High rate of constitutional chromosomal rearrangements in apparently sporadic ALS.

Of 85 patients with ALS, the authors identified 3 patients with balanced translocations and 2 patients with pericentric inversions, all affecting distinct chromosomal loci. The high rate of constitutional aberrations (5.9%) suggests that ALS is, in part, associated with recombination-based rearrangements of genomic sequences.

Human carbonic anhydrase XII: cDNA cloning, expression, and chromosomal localization of a carbonic anhydrase gene that is overexpressed in some renal cell cancers.

We report the cloning and characterization of a tumor-associated carbonic anhydrase (CA) that was identified in a human renal cell carcinoma (RCC) by serological expression screening with autologous antibodies. The cDNA sequence predicts a 354-amino acid polypeptide with a molecular mass of 39,448 Da that has features of a type I membrane protein. The predicted sequence includes a 29-amino acid signal sequence, a 261-amino acid CA domain, an additional short extracellular segment, a 26-amino acid hydrophobic transmembrane domain, and a hydrophilic C-terminal cytoplasmic tail of 29 amino acids that contains two potential phosphorylation sites. The extracellular CA domain shows 30-42% homology with known human CAs, contains all three Zn-binding histidine residues found in active CAs, and contains two potential sites for asparagine glycosylation. When expressed in COS cells, the cDNA produced a 43- to 44-kDa protein in membranes that had around one-sixth the CA activity of membranes from COS cells transfected with the same vector expressing bovine CA IV. We have designated this human protein CA XII. Northern blot analysis of normal tissues demonstrated a 4.5-kb transcript only in kidney and intestine. However, in 10% of patients with RCC, the CA XII transcript was expressed at much higher levels in the RCC than in surrounding normal kidney tissue. The CA XII gene was mapped by using fluorescence in situ hybridization to 15q22. CA XII is the second catalytically active membrane CA reported to be overexpressed in certain cancers. Its relationship to oncogenesis and its potential as a clinically useful tumor marker clearly merit further investigation.

Human endogenous retroviral element k10 (HERV-K10): chromosomal localization by somatic hybrid mapping and fluorescence in situ hybridization.

The human endogenous retrovirus K10 (HERV-K10) was mapped to human chromosomes using HERV-K10 specific PCR primers on a somatic hybrid mapping panel. A non-random chromosomal location was demonstrated with PCR signals on chromosomes 1, 3, 4, 5, 6, 7, 10, 11, 12, 14, 15, 19, 20, 21, 22 and Y. There was a lack of PCR products on the other chromosomes, even after hybridization with a HERV-K10 specific probe. To further localize the HERV-K10 sequence we used fluorescence in situ hybridization. Chromosomes 1, 3, 6, 7, 10, 11, 12 and 22 were found to contain several HERV-K10 sequences in different regions. The presence of several integration sites on some chromosomes is consistent with previous studies demonstrating 30-50 copies of the HERV-K10 sequence per haploid genome. The mapping information reported in this study will assist the analysis of the biological significance of the HERV-K10 sequence.

Generation and characterization of a human chromosome 6-specific hncDNA library from a somatic cell hybrid.

Chromosome specific cDNA libraries are a useful source of candidate genes for disorders which have been linked to particular chromosomes. Here, we report the generation of a cDNA library made from a somatic cell hybrid retaining chromosome 6 as its only human component. In order to ascertain the chromosomal location of cDNAs the library was amplified by inter-Alu-PCR and used as probe for competitive in situ suppression (CISS). To identify human specific cDNA clones the library was screened with PD39, a highly human specific Alu consensus probe. Out of 350,000 clones 360 were found to hybridize with PD39. Nucleotide sequences were determined for 40 clones with inserts larger than 500 basepairs (bp) and a sequence comparison was performed at the National Center for Biotechnology Information using BLASTN. One clone was shown to be identical to Manganese Superoxide Dismutase (MnSOD/SOD2) which has previously been assigned to chromosome 6q25. Localization of 11 clones was determined using PCR and clone-specific primer pairs on a hybrid mapping panel DNA set. Two PCR-localized clones and five additional clones were localized by fluorescence in situ hybridization. Transcripts for five clones were identified by RT-PCR. The generation of chromosome 6-specific hncDNAs from a somatic cell hybrid should aid in the identification of disease-associated genes localized on this chromosome.

