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.     

Isolation and regional localization of DNA segments revealing polymorphic loci from human chromosome 13.

A recombinant DNA library enriched for portions of human chromosome 13 has been constructed from a hamster-human somatic cell hybrid that contained human chromosomes 13, 12, and 6p. A total of 733 phages were identified that contain human DNA inserts, and 46 single-copy subfragments have been derived and used as probes on Southern transfers of genomic DNA isolated from unrelated individuals. From this set, nine fragments revealing polymorphic loci (RFLP) in Msp I- or Taq I-digested DNA have been identified, of which three are polymorphic with both enzymes. Six of these probes have been shown to segregate concordantly with human chromosome 13 in a somatic cell hybrid mapping panel, and the RFLPs at these loci have been shown to behave as codominant Mendelian alleles. Additionally, hybridization to DNA isolated from cells containing various deletions of chromosome 13 has allowed regional localization. This recombinant DNA library will be useful in the study of retinoblastoma as well as in the study of the mechanisms responsible for abnormalities of this autosome.     

Isolation of DNA sequences on human chromosome 21 by application of a recombination-based assay to DNA from flow-sorted chromosomes

Summary By merging two efficient technologies, bivariate flow sorting of human metaphase chromosomes and a recombination-based assay for sequence complexity, we isolated 28 cloned DNA segments homologous to loci on human chromosome 21. Subregional mapping of these DNA segments with a somatic cell hybrid panel showed that 26 of the 28 cloned DNA sequences are distributed along the long arm of chromosome 21, while the other 2 hybridize with sequences on the short arm of both chromosome 21 and other chromosomes. This new collection of probes homologous to chromosome 21 should facilitate molecular analyses of trisomy 21 by providing DNA probes for the linkage map of chromosome 21, for studies of nondisjunction, for chromosome walking in clinically relevant subregions of chromosome 21, and for the isolation of genes on chromosome 21 following the screening of cDNA libraries.     

Characterization of radiation/fusion hybrids containing parts of human chromosome 10 and their use in mapping chromosome 10-specific probes.

We have characterized a panel of somatic cell hybrid cell lines which contain different portions of human chromosome 10. Genomic DNA from the somatic cell hybrids was tested for hybridization with each of an ordered set of probes used previously to construct a genetic map of chromosome 10, as well as several additional probes, previously localized by in situ hybridization. Hybridization of an unmapped probe to the cell line DNAs can be used to determine its most likely position on the chromosome relative to the mapped set of probes. Genomic DNA from two of the cell lines has been used to construct region-specific cosmid and bacteriophage libraries, and clones derived from these libraries were localized by hybridization to the panel of hybrid cell lines. Several of these probes reveal restriction fragment length polymorphisms which have been genetically mapped. Three of the probes map near the locus for multiple endocrine neoplasia type 2A, and one of these probes, BG-JC353 (D10S167), maps between RBP3 and TB14.34 (D10S34). Another probe, CRI-J282 (D10S104), is close to the FNRB locus. The panel of hybrid cell lines is thus useful for rapidly localizing unmapped probes and as a source of DNA for the construction of recombinant libraries derived from specific regions of the chromosome.     

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.     

Isolation of human chromosome 21 sequences and their application to in situ hybridization

Summary We report the isolation of 50 independent unique sequences from a human chromosome 21 library (identification code LA21 NSO1). These sequences were individually assigned to chromosome 21 using a mouse-human somatic hybrid cell line, WAVR 4d-F94a. Use of these unique clones as a mixture of probes for in situ hybridization of human metaphase chromosomes demonstrated strong signals on chromosome 21. These unique DNA sequences should provide useful tools for structural and functional analysis of human chromosome 21. The use of these sequences for the detection of Down syndrome is discussed.     

