Comprehensive copy number and gene expression profiling of the 17q23 amplicon in human breast cancer

The biological significance of DNA amplification in cancer is thought to be due to the selection of increased expression of a single or few important genes. However, systematic surveys of the copy number and expression of all genes within an amplified region of the genome have not been performed. Here we have used a combination of molecular, genomic, and microarray technologies to identify target genes for 17q23, a common region of amplification in breast cancers with poor prognosis. Construction of a 4-Mb genomic contig made it possible to define two common regions of amplification in breast cancer cell lines. Analysis of 184 primary breast tumors by fluorescence in situ hybridization on tissue microarrays validated these results with the highest amplification frequency (12.5%) observed for the distal region. Based on GeneMap'99 information, 17 known genes and 26 expressed sequence tags were localized to the contig. Analysis of genomic sequence identified 77 additional transcripts. A comprehensive analysis of expression levels of these transcripts in six breast cancer cell lines was carried out by using complementary DNA microarrays. The expression patterns varied from one cell line to another, and several overexpressed genes were identified. Of these, RPS6KB1, MUL, APPBP2, and TRAP240 as well as one uncharacterized expressed sequence tag were located in the two common amplified regions. In summary, comprehensive analysis of the 17q23 amplicon revealed a limited number of highly expressed genes that may contribute to the more aggressive clinical course observed in breast cancer patients with 17q23-amplified tumors.

The role of genetic and genomic attributes in the success of polyploids

In 1950, G. Ledyard Stebbins devoted two chapters of his book Variation and Evolution in Plants (Columbia Univ. Press, New York) to polyploidy, one on occurrence and nature and one on distribution and significance. Fifty years later, many of the questions Stebbins posed have not been answered, and many new questions have arisen. In this paper, we review some of the genetic attributes of polyploids that have been suggested to account for the tremendous success of polyploid plants. Based on a limited number of studies, we conclude: (i) Polyploids, both individuals and populations, generally maintain higher levels of heterozygosity than do their diploid progenitors. (ii) Polyploids exhibit less inbreeding depression than do their diploid parents and can therefore tolerate higher levels of selfing; polyploid ferns indeed have higher levels of selfing than do their diploid parents, but polyploid angiosperms do not differ in outcrossing rates from their diploid parents. (iii) Most polyploid species are polyphyletic, having formed recurrently from genetically different diploid parents. This mode of formation incorporates genetic diversity from multiple progenitor populations into the polyploid “species”; thus, genetic diversity in polyploid species is much higher than expected by models of polyploid formation involving a single origin. (iv) Genome rearrangement may be a common attribute of polyploids, based on evidence from genome in situ hybridization (GISH), restriction fragment length polymorphism (RFLP) analysis, and chromosome mapping. (v) Several groups of plants may be ancient polyploids, with large regions of homologous DNA. These duplicated genes and genomes can undergo divergent evolution and evolve new functions. These genetic and genomic attributes of polyploids may have both biochemical and ecological benefits that contribute to the success of polyploids in nature.

Terpenoid-based defenses in conifers: cDNA cloning, characterization, and functional expression of wound-inducible (E)-α-bisabolene synthase from grand fir (Abies grandis)

(E)-α-Bisabolene synthase is one of two wound-inducible sesquiterpene synthases of grand fir (Abies grandis), and the olefin product of this cyclization reaction is considered to be the precursor in Abies species of todomatuic acid, juvabione, and related insect juvenile hormone mimics. A cDNA encoding (E)-α-bisabolene synthase was isolated from a wound-induced grand fir stem library by a PCR-based strategy and was functionally expressed in Escherichia coli and shown to produce (E)-α-bisabolene as the sole product from farnesyl diphosphate. The expressed synthase has a deduced size of 93.8 kDa and a pI of 5.03, exhibits other properties typical of sesquiterpene synthases, and resembles in sequence other terpenoid synthases with the exception of a large amino-terminal insertion corresponding to Pro81–Val296. Biosynthetically prepared (E)-α-[3H]bisabolene was converted to todomatuic acid in induced grand fir cells, and the time course of appearance of bisabolene synthase mRNA was shown by Northern hybridization to lag behind that of mRNAs responsible for production of induced oleoresin monoterpenes. These results suggest that induced (E)-α-bisabolene biosynthesis constitutes part of a defense response targeted to insect herbivores, and possibly fungal pathogens, that is distinct from induced oleoresin monoterpene production.

