Quantitative mapping of amplicon structure by array CGH identifies CYP24 as a candidate oncogene.

We show here that quantitative measurement of DNA copy number across amplified regions using array comparative genomic hybridization (CGH) may facilitate oncogene identification by providing precise information on the locations of both amplicon boundaries and amplification maxima. Using this analytical capability, we resolved two regions of amplification within an approximately 2-Mb region of recurrent aberration at 20q13.2 in breast cancer. The putative oncogene ZNF217 (ref. 5) mapped to one peak, and CYP24 (encoding vitamin D 24 hydroxylase), whose overexpression is likely to lead to abrogation of growth control mediated by vitamin D, mapped to the other.

High resolution analysis of DNA copy number variation using comparative genomic hybridization to microarrays.

Gene dosage variations occur in many diseases. In cancer, deletions and copy number increases contribute to alterations in the expression of tumour-suppressor genes and oncogenes, respectively. Developmental abnormalities, such as Down, Prader Willi, Angelman and Cri du Chat syndromes, result from gain or loss of one copy of a chromosome or chromosomal region. Thus, detection and mapping of copy number abnormalities provide an approach for associating aberrations with disease phenotype and for localizing critical genes. Comparative genomic hybridization (CGH) was developed for genome-wide analysis of DNA sequence copy number in a single experiment. In CGH, differentially labelled total genomic DNA from a 'test' and a 'reference' cell population are cohybridized to normal metaphase chromosomes, using blocking DNA to suppress signals from repetitive sequences. The resulting ratio of the fluorescence intensities at a location on the 'cytogenetic map', provided by the chromosomes, is approximately proportional to the ratio of the copy numbers of the corresponding DNA sequences in the test and reference genomes. CGH has been broadly applied to human and mouse malignancies. The use of metaphase chromosomes, however, limits detection of events involving small regions (of less than 20 Mb) of the genome, resolution of closely spaced aberrations and linking ratio changes to genomic/genetic markers. Therefore, more laborious locus-by-locus techniques have been required for higher resolution studies. Hybridization to an array of mapped sequences instead of metaphase chromosomes could overcome the limitations of conventional CGH (ref. 6) if adequate performance could be achieved. Copy number would be related to the test/reference fluorescence ratio on the array targets, and genomic resolution could be determined by the map distance between the targets, or by the length of the cloned DNA segments. We describe here our implementation of array CGH. We demonstrate its ability to measure copy number with high precision in the human genome, and to analyse clinical specimens by obtaining new information on chromosome 20 aberrations in breast cancer.

Mutagenic activity of rhodamine dyes and their impurities as detected by mutation induction in Salmonella and DNA damage in Chinese hamster ovary cells.

Commercial rhodamine dyes 6G and B induce His+ reversion mutations in Salmonella and single-strand breaks in Chinese hamster ovary cells, as detected by alkaline sucrose sedimentation. Aroclor 1254-induced rat liver homogenate (S9) is required for production of genetic activity by these dyes. Rhodamine 6G induces both frameshift and base substitution mutations, whereas rhodamine B induces only frameshift mutations. Rhodamine 6G is genetically more active and more toxic than is rhodamine B in both the bacterial and mammalian assays. Rhodamine 6G and B induce doublings of His+ revertants in Salmonella at the doses of 0.02 and 0.52 mumol/plate and shifts in the molecular weight of Chinese hamster ovary DNA at concentrations of 9 x 10(-5) and 9 x 10(-4) M, respectively. All genetic effects assayed demonstrate dose-related increases. Further testing of the pure dyes in Salmonella revealed that rhodamine B loses most of its mutagenicity with purification, whereas rhodamine 6G does not. Impurities from commercial rhodamine B demonstrate the same extent of mutagenicity as the commercial dye.

Increased copy number at 20q13 in breast cancer: defining the critical region and exclusion of candidate genes.

Studies by comparative genomic hybridization have indicated that a major new locus for DNA amplification in breast cancer is 20q13 and suggested that this genetic event is associated with aggressive clinical behavior. We used interphase fluorescence in situ hybridization with anonymous cosmid probes and gene-specific P1 clones to determine the minimal common region of increased copy number and to study involvement of known genes at 20q13. Based on high-level copy number increases (3 to 10-fold) found with one or more probes in 5 of 14 (35%) breast cancer cell lines and in 3 of 36 (8%) primary tumors, the critical region was narrowed to approximately 1.5 megabases at 20q13.2 defined by fractional length pter values 0.81-0.84. Previously known genes were excluded as candidates, implying that this chromosomal region harbors a novel oncogene that contributes to the malignant progression of breast cancer.