Isolation of chromosome-21-specific DNA probes and their use in the analysis of nondisjunction in Down syndrome

Summary Thirteen single-copy, chromosome-21-specific DNA probes were isolated from a recombinant library made from flow-sorted chromosome 21 DNA and regionally mapped using a panel of somatic cell hybrids. Five probes mapped in the 21q21-q22.1 region, six to the 21q22.1-qter region, and one to each of the regions 21q22.1-q22.2 and 21q22.3. Two of these probes, one of which maps in the critical region for Down syndrome, have recently been shown to be expressed at high levels in Down syndrome brain tissue (Stefani et al. 1988). Following preliminary screening for restriction fragment lenght polymorphisms (RFLPs), five polymorphisms were discovered with four of the chromosome 21 DNA probes. A frequent MspI polymorphism detected by one of the probes was used in conjunction with four previously described polymorphic chromosome 21 probes to analyse the origin of nondisjunction in 33 families with a child or fetus with trisomy 21. The parental origin of the additional chromosome 21 was determined in 12 cases: in 9 (75%) of these it was derived from the mother and in the other 3 cases (25%) it was of paternal origin. Cytogenetic analysis of Q-banding heteromorphisms was informative in three of five families tested, and in each case the RFLP results were confirmed. The meiotic stage of nondisjunction was defined with confidence in five families, the results being obtained with pericentromeric RFLP or cytogenetic markers. Recombination between two nondisjoined chromosomes was demonstrated in one family and is consistent with the view that a lack of recombination between chromosome 21 homologues or failure of their conjunction is not the invariable cause of trisomy 21.     

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.     

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.     

Isolation and Mapping of Human Chromosome 21 Cosmids Using a Probe for RTVL-H Retrovirus-like Elements

We have explored the usefulness of a cloned low-repetitive DNA element, termed RTVL-H, for the region-specific isolation of DNA cosmids from chromosome 21. Hybridization of the RTVL-H element as a probe to genomic Southern blots of DNAs from a chromosome 21-specific somatic cell hybrid panel demonstrated the presence of at least seven RTVL-H-related sequences in defined regions dispersed over the chromosome. Some of the RTVL-H sequences detected by using the chromosome 21-specific somatic cell hybrid panel were subsequently isolated from a chromosome 21-specific cosmid library and mapped using somatic cell hybrids. In general, close correspondence was found between mapping results obtained using the consensus RTVL-H probe on the chromosome 21-specific somatic cell hybrid panel and the more precise mapping obtained using cosmids. The results demonstrate that sequences such as the RTVL-H element can be used for isolating region-specific sequences.     

Isolation of 37 single-copy DNA probes from human chromosome 6 and physical mapping of 11 probes by in situ hybridization

Fifty-five single-copy DNA probes were isolated from the library LL06NS01, which was constructed from a complete HindIII digest of a flow-sorted human chromosome 6. Because chromosomes from a human x Chinese hamster somatic cell hybrid were used as the starting material for the flow-sorting, the library could be expected to contain some contaminating Chinese hamster DNA as well as DNA from human chromosomes other than 6. Thirty-seven of the 55 probes, however, were shown to map to human chromosome 6 by Southern blot hybridization with DNA from a panel of somatic cell hybrids. Eleven of the probes were mapped further by in situ hybridization. Four probes were localized to the short arm of chromosome 6, six to the long arm, and one to the centromeric region.     

Genes for two autosomal recessive forms of chronic granulomatous disease assigned to 1q25 (NCF2) and 7q11.23 (NCF1).

Chronic granulomatous disease (CGD) is a heterogeneous group of inherited disorders of impaired superoxide production in phagocytes. The most common X-linked recessive form involves the CYBB locus in band Xp21.1 that encodes the membrane-bound beta subunit of the cytochrome b558 complex. Two autosomal recessive forms of CGD result from defects in cytosolic components of the phagocyte NADPH oxidase system, p47phox (NCF1) and p67phox (NCF2). By using human cDNA probes we have mapped the genes for these proteins to chromosomal sites. The combined data from Southern analysis of somatic cell hybrid lines and chromosomal in situ hybridization localize NCF1 to 7q11.23 and NCF2 to band 1q25. The NCF1 localization corrects an erroneous preliminary assignment to chromosome 10. In the mouse, the locus corresponding to NCF2 (Ncf-2) was mapped with somatic cell hybrid panels and recombinant inbred strains to mouse chromosome 1 near Xmv-21 within a region of conserved homology with human chromosome 1 region q21-q32. A second site, probably a processed pseudogene, was identified on mouse chromosome 13.     