Octamer-based genome scanning distinguishes a unique subpopulation of Escherichia coli O157:H7 strains in cattle

Multilocus-genotyping methods have shown that Escherichia coli O157:H7 is a geographically disseminated clone. However, high-resolution methods such as pulse-field gel electrophoresis demonstrate significant genomic diversity among different isolates. To assess the genetic relationship of human and bovine isolates of E. coli O157:H7 in detail, we have developed an octamer-based genome-scanning methodology, which compares the distance between over-represented, strand-biased octamers that occur in the genome. Comparison of octamer-based genome-scanning products derived from >1 megabase of the genome demonstrated the existence of two distinct lineages of E. coli O157:H7 that are disseminated within the United States. Human and bovine isolates are nonrandomly distributed among the lineages, suggesting that one of these lineages may be less virulent for humans or may not be efficiently transmitted to humans from bovine sources. Restriction fragment length polymorphism analysis with lambdoid phage genomes indicates that phage-mediated events are associated with divergence of the lineages, thereby providing one explanation for the degree of diversity that is observed among E. coli O157:H7 by other molecular-fingerprinting methods.

Detection of HIV-1 RNA by nucleic acid sequence-based amplification combined with fluorescence correlation spectroscopy

Nucleic acid sequence-based amplification (NASBA) has proved to be an ultrasensitive method for HIV-1 diagnosis in plasma even in the primary HIV infection stage. This technique was combined with fluorescence correlation spectroscopy (FCS) which enables online detection of the HIV-1 RNA molecules amplified by NASBA. A fluorescently labeled DNA probe at nanomolar concentration was introduced into the NASBA reaction mixture and hybridizing to a distinct sequence of the amplified RNA molecule. The specific hybridization and extension of this probe during amplification reaction, resulting in an increase of its diffusion time, was monitored online by FCS. As a consequence, after having reached a critical concentration of 0.1–1 nM (threshold for unaided FCS detection), the number of amplified RNA molecules in the further course of reaction could be determined. Evaluation of the hybridization/extension kinetics allowed an estimation of the initial HIV-1 RNA concentration that was present at the beginning of amplification. The value of initial HIV-1 RNA number enables discrimination between positive and false-positive samples (caused for instance by carryover contamination)—this possibility of discrimination is an essential necessity for all diagnostic methods using amplification systems (PCR as well as NASBA). Quantitation of HIV-1 RNA in plasma by combination of NASBA with FCS may also be useful in assessing the efficacy of anti-HIV agents, especially in the early infection stage when standard ELISA antibody tests often display negative results.

Quantifying single gene copy number by measuring fluorescent probe lengths on combed genomic DNA

An approach was developed for the quantification of subtle gains and losses of genomic DNA. The approach relies on a process called molecular combing. Molecular combing consists of the extension and alignment of purified molecules of genomic DNA on a glass coverslip. It has the advantage that a large number of genomes can be combed per coverslip, which allows for a statistically adequate number of measurements to be made on the combed DNA. Consequently, a high-resolution approach to mapping and quantifying genomic alterations is possible. The approach consists of applying fluorescence hybridization to the combed DNA by using probes to identify the amplified region. Measurements then are made on the linear hybridization signals to ascertain the region's exact size. The reliability of the approach first was tested for low copy number amplifications by determining the copy number of chromosome 21 in a normal and trisomy 21 cell line. It then was tested for high copy number amplifications by quantifying the copy number of an oncogene amplified in the tumor cell line GTL-16. These results demonstrate that a wide range of amplifications can be accurately and reliably quantified. The sensitivity and resolution of the approach likewise was assessed by determining the copy number of a single allele (160 kb) alteration.

t(11;22)(q23;q11.2) in acute myeloid leukemia of infant twins fuses MLL with hCDCrel, a cell division cycle gene in the genomic region of deletion in DiGeorge and velocardiofacial syndromes

