the DNAs were digested with EcoRI or HindIII, and fragments free of repetitive sequences were radioactively labeled using random hexanucleotide primers and tested as unique sequence hybridization probes. Over 6500 of the fragments were tested and of these 758 were unique sequence probes with minimal or no background hybridization. Their hybridization only to chromosome 3 was verified. These probes, which were derived from 452 independent recombinants, should provide an effective saturation of human chromosome 3.     

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.     

Isolation and field-inversion gel electrophoresis analysis of DNA markers located close to the Huntington disease gene

A radiation-induced hybrid cell line containing 10-20 million base pairs of DNA derived from the terminal part of human 4p16 in a background of hamster chromosomes has been used to construct a genomic library highly enriched for human sequences located close to the Huntington disease (HD) gene. Recombinant phage containing human inserts were isolated from this library and used as hybridization probes against two other radiation hybrids containing human fragments with chromosomal breaks in 4p16 and against a human-hamster somatic cell hybrid that retains only the 4p15-4pter part of chromosome 4. Of 121 human phage tested, 6 were mapped distal to the HD-linked D4S10 locus. Since the HD gene is located between D4S10 and the 4p telomere, all of these sequences are likely to be closer to HD than D4S10, and any one of them may be a distal flanking marker for the disease locus. Long-range restriction map analysis performed with a field-inversion gel system shows that the six new loci are distributed in different places within 4p16. Although it is not possible to establish an order for the six sequences with the FIGE data, the results demonstrate that the region detected by these probes must span at least 2000 kb of DNA.     

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.     

Generation of sequence-tagged sites from Xp22.3 by isolating commonAlu-PCR products of radiation hybrids retaining overlapping human X chromosome fragments

Several human diseases have been mapped to Xp22.3 on the distal short arm of the human X chromosome, and many genes in this area have been found to be expressed from the inactive X chromosome. To facilitate physical mapping and characterization of this interesting region, we have constructed a battery of radiation hybrids containing human X chromosomal fragments, and isolated two hybrid clones with overlapping fragments of Xp22.3. Alu-PCR on these hybrids and identification of sequences common to both hybrids allowed the isolation of six sequence-tagged sites (STSs) from Xp22.3. Five of the STSs were mapped to individual YACs comprising a recently constructed contig of this region. These novel STSs are useful markers for further physical characterization of this part of the genome. Received: 4 May 1995 / Revised: 27 September 1995     

Adaptation of the interspersed repetitive sequence polymerase chain reaction to the isolation of mouse DNA probes from somatic cell hybrids on a hamster background

A strategy for the rapid isolation of DNA probes from radiation-fusion Chinese hamster cell hybrids containing overlapping portions of the murine X chromosome based on the interspersed repetitive sequence polymerase chain reaction (IRS-PCR) previously used with human somatic cell hybrids has been developed. This specific amplification of mouse DNA on a hamster background depends on the use of primers directed to the B2 short interspersed repeat element family and the R repeat, from the long interspersed repeat element family, L1. Two sets of amplification conditions, which gave specific amplification of mouse DNA from either a mouse X-monochromosomal hybrid or irradiation-fusion hybrids having reduced X content, were defined. The mouse X-only chromosome hybrid yielded approximately 20 discrete reproducible bands, while the irradiation-fusion hybrids yielded between 1 and 10 discrete products. Comparison of different irradiation-fusion hybrids has allowed the definition of both specific and shared products corresponding to different regions within the overlapping X-chromosome fragments present within these hybrids. Use of such hybrids and the IRS-PCR technique has allowed the isolation of probes corresponding to the central region of the mouse X chromosome that contains the X-inactivation center. The method should be widely applicable to the isolation of mouse DNA sequences from mouse hybrid cell lines on either human or Chinese hamster backgrounds.     

Segregation of the Huntington disease region of human chromosome 4 in a somatic cell hybrid

We have developed an X-irradiation:cell fusion procedure that segregates segments of human chromosomes lacking selectable markers and have used this approach to construct somatic cell hybrids retaining fragments of human chromosome 4 as the only human material. To identify hybrids retaining a small chromosomal fragment in the region of the Huntington disease (HD) gene, we used Southern blot analysis to screen 72 hybrid lines for the presence or absence of seven chromosome 4 single-copy loci. These data, combined with in situ hybridization experiments, identified three hybrids of interest. One of these cell lines, C25, stably retains a 10,000- to 20,000-kb fragment of distal 4p in the vicinity of the HD gene, translocated to a hamster chromosome. Field-inversion gel electrophoresis revealed no evidence of rearrangements in the human DNA present in C25. In combination with similar radiation hybrids, C25 is a valuable tool for isolating DNA probes near the HD gene.     

Fingerprinting human chromosomes by polymerase chain reaction-mediated DNA amplification.