Isolation and localization of transcribed sequences on human chromosome 22.

Recently, we reported the generation of a heteronuclear (hn) cDNA library from a human x rodent somatic cell hybrid retaining human chromosome 22. Here, we report the characterization and localization of 12 cDNA derived clones from this library. Human-specific cDNA sequences have been selectively amplified by inter-Alu PCR. To exclude Alu transcripts, only clones with inserts larger than 500 bp were analyzed. Ten of the 12 clones were localized by PCR on chromosome 22, with four clones mapping on additional chromosomes. One PCR-localized clone and two additional clones were mapped on chromosome 22 by in situ hybridization. Transcripts in human cells were identified for seven of the eight clones analyzed by RT-PCR. None of the clones showed significant sequence similarities within the GenBank and EMBL databases, indicating that these clones represent previously unknown genes. This is the first report on the isolation of chromosome 22-specific transcripts from a human x rodent somatic cell hybrid.

Enrichment of chromosome specific hncDNAs by magnetic bead coupled Alu sequences.

We have employed a strategy for the rapid enrichment of cDNA clones from human chromosome 22 utilizing magnetic beads. Starting from a somatic cell hybrid which retains chromosome 22 in rodent background, heteronuclear (hn) RNA was transcribed into hncDNA using poly dT-primers. Using linker specific primers hncDNA was amplified by PCR. To identify chromosome 22 specific hncDNAs a highly human specific Alu consensus sequence (PD39) was biotinylated and hybridized to the PCR product of the hncDNAs in solution. Hybridized hncDNA-PD39 complexes are captured using streptavidin-coated magnetic beads. Hybridized hncDNAs are selectively amplified by PCR. To verify the chromosome specificity the hncDNA was used as probe for in situ hybridization. Following two rounds of selection with magnetic beads there was an increasingly strong hybridization signal on chromosome 22. The capturing of hncDNAs by magnetic beads as described in this study is faster and more efficient than previously described methods for the isolation of chromosome specific hncDNAs. The novel approach has been employed to generate hncDNAs highly enriched for chromosome 22 specific sequences.

Coamplification on chromosomes 7p12-13 and 9q12-13 identified by reverse chromosome painting in a glioblastoma multiforme.

DNA amplification is known to occur in approximately 50% of glioblastomas, with the epidermal growth factor receptor (EGFR) gene being the most frequently amplified. Whereas previous amplification studies have largely been limited to the analysis of known tumor-related genes, reverse chromosome painting allows us to search for as yet unidentified amplified domains. Here, we report the analysis of a glioblastoma multiforme by reverse chromosome painting. Hybridization signals were found on chromosome 7p12-13 and chromosome 9q12-13. Standard Southern blot analysis revealed amplification of the EGFR gene, which is localized on band 7p13. These findings corroborate previous reports on coamplification of sequences on different chromosomes in glioblastoma.

DNA amplifications on chromosomes 7, 9 and 12 in glioblastoma detected by reverse chromosome painting.

Biopsies and cell culture, respectively, of four human glioblastoma multiforme (WHO 4) have been evaluated for gene amplification using reverse chromosome painting. Three of the tumours showed amplified domains within chromosome bands 12q13-15. The exact localisation and extension of the amplified domains, however, varies within this region. Southern blot analysis revealed amplification of the GLI oncogene in two of the glioblastomas which were found to contain amplified domains within 12q13-15. Reverse chromosome painting also identified amplified domains within bands 7q21 and 9p23-24. Amplification within region 9p23-24 has previously not been reported in glioblastoma. The amplified domain encompassing 9p23-24 was detected in the same glioblastoma which contained an amplification unit within bands 12q13-14. These data, together with previous reports, indicate that amplifications are predominantly found on chromosomes 7, 9 and 12 in glioblastoma. In addition, this study provides further evidence that coamplification is not a rare event in glioblastoma.