We examined the MLL genomic translocation breakpoint in acute myeloid leukemia of infant twins. Southern blot analysis in both cases showed two identical MLL gene rearrangements indicating chromosomal translocation. The rearrangements were detectable in the second twin before signs of clinical disease and the intensity relative to the normal fragment indicated that the translocation was not constitutional. Fluorescence in situ hybridization with an MLL-specific probe and karyotype analyses suggested t(11;22)(q23;q11.2) disrupting MLL. Known 5′ sequence from MLL but unknown 3′ sequence from chromosome band 22q11.2 formed the breakpoint junction on the der(11) chromosome. We used panhandle variant PCR to clone the translocation breakpoint. By ligating a single-stranded oligonucleotide that was homologous to known 5′ MLL genomic sequence to the 5′ ends of BamHI-digested DNA through a bridging oligonucleotide, we formed the stem–loop template for panhandle variant PCR which yielded products of 3.9 kb. The MLL genomic breakpoint was in intron 7. The sequence of the partner DNA from band 22q11.2 was identical to the hCDCrel (human cell division cycle related) gene that maps to the region commonly deleted in DiGeorge and velocardiofacial syndromes. Both MLL and hCDCrel contained homologous CT, TTTGTG, and GAA sequences within a few base pairs of their respective breakpoints, which may have been important in uniting these two genes by translocation. Reverse transcriptase-PCR amplified an in-frame fusion of MLL exon 7 to hCDCrel exon 3, indicating that an MLL-hCDCrel chimeric mRNA had been transcribed. Panhandle variant PCR is a powerful strategy for cloning translocation breakpoints where the partner gene is undetermined. This application of the method identified a region of chromosome band 22q11.2 involved in both leukemia and a constitutional disorder.

Hybridization of single-stranded DNA targets to immobilized complementary DNA probes: comparison of hairpin versus linear capture probes

A microtiter-based assay system is described in which DNA hairpin probes with dangling ends and single-stranded, linear DNA probes were immobilized and compared based on their ability to capture single-strand target DNA. Hairpin probes consisted of a 16 bp duplex stem, linked by a T2-biotin·dT-T2 loop. The third base was a biotinylated uracil (UB) necessary for coupling to avidin coated microtiter wells. The capture region of the hairpin was a 3′ dangling end composed of either 16 or 32 bases. Fundamental parameters of the system, such as probe density and avidin adsorption capacity of the plates were characterized. The target DNA consisted of 65 bases whose 3′ end was complementary to the dangling end of the hairpin or to the linear probe sequence. The assay system was employed to measure the time dependence and thermodynamic stability of target hybridization with hairpin and linear probes. Target molecules were labeled with either a 5′-FITC, or radiolabeled with [γ-33P]ATP and captured by either linear or hairpin probes affixed to the solid support. Over the range of target concentrations from 10 to 640 pmol hybridization rates increased with increasing target concentration, but varied for the different probes examined. Hairpin probes displayed higher rates of hybridization and larger equilibrium amounts of captured targets than linear probes. At 25 and 45°C, rates of hybridization were better than twice as great for the hairpin compared with the linear capture probes. Hairpin–target complexes were also more thermodynamically stable. Binding free energies were evaluated from the observed equilibrium constants for complex formation. Results showed the order of stability of the probes to be: hairpins with 32 base dangling ends > hairpin probes with l6 base dangling ends > 16 base linear probes > 32 base linear probes. The physical characteristics of hairpins could offer substantial advantages as nucleic acid capture moieties in solid support based hybridization systems.

Identification of sample-specific sequences in mammalian cDNA and genomic DNA by the novel ligation-mediated subtraction (Limes)

The representational difference analysis (RDA) and other subtraction techniques are used to enrich sample-specific sequences by elimination of ubiquitous sequences existing in both the sample of interest (tester) and the subtraction partner (driver). While applying the RDA to genomic DNA of cutaneous lymphoma cells in order to identify tumor relevant alterations, we predominantly isolated repetitive sequences and artificial repeat-mediated fusion products of otherwise independent PCR fragments (PCR hybrids). Since these products severely interfered with the isolation of tester-specific fragments, we developed a considerably more robust and efficient approach, termed ligation-mediated subtraction (Limes). In first applications of Limes, genomic sequences and/or transcripts of genes involved in the regulation of transcription, such as transforming growth factor β stimulated clone 22 related gene (TSC-22R), cell death and cytokine production (caspase-1) or antigen presentation (HLA class II sequences), were found to be completely absent in a cutaneous lymphoma line. On the assumption that mutations in tumor-relevant genes can affect their transcription pattern, a protocol was developed and successfully applied that allows the identification of such sequences. Due to these results, Limes may substitute/supplement other subtraction/comparison techniques such as RDA or DNA microarray techniques in a variety of different research fields.

