Construction and characterization of band-specific DNA libraries

Summary A universally primed polymerase chain reaction was developed to amplify DNA dissected from GTG-banded human chromosomes. The amplification products are cloned into plasmid vectors, which allow the rapid characterization of recombinant clones. Starting from 20–40 chromosome fragments, several thousand independent clones detecting single-copy sequences can be obtained. Although these libraries comprise only a few percent of the dissected DNA, they provide narrowly spaced anchor clones for the molecular characterization of chromosome bands and the identification of gene sequences. Here we describe the construction and characterization of DNA libraries for the Langer-Giedion syndrome chromosome region (LGCR, 8q23–24.1), Wilms tumor chromosome region 1 (WT1, 11p13), Prader-Willi syndrome/Angelman syndrome chromosome region (PWCR/ANCR, 15q11.2–12), meningioma chromosome region (MGCR, 22q12–13), and fragile X chromosome region (FRAXA, Xq27.3).     

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.     

Two type II keratin genes are localized on human chromosome 12

Summary Human genomic DNA containing two type II keratin genes, one coding for keratin 1 (K1, a 68-kD basic protein) and another closely linked type II gene 10–15 kb upstream (K?, gene product unknown), was isolated on a single cosmid clone. EcoRI restriction fragments of the cosmid were subcloned into pGEM-3Z, and specific probes comprising the C-terminal coding and 3 noncoding regions of the two genes were constructed. The type II keratin genes were localized by in situ hybridization of the subcloned probes to normal human lymphocyte chromosomes. In a total of 70 chromosome spreads hybridized with the K? probe (gHK?-3, PstI, 800 bp), 36 of the 105 grains observed were on chromosome 12, and 32 of these were clustered on the long arm near the centromere (12q11–13). In 100 labeled metaphases hybridized with the K1 probe (gHK1–3, BamHI-PstI, 2100 bp), 53 grains localized to chromosome 12 and 46 of these were found in the same region (q11–13). Therefore, both the gene for human keratin 1, a specific marker for terminal differentiation in mammalian epidermis, and another closely linked unknown type II keratin gene (K?, 10–15 kb upstream of K1) are on the long arm (q11–13) of human chromosome 12.     

Topography of genetic loci in the nuclei of cells of colorectal carcinoma and adjacent tissue of colonic epithelium

To determine the influence of increased gene expression and amplification in colorectal carcinoma on chromatin structure, the nuclear distances between pairs of bacterial artificial chromosome (BAC) clones with genomic separation from 800 to 29,000 kb were measured and compared between the tumor and parallel epithelial cells of six patients. The nuclear distances were measured between the loci in chromosomal bands 7p22.3–7p21.3; 7q35–7q36.3; 11p15.5–11p15.4; 20p13; 20p12.2; 20q11.21 and 20q12 where increased expression had been found in all types of colorectal carcinoma. The loci were visualized by three-dimensional fluorescence in situ hybridization using 22 BAC clones. Our results show that for short genomic separations, mean nuclear distance increases linearly with increased genomic separation. The results for some pairs of loci fell outside this linear slope, indicating the existence of different levels of chromatin folding. For the same genomic separations the nuclear distances were frequently shorter for tumor as compared with epithelial cells. Above the initial growing phase of the nuclear distances, a plateau phase was observed in both cell types where the increase in genomic separation was not accompanied by an increase in nuclear distance. The ratio of the mean nuclear distances between the corresponding loci in tumor and epithelium cells decreases with increasing amplification of loci. Our results further show that the large-scale chromatin folding might differ for specific regions of chromosomes and that it is basically preserved in tumor cells in spite of the amplification of many loci.Communicated by T. Hassold     

Gene amplification profiling of esophageal squamous cell carcinomas by DNA array CGH

Gene amplification is one of the basic mechanisms that lead to overexpression of oncogenes. DNA array comparative genomic hybridization (CGH) has great potential for comprehensive analysis of both a relative gene-copy number and altered chromosomal regions in cancers, which enables us to identify new amplified genes and unstable chromosomal loci. We examined the amplification status in 32 esophageal squamous cell carcinomas (ESCCs) and 13 ESCC cell lines on 51 frequently amplified loci in a variety of cancers by both DNA array CGH and Southern blot analyses. The 1p34 locus containing MYCL1, 2p24 (MYCN), 7p12 (EGFR), and 12q14 (MDM2) were amplified in one of the 32 cases (3%), and the 17q12 locus (ERBB2) and 8p11 (FGFR1) in two of the 32 cases (6%), while only the 11q13 locus (Cyclin D1, FGF4, and EMS1) was frequently amplified (28%, 9/32), demonstrating this locus to be a major target in ESCCs. One locus, 8q24 (c-MYC) was found to be amplified only in the cell lines. Eight out of 51 loci (15.7%) were found to be amplified in at least one of the 32 primary ESCCs or the 13 ESCC cell lines, suggesting that chromosomal loci frequently amplified in a type of human cancer may also be amplified in other types of cancers. This paper is the first report of an application of DNA array CGH to ESCCs.     