A straightforward approach to isolate DNA sequences with potential linkage to the retinoblastoma locus

Summary From a human-Chinese hamster somatic cell hybrid a clone was derived containing chromosome 13 in duplicate as its only human material. This clone was used to construct a human chromosome 13-specific recombinant DNA-library. Overlapping Sau3AI DNA sequences (11.9–17.2 kb) from the cell hybrid were inserted into the lambda phage vector EMBL4. From eleven recombinants having a human insert thirteen putative unique DNA sequences were isolated and cloned into the plasmid vector pBR329. A human-mouse hybrid containing a human chromosome 13 with a deletion of 13q14 and lacking its undeleted homologue was constructed to be used in a selection procedure for DNA sequences belonging to band q14. Three probes originating from two different phages were assigned to 13q14 because they did not hybridise to DNA from this cell hybrid. One of these 13q14 probes detects a low frequency (2/44) Msp I restriction fragment length polymorphism. The probes are now being used for screening a cosmid library to find adjacent polymorphic sequences with a RFLP information content suitable for application in the diagnosis of hereditary retinoblastoma.Preliminary reports of these experiments were presented at the 8th International Workshop on Human Gene Mapping, Helsinki, August 4–10, 1985, and the 13th International Congress of Biochemistry, Amsterdam, August 25–30, 1985 (Scheffer et al. 1985a,b)     

Isolation and regional localization by in situ hybridization of a unique gene segment to chromosome 21

V chromosome 21 (ch. 21) flow-sorted library was screened for the presence of unique DNA segments which are specific for the 21st chromosome. By combining the techniques of somatic cell genetics and in situ hybridization, we have identified several of these recombinant probes and have regionally mapped one of them to the distal half of the long arm of chromosome 21 (q22.1- greater than qter). This represents the first report of the sublocalization of a unique DNA segment to chromosome 21 by in situ hybridization.     

Isolation of anonymous DNA markers for human chromosome 22q11 from a flow-sorted library,and mapping using hybrids from patients with DiGeorge syndrome

Summary DiGeorge syndrome (DGS) is a human developmental defect of the structures derived from the third and fourth pharyngeal pouches. It apparently arises due to deletion of 22q11. We describe a strategy for the isolation of DNA probes for this region. A deleted chromosome 22, which includes 22q11, was flow-sorted from a lymphoblastoid cell line of a patient with cat eye syndrome and used as the source of DNA. A DNA library was constructed from this chromosome by cloning into the EcoR1 site of the vector Lambda gt10. Inserts were amplified by PCR and mapped using a somatic cell hybrid panel of this region. Out of 32 probes, 14 were mapped to 22q11. These probes were further sublocalised within the region by dosage analysis of DGS patients, and by the use of two new hybrid cell lines which we have produced from DGS patients. One of these lines (7939B662) contains the altered human chromosome segregated from its normal homologue. This chromosome 22 contains an interstitial deletion in 22q11, and will be useful for localising further probes to the DGS region.     

PCR amplification of chromosome-specific alpha satellite DNA: definition of centromeric STS markers and polymorphic analysis.

Alpha satellite DNA is a tandemly repetitive DNA family found at the centromere of every human chromosome. Chromosome-specific subsets have been isolated for over half the chromosomes and have prove useful as markers for both genetic and physical mapping. We have developed specific oligonucleotide primer sets for polymerase chain reaction (PCR) amplification of alpha satellite DNA from chromosomes 3, 7, 13/21, 17, X, and Y. For each set of primers, PCR products amplified from human genomic DNA are specific for the centromere of the target chromosome(s), as shown by somatic cell hybrid mapping and by fluorescence in situ hybridization. These six subsets represent several evolutionarily related alpha satellite subfamilies, suggesting that specific primer pairs can be designed for most or all chromosomal subsets in the genome. The PCR products from chromosome 17 directly reveal the polymorphic nature of this subset, and a new DraI polymorphism is described. The PCR products from chromosome 13 are also polymorphic, allowing in informative cases genetic analysis of this centromeric subset distinguished from the highly homologous chromosome 21 subset. These primer sets should allow placement of individual centromeres on the proposed STS map of the human genome and may be useful for somatic cell hybrid characterization and for making in situ probes. In addition, the ability to amplify chromosome-specific repetitive DNA families directly will contribute to the structural and functional analysis of these abundant classes of DNA.     

