The human chromosome content in human x rodent somatic cell hybrids analyzed by a screening technique using Alu PCR

The ubiquitous nature of the Alu sequence throughout the human genome forms the basis of an assay we present here for analyzing the human chromosome content of human x rodent somatic cell hybrids. A human-specific Alu primer was used both to amplify sequences and to 32P label the products in a polymerase chain reaction (PCR) technique. Unlabeled inter-Alu PCR products from two series of human x rodent hybrids were used to prepare dot blots which were probed with labeled inter-Alu products prepared from between 10(3) and 10(4) hybrid cells. In the first series we demonstrate that a labeled inter-Alu probe from the hybrid DL18ts, containing a single chromosome 18, on a dot blot hybridized only with those inter-Alu products containing chromosome 18. Similar specificity for human chromosome 5 was shown when a Southern blot of the PCR products was hybridized with a probe made from the hybrid HHW 213, which contains only chromosome 5p. Using a dot blot from a second series of control hybrids, 15 of which contained single human chromosomes, hybridization of a labeled probe from the hybrid 18X4-1 was shown to react specifically with the controls that expressed chromosome 18. Application of the technique reported here allows simple and rapid characterization of the human chromosome content in human x rodent hybrids.     

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.     

Assignment of Alu-repetitive sequences to large restriction fragments from human chromosomes 6 and 22

We have employed a pulsed field gel electrophoresis and Alu hybridization approach for identification of large restriction fragments on chromosome 6 and 22. This technique allows large portions of selected human chromosomes to be visualized as discrete hybridization signals. Somatic cell hybrid DNA which contains chromosome 6 or chromosome 22 was restricted with either Notl or Mlul. The restriction fragments were separated by pulsed field gel electrophoresis (PFGE) and hybridized against an Alu repetitive sequence (Blur 8). The hybridization signals result in a fingerprint-like pattern which is unique for each chromosome and each restriction enzyme. In addition, a continuous pattern of restriction fragments was demonstrated by gradually increasing puls times. This approach will also be suitable to analyze aberrant human chromosomes retained in somatic cell hybrids and can be used to analyze flow sorted human chromosomes. To this end, our method provides a valuable alternative to standard cytogenetic analysis.     

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。     

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.     

Identification of Neurofibromatosis 1 (NF1) Homologous Loci by Direct Sequencing, Fluorescence in Situ Hybridization, and PCR Amplification of Somatic Cell Hybrids

Using fluorescence in situ hybridization (FISH), we have identified seven NF1-related loci, two separate loci on chromosome 2, at bands 2q21 and 2q33-q34, and one locus each on five other chromosomes at bands 14q11.2, 15q11.2, 18p11.2, 21q11.2-q21, and 22q11.2. Application of PCR using NF1 primer pairs and genomic DNA from somatic cell hybrids confirmed the above loci, identified additional loci on chromosomes 12 and 15, and showed that the various loci do not share homology beyond NF1 exon 27b. Sequenced PCR products representing segments corresponding to NF1 exons from these loci demonstrated greater than 95% sequence identity with the NF1 locus. We used sequence differences between bona fide NF1 and NF1 -homologous loci to strategically design primer sets to specifically amplify 30 of 36 exons within the 5′ end of the NF1 gene. These developments have facilitated mutation analysis at the NF1 locus using genomic DNA as template.     

Isolation of region-specific and polymorphic markers from chromosome 17 by restricted Alu polymerase chain reaction

We demonstrate that the digestion of template DNAs with restriction endonucleases prior to Alu polymerase chain reaction ("restricted Alu-PCR") reduces the complexity of the Alu-primed amplification patterns of human DNA in somatic cell hybrids and allows a direct informative comparison of these patterns. A comparison of restricted Alu-PCR patterns of a monochromosomal hybrid retaining a human chromosome 17 (MH22-6) and a hybrid retaining a human chromosome 17 deleted for band p11.2 (DH110-D1) revealed four Alu-PCR products that were present in the former but absent in the latter hybrid. Hybridization of these fragments to the total Alu-PCR amplification products of the two hybrids confirmed their absence in DH110-D1 amplification products. Hybridization to a panel of somatic cell hybrids indicated that two of these fragments were deleted in the hybrid DH110-D1 and mapped to 17p11.2, as expected. However, two additional fragments were not deleted in the hybrid DH110-D1 and mapped to other regions of chromosome 17. An insertion-deletion polymorphism was associated with one of the latter fragments, which may be the mechanism for the lack of its amplification in the hybrid DH110-D1. Restricted Alu-PCR should enhance the applications of Alu-PCR and provides a new method for the identification of chromosome-specific polymorphic markers.     