We describe here a method for DNA fingerprinting of human chromosomes by Alu-polymerase chain reaction (PCR) amplification of DNA from monochromosomal hybrids, following digestion with restriction endonucleases. DNA digestion with restriction enzymes prior to PCR amplification reduces the total number of amplified fragments. The number and pattern of bands of PCR products observed in an electrophoretic medium are chromosome specific and provide a "fingerprint signature" for individual human chromosomes. Using this approach, we have produced fingerprints for human chromosomes 2, 5, 7, 9, and 12. The applicability of this approach to chromosome identification was assessed by comparing the fingerprints obtained for two different hybrids containing chromosome 7. DNA fragments specific for the long and the short arms of human chromosome 12 have also been identified. In addition, Alu-PCR-generated DNA fragments, specific for different chromosomes, were used to probe Southern blots of a hybrid cell panel to identify human chromosomes present in hybrid cell lines. The chromosomal specificity of these probes permits the identification of intact as well as rearranged chromosomes composed of segments arising from more than one chromosome.     

Isolation and regional mapping of DNA sequences unique to human chromosome 21.

To isolate DNA sequences unique to chromosome 21 we have used a recombinant-DNA library, constructed from a mouse-human somatic-cell hybrid line containing chromosome 21 as the only human chromosome. Individual recombinant phage containing human DNA inserts were identified by their hybridization to total human DNA sequences and by their failure to hybridize to total mouse DNA sequences. A repeat-free human DNA fragment was then subcloned from each of 14 such recombinant phage. An independent somatic-cell hybrid was used to assign all 14 subcloned fragments to chromosome 21. Thirteen of the fragments have been regionally mapped using a somatic-cell hybrid containing a human 21 translocation chromosome. Two probes map proximal to the 21q21.2 translocation breakpoint, and 11 probes map distal to this breakpoint, placing them in the region 21q21.2-21q22. One of seven probes used to screen for restriction-fragment-length polymorphisms recognized polymorphic DNA fragments when hybridized to genomic DNA from unrelated individuals. These 14 unique probes provide useful tools for studying the structure and function of human chromosome 21 as well as for investigating the molecular biology of Down syndrome.     

Alu-PCR: characterization of a chromosome 6-specific hybrid mapping panel and cloning of chromosome-specific markers.

The Alu-polymerase chain reaction (Alu-PCR) was applied to selectively amplify DNA sequences from human chromosome 6 using a single primer (A1) directed to the human Alu consensus sequence. A specific amplification pattern was demonstrated for a panel of eight somatic cell hybrids containing different portions of chromosome 6. This PCR pattern permits the identification of submicroscopic DNA alterations and can be utilized as a reference for additional chromosome 6-specific hybrids. To obtain new chromosome 6-specific markers we established two libraries from PCR-amplified sequences using two somatic cell hybrids (MCH381.2D and 640-5A). Out of a total of 109 clones that were found to be chromosome 6 specific, 13 clones were regionally assigned. We also included a procedure that allows the isolation of chromosome 6-specific markers from hybrids that contain human chromosomes other than 6. Our results will contribute to the molecular characterization of chromosome 6 by fostering characterization of somatic cell hybrids and by the generation of new regionally assigned DNA markers.     

An expanded mouse-human hybrid cell panel for mapping human chromosome 16

A mouse/human hybrid cell panel of human chromosome 16 has been extended to a total of 31 hybrids. These hybrids were derived from constitutional translocations and deletions ascertained during clinical cytogenetic studies. This panel of hybrids, together with four fragile sites, have the potential to divide chromosome 16 into 38 regions. Rapid detailed physical mapping of gene probes or anonymous DNA probes is possible using this hybrid panel. This hybrid cell panel also allows the physical mapping of other chromosomes with three breakpoints on chromosomes 1, 4, 11 and 13 and two on chromosomes 3, 10 and 18.     

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.     

Differential staining of human and murine chromatin in situ by hybridization with species-specific satellite DNA probes.

Human and murine chromatin was differentially labeled by hybridization with DNA probes that bind to species-specific satellite DNA. The targets for in situ hybridization were the mouse-specific major or gamma satellite DNA and the human alpha satellite DNA. These sequences typically are localized at or near the chromosome centromeres, and remain their tight localization throughout the cell cycle. DNA probes were synthesized in vitro by primer directed DNA amplification using the polymerase chain reaction. In typical applications like the differentiation of cells derived from chimeric animals or the characterization of chromosomes in somatic cell hybrids, the two DNA probes are differently labeled and detected using label-specific reagents that fluoresce at different wavelengths. The rapid technique for chromatin discrimination described here combines high specificity with unprecedented signal intensity.     

Localization of 616 human chromosome 3-specific cosmids using a somatic cell hybrid deletion mapping panel.