Independent amplification and frequent co-amplification of three nonsyntenic regions on the long arm of chromosome 20 in human breast cancer.

DNA amplification at 20q13.2 is common in breast cancer, correlates with poor prognosis, and may reflect location of an important oncogene. Recently, other regions along 20q were also found to undergo amplification. Here, amplification levels and patterns of co-amplification were analyzed by interphase fluorescence in situ hybridization at 14 loci along 20q in 146 uncultured breast carcinomas and 14 cell lines. Three regions were independently amplified in uncultured tumors: RMC20C001 region at 20q13.2 (highly amplified in 9.6% of the cases), PTPN1 region 3 Mb proximal (6.2%), and AIB3 region at 20q11 (6.2%). Co-amplifications involving two or three of these regions were seen in 11 of the 19 highly amplified tumors. The results suggest that three distinct nonsyntenic regions along 20q may be important and that complex chromosomal rearrangements underlie their frequent co-amplification in breast cancer.

Amplification of chromosomal region 20q13 in invasive breast cancer: prognostic implications.

Amplification of the chromosome 20q13 region was recently discovered in breast cancer by comparative genomic hybridization and subsequently further defined by fluorescence in situ hybridization with specific probes. The target gene of the amplification remains unknown. Here, fluorescence in situ hybridization with a cosmid probe for the minimal region of amplification (RMC20C001) was used to study 20q13 amplification in 132 primary breast carcinomas and 11 metastases. The size of the amplicon was studied with four flanking probes. Thirty-eight (29%) primary tumors and 3 (27%) metastases showed increased copy number of the RMC20C001 probe (>1.5-fold relative to the p-arm control). Nine (6.8%) of the primary tumors were highly (>3-fold) amplified. Although the size and location of the amplified region varied from one tumor to another, only the RMC20C001 probe was consistently amplified. 20q13 amplification was significantly associated with a high histological grade (P = 0.01), DNA aneuploidy (P = 0.01), and high S-phase fraction (P = 0.0085). High-level amplification was also associated with short disease-free survival of patients with node-negative breast cancer (P = 0.002). We conclude that high-level 20q13 amplification may be an indicator of poor clinical outcome in node-negative breast cancer and that this chromosomal region is likely to contain a gene with an important role in breast cancer progression. A large definitive study is warranted to assess the independent prognostic value of 20q13 amplification.

The genomic nucleotide sequences of two differentially expressed actin-coding genes from the sea star Pisaster ochraceus.

The genomic sequences of two differentially expressed actin genes from the sea star Pisaster ochraceus are reported. The cytoplasmic actin gene (Cy) is expressed in eggs and early development. The muscle actin gene (M) is expressed in tube feet and testes. Both genes contain an 1125-nucleotide coding region interrupted by three introns at codons 41, 121 and 204. Gene M contains two additional introns at codons 150 and 267. The intron position at codon 150, although present in higher vertebrate actins, has not been reported in actin genes from invertebrates. The M gene coding region has 89.5% nucleotide homology to the Cy gene, and differs from the Cy actin gene in 13 of 375 amino acids (aa), 11 of which are found in the C-terminal half of the gene. The C-terminal half of the M gene contains a significant number of muscle isotype codons. Even though there is only 1 aa change in the first 150 codons, there have been limited substitutions at many four-fold degenerate sites which may indicate selection pressure upon the secondary structure of the mRNA and/or a biased codon usage. Variant CCAAT, TATA, and poly(A)-addition signals have been identified in the 5' and 3' flanking regions. The presence of 5' and 3' splice junction sequences in the 5' flanking region of the Cy gene suggests the potential for an intron there.

Gene arrangement in sea star mitochondrial DNA demonstrates a major inversion event during echinoderm evolution.