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.     

Development of a sequence-tagged site for the centromere of chromosome 10: its use in cytogenetic and physical mapping

We sequenced the alphoid centromere probe p10RP8 (D10Z1), aligned it to three published consensus sequences, and developed a sequence-tagged site (STS), sJRH-2, based upon oligonucleotide primers having two 3 mismatches with these consensus sequences. Polymerase chain reaction (PCR) amplification using genomic DNA from a somatic cell hybrid panel representing all human chromosomes demonstrated amplification from only those cell lines containing chromosome 10. Fluorescence in situ hybridization of the amplified product demonstrated intense and specific hybridization of the PCR product to 10p11.1-q11.1. A human genomic yeast artificial chromosome (YAC) library was screened using the sJRH-2 PCR assay, and five clones were identified. Sequence analysis of one chimeric clone (consisting of DNA segments derived from chromosomes 5p and 10cen) confirmed specificity of the STS for the centromere of chromosome 10. sJRH-2 provides a convenient cytogenetic marker for chromosome 10, which will also be useful for physical mapping of the pericentromeric region of chromosome 10, a region that harbors the gene(s) for three forms of multiple endocrine neoplasia (types 2A, 2B, and familial medullary thyroid carcinoma). The GenBank accession number for the p10RP8 sequence is X63622.     

High accuracy genotyping directly from genomic DNA using a rolling circle amplification based assay

Background

Rolling circle amplification of ligated probes is a simple and sensitive means for genotyping directly from genomic DNA. SNPs and mutations are interrogated with open circle probes (OCP) that can be circularized by DNA ligase when the probe matches the genotype. An amplified detection signal is generated by exponential rolling circle amplification (ERCA) of the circularized probe. The low cost and scalability of ligation/ERCA genotyping makes it ideally suited for automated, high throughput methods.

Results

A retrospective study using human genomic DNA samples of known genotype was performed for four different clinically relevant mutations: Factor V Leiden, Factor II prothrombin, and two hemochromatosis mutations, C282Y and H63D. Greater than 99% accuracy was obtained genotyping genomic DNA samples from hundreds of different individuals. The combined process of ligation/ERCA was performed in a single tube and produced fluorescent signal directly from genomic DNA in less than an hour. In each assay, the probes for both normal and mutant alleles were combined in a single reaction. Multiple ERCA primers combined with a quenched-peptide nucleic acid (Q-PNA) fluorescent detection system greatly accellerated the appearance of signal. Probes designed with hairpin structures reduced misamplification. Genotyping accuracy was identical from either purified genomic DNA or genomic DNA generated using whole genome amplification (WGA). Fluorescent signal output was measured in real time and as an end point.

Conclusions

Combining the optimal elements for ligation/ERCA genotyping has resulted in a highly accurate single tube assay for genotyping directly from genomic DNA samples. Accuracy exceeded 99 % for four probe sets targeting clinically relevant mutations. No genotypes were called incorrectly using either genomic DNA or whole genome amplified sample.     

A de novo unbalanced reciprocal translocation identified as paternal in origin in the Prader-Willi syndrome

Summary Interstitial cytogenetic deletions involving the paternally derived chromosome 15q11–13 have been described in patients with the Prader-Willi syndrome (PWS). We report a child with PWS and a de novo unbalanced karyotype –45,XY,–9,–15,+der(9)t(9;15)(q34;q13). Molecular studies with the DNA probe pML34 confirmed that only a single Prader Willi critical region (PWCR: 15q11.2-q12) copy was present. Hybridisation of patient and parental DNA with the multi-allelic probe CMW1, which maps to pter-15q13, showed that the chromosome involved in the translocation was paternal in origin. This is the first example of a paternally-derived PWCR allele loss caused by an unbalanced translocation that has arisen de novo.     

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)     

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.     

Chromosomal localization of genes by scanning electron microscopy using in situ hybridization with biotinylated probes: Y chromosome repetitive sequences

Summary The feasibility of using scanning electron microscopy (SEM) to identify the position of specific DNA sequences was examined using a Y chromosome specific probe (pHY2.1). Tests were carried out on chromosome spreads hybridizedin situ with biotinylated pHY2.1. Chromosomal sites of hybridization of the probe were localized by an indirect immunohistochemical procedure which resulted in a gold product which could be amplified by silver precipitation. In the SEM, the specific location of the probe was easily identified due to the enhanced signal produced by the gold—silver complex. The probe was localized both on the long arm of the Y chromosome and within interphase nuclei. It was found that SEM was more sensitive than light microscopy since the probe could be identified without silver amplification. With refinements to the technique, SEM could provide a useful method for high resolution localizing of unique DNA sequences (i.e. single copy genes).     