A total of 5700 human chromosome 3-specific cosmid clones was isolated from a series of cosmid libraries constructed from somatic cell hybrids whose only human component was an entire chromosome 3 or a chromosome 3 containing an interstitial deletion removing 50% of long arm sequences. Several unique sequence chromosome 3-specific hybridization probes were isolated from each of 616 of these cosmids. These probes were then used to localize the cosmids by hybridization to a somatic cell hybrid deletion mapping panel capable of resolving chromosome 3 into nine distinct subregions. All 616 of the cosmids were localized to either the long or short arm of chromosome 3 and 63% of the short arm cosmids were more precisely localized. We have identified a total of 87 cosmids that contain fragments that are evolutionarily conserved. Fragments from these cosmids should prove useful in the identification of new chromosome 3-specific genes as well as in comparative mapping studies. The localized cosmids should provide excellent saturation of human chromosome 3 and facilitate the construction of physical and genetic linkage maps to identify various disease loci including Von Hippel Lindau disease and renal and small cell lung carcinoma.     

Regional localization of 18 human X-linked DNA sequences

A series of human probes with unique sequences has been isolated from a recombinant phage library constructed with DNA obtained from a human-hamster hybrid cell line. This cell line contained the X chromosome as the only human component. For 18 of these probes, a human X-chromosome origin has been confirmed and they have been regionally assigned by a combination of techniques: dosage studies utilizing DNA from human fibroblasts carrying X-chromosome duplications and deletions; the presence or absence of hybridization to digested DNA from hybrid lines carrying fragments of the X chromosome; and in situ hybridization to metaphase chromosomes. The use of dosage as a means to regionally assign probes significantly improves resolution of the X chromosome.     

A cosmid library specific for human Chromosome regions 6p21.3 and 6q27

We have explored the potential of irradiation-fusion gene transfer (IFGT) hybrids as a source of well-defined human chromosome fragments from which probes can be derived. Extensive characterization of the IFGT hybrid 4J4 with a full panel of markers from Chromosome (Chr) 6 showed that the human DNA content derives largely from 6p21.3 and 6q27. A cosmid library has been constructed from 4J4 DNA, and 370 recombinants containing human DNA have been isolated and overlapping clones ordered into 20 contigs. Regional localization of representative clones from each contig, determined by fluorescent in situ hybridization (FISH), places 13 contigs in 6q27 and 6 in 6p21.3. Preliminary screening of cDNA libraries with selected cosmids has identified two expressed sequences. Since there are a number of medically important genes in both these regions of human Chr 6 with several disease loci linked to the HLA-A region in 6p21.3 and various tumor suppressor genes to 6q27, this library will provide a valuable resource to aid the isolation of candidate genes for these diseases. In addition, unique markers for detailed physical and genetic mapping of these regions of human Chr 6 can be easily obtained.     

Toward a complete linkage map of the human X chromosome: regional assignment of 16 cloned single-copy DNA sequences employing a panel of somatic cell hybrids.

Closely linked restriction fragment length polymorphisms (RFLPs) are potentially useful as diagnostic markers of genetic defects, and, in principle, RFLPs can be employed to construct a complete linkage map of the human genome. On the X chromosome, linkage studies are particularly rewarding because in man more than 120 X-linked genes are known. Thus, it is probable that each X-specific RFLP will be of use as a genetic marker of one or several X-linked disorders. To facilitate the search for closely linked RFLPs, we have regionally assigned 16 cloned DNA sequences to various portions of the human X chromosome, employing a large panel of somatic cell hybrids. These probes have been used to correlate genetic and physical distances on Xp, and it can be extrapolated from these data that the number and distribution of available Xq sequences will also suffice to span the long arm of the X chromosome.     

Localization of three DNA segments encompassing tRNA genes to human chromosomes 1, 5, and 16: proposed mechanism and significance of tRNA gene dispersion

The chromosomal locations of three cloned human DNA fragments encompassing tRNA genes have been determined by Southern analysis of human-rodent somatic cell hybrid DNAs with subfragments from these cloned genes and flanking sequences used as hybridization probes. These three DNA segments have been assigned to human chromosomes 1, 5, and 16, and homologous sequences are probably located on chromosome 14 and a separate locus on chromosome 1. These studies, combined with previous results, indicate that tRNA genes and pseudogenes are dispersed on at least seven different human chromosomes and suggest that these sequences will probably be found on most, if not all, human chromosomes. Short (8-12 nucleotide) direct terminal repeats flank many of the dispersed tRNA genes. The presence of these flanking repeats, combined with the dispersion of tRNA genes throughout the human genome, suggests that many of these genes may have arisen by an RNA-mediated retroposition mechanism. The possible functional significance of this gene dispersion is considered.     

Direct nonradioactive in situ hybridization of somatic cell hybrid DNA to human lymphocyte chromosomes

Biotinylated DNA from various human-rodent hybrids was hybridized to human lymphocyte spreads after preannealing of the repeated sequences with sonicated total human DNA. Fluorescent labeling was achieved by successive treatments with fluorescein-labeled avidin and biotinylated antiavidin antibody. The use of labeled total DNA from hybrids with known chromosome composition permits the fluorescent staining-("painting") of specific chromosomes, or parts thereof, in human lymphocyte metaphases. Alternatively, the human chromosome content of cell hybrids with unknown chromosome composition is directly assessed from the labeling pattern of human lymphocyte spreads using the total hybrid DNA as probe.