Generation of a chromosome-22-specific c-DNA library as confirmed by FISH analysis.

We have recently developed a strategy for the rapid enrichment of c-DNA fragments from selected human chromosomes. Heteronuclear RNA (hn-RNA) is isolated from a somatic cell hybrid that retains a single human chromosome in a rodent background. Following c-DNA synthesis, human sequences are selectively amplified by the Alu polymerase chain reaction (Alu-PCR). Here we have applied this protocol for the selective isolation of novel c-DNAs encoded by chromosome 22. Fluorescence in situ hybridization has been used to confirm the chromosome-22-specific origin of the c-DNA fragments. Controls show DNAse-free RNase-treated hn-RNA results in no c-DNAs or Alu-PCR products. As demonstrated by competitive in situ suppression hybridization (CISS), the majority of the Alu-PCR products from hybrid GM 10027 are located on chromosome 22. Without competition, hybridization signals have also been identified on other human chromosomes. These unspecific hybridization signals result from Alu sequences and can successfully be reduced by competition with cot 1 DNA. This is the first report of the use of CISS for the localization of chromosome-specific c-DNAs.

Immunophenotyping of mitotic cells from long-term cultures of chorionic villi.

Chromosomal mosaicism in chorionic villus samples (CVS) may arise from different sources, such as clonal diversity within the chorionic tissue or contamination with maternal cells. To determine the origin of karyotyped cells, we compared the immunocytochemical features of mitotic cells in CVS long-term cultures with histological sections of their tissue of origin, i.e. chorionic villi. Immunolabelling of intermediate filaments specific for epithelial cells (cytokeratin) and mesenchymal cells (vimentin) established that mitoses yielded from CVS long-term cultures indeed stem from independently growing clones derived from both the epithelial and mesenchymal parts of the chorionic villi. Thus, mosaicism in CVS cultures may reflect true genetic heterogeneity within the biopsy. However, epithelial chorionic cells undergo in vitro metaplasia leading to co-expression of cytokeratins and vimentin. Fetal-specific immune markers (beta-HCG and SP1-glycoprotein) are not reliably expressed in CVS cell culture.

Assignment of casein kinase 2 alpha sequences to two different human chromosomes.

Human casein kinase 2 alpha gene (CK-2-alpha) sequences have been localized within the human genome by in situ hybridization and somatic cell hybrid analysis using a CK-2 alpha cDNA as a probe. By in situ hybridization, the CK-2 alpha cDNA could be assigned to two different loci, one on 11p15.1-ter and one on 20p13. The existence of two separate chromosomal loci suggests that CK-2 alpha is a member of a gene family. Only the locus on chromosome 11 was confirmed by somatic cell hybrid analysis. The analysis was based on the presence of a CK-2-alpha-specific 20-kb fragment. However, the CK-2 alpha cDNA hybridizes to several additional fragments in total human DNA.

Large-scale physical mapping within the region 22q12.3-13.1 in meningioma.

The lack of physical mapping data strongly restricts the analysis of the meningioma chromosomal region that was assigned to the bands 22q12.3-qter. Recently, we reported a new marker D22S16 for chromosome 22 that was assigned to the region 22q13-qter by in situ hybridization. Utilizing somatic cell hybrids we now sublocalized the marker D22S16 within the band region 22q12-13.1, thus placing it in the vicinity of the gene for the platelet derived growth factor (PDGFB). A physical map was established for the regions surrounding the PDGFB gene and the D22S16 marker. By means of pulsed-field gel electrophoresis (PFGE) D22S16 and PDGFB were found to be physically linked within 900 kb. We also identified two CpG clusters bordering the PDGFB gene. For the enzyme NotI, a variation of the PDGFB restriction pattern was found between different individuals. PFGE analysis of the two loci (PDFGB and D22S16) failed to identify major rearrangements in meningioma.

Isolation and characterization of DNA sequences from flow-sorted human chromosome 22 libraries.