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.     

Rapid isolation of DNA probes within specific chromosome regions by interspersed repetitive sequence polymerase chain reaction

A method was recently developed for the specific amplification of human DNA sequences from interspecific somatic cell hybrids by the polymerase chain reaction (PCR) using primers directed to Alu, a short interspersed repeat element (SINE). We now show human-specific amplification using a primer to the 3' end of the human long interspersed repeat element L1Hs (LINE). A monochromosomal hybrid containing an intact human X chromosome yielded approximately 25 discrete products, ranging in size from 800 to 4500 bp. Combination of a single Alu primer and the L1Hs primer yielded a large number of smaller products (300-1000 bp) distinct from those observed with either primer alone. Inspection of ethidium bromide-stained gels showed one Alu-Alu and three Alu-L1Hs products which were present in an intact X chromosome but absent in a hybrid containing an X chromosome deleted for the single metaphase band q28. These four fragments were isolated from the gel and used as probes on Southern blots which confirmed their localization to Xq28. These results demonstrate that primers can be constructed to a variety of interspersed repetitive sequences (IRS) and used individually or in combination for the rapid isolation of DNA fragments from defined chromosomal regions by IRS-PCR.     

14个染色体区带特异性探针池的构建

本文运用人类染色体显微切割和ＰＣＲ技术,成功地构建了１４个染色体区带特异性探针池,并通过染色体原位杂交证明它们均分别来源于相应的被切割的染色体区带。     

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.     

Assignment of a serotonin 5HT-2 receptor gene (HTR2) to human chromosome 13q14-q21 and mouse chromosome 14

A gene for serotonin 5HT-2 receptor (HTR2) is assigned to human chromosome 13 by somatic cell hybrids and to region 13q14-q21 by in situ hybridization. It is assigned to mouse chromosome 14 by somatic cell hybrid analysis.     

Characterization and regional mapping of new anonymous chromosome 20-specific DNA markers isolated from a flow-sorted DNA library

Employing the flow-sorted chromosome 20-specific DNA library LL20NS01, we isolated seven novel unique poly- and monomorphic DNA markers specific to human chromosome 20. Initially, 201 phage clones were analyzed regarding insert size and repetitivity. By testing 14 single- and low-copy number clones for their ability to detect RFLPs, three polymorphisms were revealed by two probes, pFMS22-1.4 [D20S22] and pFMS76 [D20S23]. Seven of twenty probes (35%) were assigned to chromosome 20 using a somatic cell hybrid DNA panel. Five of them were regionally mapped by in situ hybridization. Three DNA markers, pFMS51 [D20S29], pFMS76 [D20S23], and pFMS106 [D20S30], were assigned to 20p11.2-p12, and two markers, pFMS22-1.4 [D20S22] and pFMS135 [D20S31], to 20q12-q13.3. Our new chromosome 20-specific DNA markers should be useful for the molecular characterization of this rather underpopulated human chromosome.     

Chromosomal localization of three human poly(A)-binding protein genes and four related pseudogenes

In humans, the poly(A)-binding proteins (PABPs) comprise a small nuclear isoform and a conserved gene family that displays at least three functional proteins: PABP1, inducible PABP (iPABP), and PABP3, plus four pseudogenes (1, 2, 3, and PABP4). In situ hybridization of PABP3 cDNA as the probe on metaphasic chromosomes have revealed five possible loci for this gene family at 2q21-q22, 13q11-q12, 12q13.3-q15, 8q22, and 3q24-q25. Amplifications of specific DNA fragments from a human-rodent somatic cell hybrid panel have allowed us to associate PABP1 and PABP3 with 8q22 and 13q11-q12, respectively. The iPABP gene has been assigned to chromosome 1. This result, compared with radiation hybrid database information, strengthens the location of this gene to 1p32-p36. The pseudogenes PABP4, 1, and 2 have been assigned to chromosomes 15, 4, and 14, respectively. Three loci detected on chromosome spreads are not associated with any amplified fragment. They might represent other related PABP genes not yet identified.     

Isolation of a human chromosome 14-only somatic cell hybrid: analysis using Alu and LINE-based PCR.