The mitochondrial (mt) DNA from the sea star Pisaster ochraceus has been isolated, restriction-mapped, and cloned into plasmid vectors. Both ribosomal RNA genes, the genes for 12 of the 13 mitochondrial proteins, and 11 of the tRNA genes have been localized by DNA sequence analyses. The sequence arrangement of the genes is markedly different from that seen in sea urchin mitochondrial DNA. A segment of the DNA molecule extending from tRNA(pro), including the tRNA cluster, ND1, ND2, and 16S genes, is inverted in relation to the sea urchin genome. The resulting gene order in the sea star is 12S, 16S, ND2, tRNA cluster, COI. As a result of the inversion, the transcriptional polarity of ND1, ND2, and 16S genes are opposite to that of the 12S and COI genes. The arrangement and transcriptional polarity of the other genes mapped here is the same as seen in urchin.

Mutagenicity of paint removers containing dichloromethane.

A volatile component of commercially available paint and varnish removers was mutagenic in strains of Salmonella typhimurium TA1535, TA100 and TA98. Levels of dichloromethane in exposure chambers were determined by gas chromatography and were related directly to mutational dose-effect curves observed for the products.

Suitability of the modified fluctuation assay for evaluating the mutagenicity of unconcentrated drinking water.

Filter-sterilized, unconcentrated tap water induced mutagenic responses (p less than 0.01) in Salmonella strain TA100 in fluctuation assays, usually with dose-related increases in positive tubes. Additional experiments were performed to study possible artifacts that could lead to falsely positive results. Determinations of bacterial survival revealed that cell populations in the tubes containing tap water were larger than in the controls. Since spontaneous mutation is a function of cell generation, the increased numbers of bacteria appeared to be responsible for the higher numbers of mutants observed. Therefore, the positive responses must be regarded as artifactual. This study suggests that survival determination should be a routine part of this method, and care should be exercised in the interpretation of positive results.

Genetic activity of actinomycin D in Saccharomyces cerevisiae but not in Escherichia coli.

The potential of actinomycin D for induction of forward mutation (ADE-), reversion (TRP+), gene conversion, and mitotic recombination, was examined using haploid and diploid strains of yeast Saccharomyces cerevisiae. No increase in forward or reverse mutations or gene conversion was detected, but actinomycin D induced up to 13-fold increases in mitotic recombinants and a 2-fold increase in numbers of aberrant colonies, in a non-selective assay for genetic activity. Actinomycin D was non-mutagenic in a fluctuation test using Escherichia coli strain WP2 UvrA-. This furnishes an example of a mutagen which is negative in bacteria but has genetic activity in yeast, emphasizing the need for using a battery of microbial tests for determining the genetic activity of any given chemical.

Genotoxic activity of pulp mill effluent in Salmonella and Saccharomyces cerevisiae assays.

An XAD-2 resin concentrate of chlorination-stage pulp mill effluent was found to induce mutations in Salmonella typhimurium strains TA1535, TA100 and TA98 but not in strains TA1537 or TA1538. The presence of either S9 mix, S9 mix without cofactors, or heat-inactivated S9 mix, reduced the mutagenic effects. Dose-related increases in gene conversion, mitotic recombination and aberrant colony formation in Saccharomyces cerevisiae strain D7 also were found.

Detection of the mutagenic activity of lead chromate using a battery of microbial tests.

The potential mutagenicity of the carcinogen lead chromate was tested by the following battery of microbial tests: the Escherichia coli PolA+/PolA- survival test; the Salmonella/microsome His+ reversion assay; the E. coli Trp+ reversion test as a plate assay; the E. coli Gal+ forward mutation test; and the Saccharomyces cerevisiae assay for mitotic recombination. Lead chromate is mutagenic in Salmonella and in Saccharomyces and is thus identified as a microbial mutagen by this battery. Metabolic activation by rat liver homogenate (S9) is not required for the mutagenic activity of lead chromate. The most statistically significant, positive result is found with a supplementary assay, the E. coli fluctuation test. To determine whether the lead ion and/or the chromate ion were responsible for the mutagenicity observed, lead chloride and chromium trioxide (chromic acid) were also tested. In E. coli fluctuation test, the ranges of maximal mutagenicity for chromium trioxide and lead chromate overlap at the concentration 10(-5)M, whereas lead chloride shows no mutagenicity and little lethality at concentrations up to 10(-3)M. Thus, it appears that the chromate ion is responsible for the mutagenicity of lead chromate.

Mutagenic activity of diallate and triallate determined by a battery of in vitro mammalian and microbial tests.