Development of a somatic cell hybrid mapping panel and molecular probes for human chromosome 3

A somatic cell hybrid mapping panel and molecular probes have been developed for human chromosome 3. This panel defines 11 regions for the short and long arms of the chromosome. Four hundred thirty-two probes have been mapped using these hybrids. One hundred thirty-one of these probes were derived from EcoRI and HindIII flow-sorted libraries. The remaining 301 probes were isolated from NotI boundary and random (partial MboI) libraries constructed from a hybrid that provided a relative enrichment in 3p DNA sequences. For some regions of the chromosome, significant differences in the distribution of probes were noted. This was observed for both the unique sequence flow-sorted and NotI probes. These differences are in agreement with previous suggestions that Giemsa light bands are GC-rich, and therefore gene-rich (especially housekeeping genes), and that the Giemsa dark bands may contain DNA that is more highly condensed. The isolation of probes from different types of libraries, or by different screening strategies, appears to reduce deficiencies that might arise from the use of probes derived with a more limited approach. These hybrids and probes should facilitate the construction of physical and genetic linkage maps to identify various disease loci involving chromosome 3.     

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.     

Production of a highly asymmetric somatic hybrid between rice and Zizania latifolia (Griseb): evidence for inter-genomic exchange

 A highly asymmetric and fertile somatic hybrid plant was obtained via protoplast fusion in an intergenric combination. Gamma-ray-irradiated Zizania latifolia (Griseb). Turcz. ex Stapf mesophyll protoplasts were electrofused with idoacetamide-inactivated rice protoplasts derived from a 2-month-old suspension cell culture. Two of the six putative hybrid calli regenerated plants. Cytological observation showed that the somatic chromosome numbers of both plants were the same as the rice parent (2n=24). Nevertheless, the hybrid nature and inter-genomic exchange events of one of the plants, i.e. SH6 (SH for somatic hybrid), were confirmed by Southern analysis using both total genomic DNA and moderate-copy, Z. latifolia-abundant DNA sequences as probes; in both cases, parental specific and/or new intergenomic recombinant hybridization fragments were detected. In both plant and seed morphology, the hybrid (SH6) was distinct from its rice parental cultivar, as well as from the wild donor species, Z. latifolia. Received: 15 August 1998 / Accepted: 30 September 1998     

Characterization of a set of X-linked sequences and of a panel of somatic cell hybrids useful for the regional mapping of the human X chromosome

Summary We have characterized 19 DNA fragments originating from the human X chromosome. Most of them have been isolated from an X chromosome genomic library (Davies et al. 1981) using a systematic screening procedure. These DNA probes have been used to search for restriction fragment length polymorphisms (RFLP). The frequency of restriction polymorphisms (1 per 350 bp analysed) was lower than expected from data obtained with autosomal fragments. The various probes have been mapped within 12 subchromosomal regions using a panel of human-rodent hybrid cell lines. The validity of the panel was established by hybridization experiments performed with 27 X-specific DNA probes, which yielded information on the relative position of translocation break-points on the X chromosome. The DNAs from the various hybrid lines are blotted onto a reusable support which allows one to quickly map any new X-specific DNA fragment. The probes already isolated should be of use to map unbalanced X chromosome aberrations or to characterize new somatic cell hybrid lines. The probes which detect RFLPs define new genetic markers which will help to construct a detailed linkage map of the human X chromosome, and might also serve for the diagnosis of carriers or prenatal diagnosis.