Interspecific somatic cell hybrids containing single human chromosomes are valuable reagents for localization of cloned genes and DNA fragments to specific chromosomes, for the development of chromosome-specific libraries, and for generation of hybrid cell lines containing subchromosomal regions. A CHO somatic cell hybrid containing a single, intact human chromosome 14 (MHR14) was developed and confirmed by LINE PCR amplification gel pattern, by Alu-517 PCR product dot blot hybridization, and by cytogenetic analysis. MHR14 will serve as the chromosome source for the development of a radiation map of human chromosome 14.     

Neural retina-specific leucine zipper gene NRL (D14S46E) maps to human chromosome 14q11.1-q11.2.

The product of a neural retina-specific gene, NRL, belongs to the "leucine zipper" family of DNA-binding proteins and has a strong similarity to the v-maf oncogene product. The NRL gene maps to human chromosome 14 by Southern blot analysis of genomic DNA from a human-rodent somatic cell hybrid panel. In situ hybridization to metaphase chromosomes has further sublocalized the gene to the region 14q11.1-q11.2. D14S46E has now been assigned to the NRL gene. Because of its specific pattern of expression, NRL is a candidate gene for retinal diseases.     

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.     

Ordering markers in the region of the ataxia-telangiectasia gene (11q22-q23) by fluorescence in situ hybridization (FISH) to interphase nuclei

Fluorescence in situ hybridization (FISH) to interphase nuclei was performed to order probes corresponding to bands 11q22-q23 where the ataxia-telangiectasia (AT) gene(s) have been located. Cosmid probes and one phage probe previously localized to this chromosome 11 region by FISH to metaphase chromosomes, were hybridized to interphase nuclei of the somatic cell hybrid J1a, which contains chromosome 11 as the only human chromosome. Two-color FISH was used with a centromeric reference probe marker. The following order was obtained: cen-D11S385 (CJ52.75)-CJ52.3-D11S384 (CJ52.193) CJ52.114-D11S424 (CJ52.77)-D11S132-NCAM-D11S351 (CJ52.208)-tel. The validity of using the centromeric probe was illustrated by showing that a probe corresponding to 11p13 hybridized more closely to the centromere than a probe corresponding to 11q22-q23, and by using cosmids hybridized three by three.     

Complementation of repair gene mutations on the hemizygous chromosome 9 in CHO: a third repair gene on human chromosome 19

A human DNA repair gene, ERCC2 (Excision Repair Cross Complementing 2), was assigned to human chromosome 19 using hybrid clone panels in two different procedures. One set of cell hybrids was constructed by selecting for functional complementation of the DNA repair defect in mutant CHO UV5 after fusion with human lymphocytes. In the second analysis, DNAs from an independent hybrid panel were digested with restriction enzymes and analyzed by Southern blot hybridization using DNA probes for the three DNA repair genes that are located on human chromosome 19: ERCC1, ERCC2, and X-Ray Repair Cross Complementing 1 (XRCC1). The results from hybrids retaining different portions of this chromosome showed that ERCC2 is distal to XRCC1 and in the same region of the chromosome 19 long arm (q13.2-q13.3) as ERCC1, but on different MluI macrorestriction fragments. Similar experiments using a hybrid clone panel containing segregating Chinese hamster chromosomes revealed the hamster homologs of the three repair genes to be part of a highly conserved linkage group on Chinese hamster chromosome number 9. The known hemizygosity of hamster chromosome 9 in CHO cells can account for the high frequency at which genetically recessive mutations are recovered in these three genes in CHO cells. Thus, the conservation of linkage of the repair genes explains the seemingly disproportionate number of repair genes identified on human chromosome 19.     

Regional assignment of 41 human DNA fragments on chromosome 7 by means of a somatic cell hybrid panel

Summary To detect new restriction fragment length polymorphisms that would cover human chromosome 7 with a network of genetic landmarks, a chromosome 7-specific phage gene library was screened for human single-copy fragments. With use of a somatic cell hybrid panel containing defined regions of human chromosome 7, 41 cloned human single-copy sequences were assigned to five regions of this chromosome. Of special importance are the cell hybrid clones GM1059Rag5 and 7851Rag10-1, derived from human cells with interstitial deletions spanning the bands 7q22-q32, within which the cystic fibrosis gene is located. Twelve new probes are described in 7q22-q32, five of which detect a total of six RFLPs.