Characterization of double minute chromosomes’ DNA content in a human high grade astrocytoma cell line by using comparative genomic hybridization and fluorescence in situ hybridization

The presence of double minute chromosomes (dmin) in cancer cells is known to be correlated with gene amplifications. In human high grade astrocytomas or glioblastomas, about 50% of cytogenetically characterized cases display dmin. G5 is a cell line which has been established from a human glioblastoma containing multiple dmin. In order to identify the DNA content of these dmin, three techniques were successively used: conventional cytogenetic analysis, comparative genomic hybridization (CGH), and fluorescent in situ hybridization (FISH). The karyotype of G5 cells showed numerical chromosome changes (hypertriploidy), several marker chromosomes, and multiple dmin. CGH experiments detected two strong DNA amplification areas located in 9p21-22 and 9p24, as well as an underrepresentation of chromosomes 6, 10, 11, 13, 14, and 18q. By using FISH with a chromosome 9-specific painting probe to metaphase chromosomes of the G5 cell line, dmin were shown to contain DNA sequences originating from chromosome 9. This study demonstrates the usefulness of a combination of classical karyotyping, CGH, and FISH to identify the chromosomal origin of amplified DNA sequences in dmin. Received: 30 October 1994 / Revised: 25 February 1996     

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

Summary 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.     

Novel DNA sequences at chromosome 10q26 are amplified in human gastric carcinoma cell lines: molecular cloning by competitive DNA reassociation.

Molecular cloning of genomic sequences altered in cancer cells is believed to lead to the identification of new genes involved in the initiation and progression of the malignant phenotype. DNA amplification is a frequent molecular alteration in tumor cells, and is a mode of proto-oncogene activation. The cytologic manifestation of this phenomenon is the appearance of chromosomal homogeneously staining regions (HSRs) or double minute bodies (DMs). The gastric carcinoma cell line KATO III is characterized by a large HSR on chromosome 11. In-gel renaturation analysis confirmed the amplification of DNA sequences in this cell line, yet none of 42 proto-oncogenes that we tested is amplified in KATO III DNA. We employed the phenol-enhanced reassociation technique (PERT) to isolate 21 random DNA fragments from the amplified domain, and used 6 of them to further clone some 150 kb from that genomic region. While in situ hybridization performed with some of these sequences indicated that in KATO III they are indeed amplified within the HSR on chromosome 11, somatic cell hybrid analysis and in situ hybridization to normal lymphocyte chromosomes showed that they are derived from chromosome 10, band q26. The same sequences were found to be amplified in another gastric carcinoma cell line, SNU-16, which contains DMs, but were not amplified in other 70 cell lines representing a wide variety of human neoplasms. One of these sequences was highly expressed in both KATO III and SNU-16. Thus, the cloned sequences supply a starting point for identification of novel genes which might be involved in the pathogenesis of gastric cancers, and are located in a relatively unexplored domain of the human genome.     

Assignment of anonymous DNA probes to specific intervals of human chromosomes 16 and X

Summary Anonymous DNA probes mapping to human chromosome 16 and the distal region of the human X chromosome were isolated from a genomic library constructed using lambda EMBL3 and DNA from a mouse/human hybrid. The hybrid cell contained a der(16)t(X;16)(q26;q24) as the only human chromosome. Fifty clones were isolated using total human DNA as a hybridisation probe. Forty six clones contained single copy DNA in addition to the repetitive DNA. Pre-reassociation with sonicated human DNA was used to map these clones by a combination of Southern blot analysis of a hybrid cell panel containing fragments of chromosomes 16 and X and in situ hybridisation. One clone mapped to 16pter 16p13.11, one clone to 16p13.316p13.11, four clones to 16p13.316p13.13, two clones to 16p13.1316p13.11, one clone to 16p13.11, seven clones to 16p13.1116q12 or 16q13, four clones to 16q12 or 16q13, three clones to 16q1316q22.1, four clones to 16q22.10516q24, and nineteen clones to Xq26Xqter. Two clones mapping to 16p13 detected RFLPs. VK5 (D16S94) detected an MspI RFLP, PIC 0.37. VK20 (D16S96) detected a TaqI RFLP, PIC 0.37 and two MspI RFLPs, PIC 0.30 and 0.50. The adult polycystic kidney disease locus (PKD1) has also been assigned to 16p13. The RFLPs described will be of use for genetic counselling and in the isolation of the PKD1 gene. Similarly, the X clones may be used to isolate RFLPs for genetic counselling and the isolation of genes for the many diseases that map to Xq26qter.     