Diallate and Triallate are carbamate herbicides used mainly for the pre-emergence control of wild oats in various crops. The genetic activity of these compounds was studied using a battery of microbial and mammalian in vitro tests. In the Salmonella/mammalian-microsome assay, Diallate and Triallate show dose-related increases without metabolic activation in strains TA1535, TA100 and TA98, indicating that these compounds cause both frameshift and base-substitution mutations. Mutagenicity of both herbicides was enhanced greatly by incubation with Aroclor 1254 induced rat-liver S9. Genetic activity in mammalian cells was determined using a number of in vitro tests with Chinese hamster ovary (CHO) cells combined with metabolic activation as described above. Both Diallate and Triallate caused dose-related decreases in colony-forming ability, with concomitant dose-related increases in the frequencies of cells with chromosome damage and in the number of sister-chromatid exchanges. However, only Diallate caused a reduction in DNA molecular weight as determined by alkaline sucrose gradient (ASG) sedimentation. DNA damage was negligible even at concentrations of Triallate that reduced colony-forming ability to zero. This suggests that the lesions in DNA detected by the ASG technique are not necessarily related to those that produce chromosomal damage. These data, taken together, strongly implicate both Diallate and Triallate as capable of causing mutations in mammals. However the risk to man in terms of inherited disease or cancer remains to be established by appropriate in vivo methodology.

Chlorination of ozonated soil fulvic acid: mutagenicity studies in Salmonella.

Samples of soil fulvic acid (SFA) were ozonated and subsequently chlorinated under acidic or slightly basic conditions. The residues were tested for His+ reversion in a fluctuation assay, using Salmonella typhimurium TA100 as the tester strain. The ozonated/chlorinated samples were mutagenic, but activity was dependent on the amount of ozone utilized and the pH of the reaction medium. Although increases in cell concentrations were also induced by some mutagenic samples, this alone did not account for the mutagenicity observed. Unchlorinated samples displayed insignificant activity.

Design considerations for array CGH to oligonucleotide arrays.

BACKGROUND: Representational oligonucleotide microarray analysis has been developed for detection of single nucleotide polymorphisms and/or for genome copy number changes. In this process, the intensity of hybridization to oligonucleotides arrays is increased by hybridizing a polymerase chain reaction (PCR)-amplified representation of reduced genomic complexity. However, hybridization to some oligonucleotides is not sufficiently high to allow precise analysis of that portion of the genome. METHODS: In an effort to identify aspects of oligonucleotide hybridization affecting signal intensity, we explored the importance of the PCR product strand to which each oligonucleotide is homologous and the sequence of the array oligonucleotides. We accomplished this by hybridizing multiple PCR-amplified products to oligonucleotide arrays carrying two sense and two antisense 50-mer oligonucleotides for each PCR amplicon. RESULTS: In some cases, hybridization intensity depended more strongly on the PCR amplicon strand (i.e., sense vs. antisense) than on the detection oligonucleotide sequence. In other cases, the oligonucleotide sequence seemed to dominate. CONCLUSION: Oligonucleotide arrays for analysis of DNA copy number or for single nucleotide polymorphism content should be designed to carry probes to sense and antisense strands of each PCR amplicon to ensure sufficient hybridization and signal intensity.

The effect of 2-micron DNA on survival and mutagenesis in Saccharomyces cerevisiae.

Strains of Saccharomyces cerevisiae, with and without endogenous 2-microns DNA, were studied in experiments designed to determine the effect of this plasmid on survival and mutagenesis in yeast. Comparison of the two strains exposed to ultraviolet light, 4-nitroquinoline oxide, or methyl methanesulfonate (MMS), revealed that the presence of 2-microns DNA slightly enhanced survival after exposure to each agent. Spontaneous frequencies of mutations (histidine reversion, canavanine resistance, and mitochondrial petites, but not adenine auxotrophy) were reduced by the presence of 2-microns DNA. MMS-induced His+ reversion was weak, and both strains responded similarly. No difference was found between the two strains when induced forward mutation to canavanine resistance was examined. The extent of induction of mitochondrial petites was about the same in both strains. Therefore, it appears that under these experimental conditions with these mutagens, 2-microns DNA has an effect on spontaneous mutation and survival after DNA damage but not on induced mutagenesis in S. cerevisiae.