Two flow-sorted chromosome 22 libraries were used to isolate DNA sequences specific for chromosome 22. 45-phage DNAs were probed against human genomic DNA. 12 of them showed unique or low-copy character. Using digested DNA from rodent-human hybrid cell lines, 3 of the 12 recombinants were assigned unique to chromosome 22 and regionally mapped. 1 clone mapped to 22pter-q11, 1 clone to 22q12-qter and 1 clone, for which in situ hybridization was performed, to 22q13.1. 2 low-copy probes, 1 of them displaying polymorphisms in MspI and TaqI digests of individual DNAs, must have similar sequences on 22 and additional chromosomes. Furthermore, a highly repetitive DNA representing a compound locus of some hundred kilobases on chromosome 22 was isolated. These 6 probes may provide useful tools for studying the structure and function of this small chromosome involved in a relatively high number of inherited and acquired diseases.

Differential activity of two oncogenes from chromosome #7 in human glioblastoma cell lines.

Human glioblastoma cell lines are known to develop polysomy of cytogenetically intact chromosomes #7 and overexpression of the erbB oncogene (7p12-p14) at a level even higher than is to be expected from the number of #7 chromosomes. The met oncogene, however, which is also located on chromosome #7 (7q31-q32), was shown not to be overexpressed in a panel of 7-polysomic glioblastoma cell lines overexpressing erbB. Molecular analysis of the cell line HeRo gave proof that there is no detectable amplification or rearrangement of the erbB gene which could be responsible for its overexpression. These findings favor the assumption of differential regulation of the met and erbB oncogenes, e.g. by means of insufficient activity of a trans-acting erbB suppressor gene possibly located on a chromosome with a low copy number.

Regional localization and molecular characterization of a DNA sequence on the long arm of chromosome 22.

A human genomic DNA fragment, p22hom13 (D22S16), was isolated from a chromosome 22-specific library. After elimination of repetitive sequences, a single copy BamHI-EcoRI fragment was subcloned into pTZ18. By using mouse/human somatic cell hybrids and in situ hybridization, the new DNA probe was mapped to chromosome 22q13-qter. Its application in the analysis of the distal part of chromosome 22 and its diagnostic use in translocations are discussed.

Characterization of a continuous cell line (MHH-NB-11) derived from advanced neuroblastoma.

A continuous cell line was established from an explanted tumor biopsy obtained from a patient with advanced neuroblastoma, which showed no response to chemotherapy. This cell line MHH-NB-11 retained most properties of the original immature tumor, even after xenotransplantation into nude mice. The cell line consisted of small dense cells with scant cytoplasm and thin; long processes and expressed neuron-specific enolase and synaptophysin, but neither GFAP nor S-100 protein. Karyotyping showed karyograms with 49 to 54 chromosomes, with a modal at 52. Most cells had trisomy 2,7,8,20, but only few structural aberrations were observed. Two of four chromosomes 1 showed a rearrangement of the terminal 1p segment, and all cells had a long HSR on the long arm of one of the chromosomes 13. This region hybridized in situ with the N-myc probe pNB-1. N-myc was amplified 20-fold in this neuroblastoma cell line as determined in Southern blot analysis. This cell line should be a useful tool in vitro or as a xenograft model for neuroblastoma research.

A novel centromeric repetitive DNA from human chromosome 22.

A recombinant DNA clone localized in the centromeric region of chromosome 22 was isolated from a flow-sorted human chromosome 22 DNA library. When the original insert of about 1.9 kb was used to probe Southern blots of EcoRI-digested genomic DNA it revealed at least 40 fragments. A comparable pattern was obtained with each of the three subclones (800, 700, and 380 bp). In situ hybridization showed signals clustered in the region 22cen. DNA sequence analysis using the 380 bp fragment subcloned in pTZ18/19 (p22hom48.4) revealed eight copies of a 48 bp repeat and the size of hybridizing restriction fragments indicated that this tandemly repeated sequence is spread over a region of a few hundred kilobases. Whereas this novel DNA, termed D22Z3, displayed no sequence homology to rodent and monkey genomes cross-homology was discernible for DNA from two great ape species.