Identification and cloning of a novel amplified DNA sequence in human malignant fibrous histiocytoma derived from a region of chromosome 12 frequently rearranged in soft tissue tumors.

Amplification of cellular oncogenes occurs frequently in several human cancers and is an important mechanism of increased gene expression. Identification of amplified genes in tumor cells has proved to be a useful approach for understanding genetic alterations in cancer. Previous procedures for isolating probes from amplified DNA sequences have relied on tissue culture cells, limiting the range of tumors that can be studied and raising questions of in vitro artifact. We have circumvented these problems by combining in gel renaturation of amplified sequences with the polymerase chain reaction. Using this approach, we have identified and partially cloned a DNA amplification unit from biopsies of human malignant fibrous histiocytoma. This amplification unit is derived from chromosome 12q13-14, a site commonly involved in rearrangements in soft tissue tumors, and contains at least one transcribed region (designated SAS, for sarcoma amplified sequence).     

Regional assignment of the human uroporphyrinogen III synthase (UROS) gene to chromosome 10q25.2→q26.3

Summary Uroporphyrinogen III synthase [UROS; hydroxymethylbilane hydro-lyase (cyclizing), EC 4.2.1.75] is the fourth enzyme in the human heme biosynthetic pathway. The recent isolation of the cDNA encoding human UROS facilitated its chromosomal localization. Human UROS sequences were specifically amplified by the polymerase chain reaction (PCR) from genomic DNA of two independent panels of human-rodent somatic cell hybrids. There was 100% concordance for the presence of the human UROS PCR product and human chromosome 10. For each of the other chromosomes, there was 19%–53% discordance with human UROS. The chromosomal assignment was confirmed by Southern hybridization analysis of DNA from somatic cell hybrids with the full-length UROS cDNA. Using human-rodent hybrids containing different portions of human chromosome 10, we assigned the UROS gene to the region 10q25.2 q26.3.     

Integration of DNA copy number alterations and transcriptional expression analysis in human gastric cancer

Background

Genomic instability with frequent DNA copy number alterations is one of the key hallmarks of carcinogenesis. The chromosomal regions with frequent DNA copy number gain and loss in human gastric cancer are still poorly defined. It remains unknown how the DNA copy number variations contributes to the changes of gene expression profiles, especially on the global level.

Principal Findings

We analyzed DNA copy number alterations in 64 human gastric cancer samples and 8 gastric cancer cell lines using bacterial artificial chromosome (BAC) arrays based comparative genomic hybridization (aCGH). Statistical analysis was applied to correlate previously published gene expression data obtained from cDNA microarrays with corresponding DNA copy number variation data to identify candidate oncogenes and tumor suppressor genes. We found that gastric cancer samples showed recurrent DNA copy number variations, including gains at 5p, 8q, 20p, 20q, and losses at 4q, 9p, 18q, 21q. The most frequent regions of amplification were 20q12 (7/72), 20q12–20q13.1 (12/72), 20q13.1–20q13.2 (11/72) and 20q13.2–20q13.3 (6/72). The most frequent deleted region was 9p21 (8/72). Correlating gene expression array data with aCGH identified 321 candidate oncogenes, which were overexpressed and showed frequent DNA copy number gains; and 12 candidate tumor suppressor genes which were down-regulated and showed frequent DNA copy number losses in human gastric cancers. Three networks of significantly expressed genes in gastric cancer samples were identified by ingenuity pathway analysis.

Conclusions

This study provides insight into DNA copy number variations and their contribution to altered gene expression profiles during human gastric cancer development. It provides novel candidate driver oncogenes or tumor suppressor genes for human gastric cancer, useful pathway maps for the future understanding of the molecular pathogenesis of this malignancy, and the construction of new therapeutic targets.     

DNA sequences amplified in cancer cells: an interface between tumor biology and human genome analysis.

There is growing evidence that amplification of specific genes is associated with tumor progression. While several proto-oncogenes are known to be activated by amplification, it is clear that not all the genes involved in DNA amplification in human tumors have been discovered. Our approach to the identification of such genes is based on the 'reverse genetics' methodology. Anonymous amplified DNA fragments are cloned by virtue of their amplification in a given tumor. These sequences are mapped in the normal genome and hence define a new genetic locus. The amplified domain is isolated by long-range cloning and analyzed along three lines of investigation: new genes are sought that can explain the biological significance of the amplification; the structure of the domain is studied in normal cells and in the amplification unit in the cancer cell; attempts are made to identify molecular probes of diagnostic value within the amplified domain. This application of genome technology to cancer biology is demonstrated in our study of a new genomic domain at chromosome 10q26 which is amplified specifically in human gastric carcinomas.