Purification and Molecular Genetic Characterization of ZPU1, a Pullulanase-Type Starch-Debranching Enzyme from Maize1

This study identified and purified specific isoamylase- and pullulanase-type starch-debranching enzymes (DBEs) present in developing maize (Zea mays L.) endosperm. The cDNA clone Zpu1 was isolated based on its homology with a rice (Oryza sativa L.) cDNA coding for a pullulanase-type DBE. Comparison of the protein product, ZPU1, with 18 other DBEs identified motifs common to both isoamylase- and pullulanase-type enzymes, as well as class-specific sequence blocks. Hybridization of Zpu1 to genomic DNA defined a single-copy gene, zpu1, located on chromosome 2. Zpu1 mRNA was abundant in endosperm throughout starch biosynthesis, but was not detected in the leaf or the root. Anti-ZPU1 antiserum specifically recognized the approximately 100-kD ZPU1 protein in developing endosperm, but not in leaves. Pullulanase- and isoamylase-type DBEs were purified from extracts of developing maize kernels. The pullulanase-type activity was identified as ZPU1 and the isoamylase-type activity as SU1. Mutations of the sugary1 (su1) gene are known to cause deficiencies of SU1 isoamylase and a pullulanase-type DBE. ZPU1 activity, protein level, and electrophoretic mobility were altered in su1-mutant kernels, indicating that it is the affected pullulanase-type DBE. The Zpu1 transcript levels were equivalent in nonmutant and su1-mutant kernels, suggesting that coordinated regulation of ZPU1 and SU1 occurs posttranscriptionally.

Amygdala-enriched genes identified by microarray technology are restricted to specific amygdaloid subnuclei

Microarray technology represents a potentially powerful method for identifying cell type- and regionally restricted genes expressed in the brain. Here we have combined a microarray analysis of differential gene expression among five selected brain regions, including the amygdala, cerebellum, hippocampus, olfactory bulb, and periaqueductal gray, with in situ hybridization. On average, 0.3% of the 34,000 genes interrogated were highly enriched in each of the five regions, relative to the others. In situ hybridization performed on a subset of amygdala-enriched genes confirmed in most cases the overall region-specificity predicted by the microarray data and identified additional sites of brain expression not examined on the microarrays. Strikingly, the majority of these genes exhibited boundaries of expression within the amygdala corresponding to cytoarchitectonically defined subnuclei. These results define a unique set of molecular markers for amygdaloid subnuclei and provide tools to genetically dissect their functional roles in different emotional behaviors.

Construction of neocentromere-based human minichromosomes by telomere-associated chromosomal truncation

Neocentromeres (NCs) are fully functional centromeres that arise ectopically in noncentromeric regions lacking α-satellite DNA. Using telomere-associated chromosome truncation, we have produced a series of minichromosomes (MiCs) from a mardel(10) marker chromosome containing a previously characterized human NC. These MiCs range in size from ≈0.7 to 1.8 Mb and contain single-copy intact genomic DNA from the 10q25 region. Two of these NC-based Mi-Cs (NC-MiCs) appear circular whereas one is linear. All demonstrate stability in both structure and mitotic transmission in the absence of drug selection. Presence of a functional NC is shown by binding a host of key centromere-associated proteins. These NC-MiCs provide direct evidence for mitotic segregation function of the NC DNA and represent examples of stable mammalian MiCs lacking centromeric repeats.

Recursive partitioning for tumor classification with gene expression microarray data

Precise classification of tumors is critically important for cancer diagnosis and treatment. It is also a scientifically challenging task. Recently, efforts have been made to use gene expression profiles to improve the precision of classification, with limited success. Using a published data set for purposes of comparison, we introduce a methodology based on classification trees and demonstrate that it is significantly more accurate for discriminating among distinct colon cancer tissues than other statistical approaches used heretofore. In addition, competing classification trees are displayed, which suggest that different genes may coregulate colon cancers.

An antisense approach to phenotype-based gene cloning in Tetrahymena

We report a pioneering approach using Tetrahymena thermophila that permits rapid identification of genes based on their null or hypomorphic phenotypes. This technique involves cell transformation with a library of plasmids that encode 26S ribosomal subunits containing short insertions. The insertions correspond to antisense sequences for a large number of genes. The majority of cells each acquires a single antisense sequence, which silences a single genomic locus. Because the insertion site within the ribosomal sequence is known, the silenced gene is easily amplified. We demonstrate that this approach can be used to identify genes required for dense core granule exocytosis.

Molecular cytogenetic delineation of a novel critical genomic region in chromosome bands 11q22.3-923.1 in lymphoproliferative disorders.

Aberrations of the long arm of chromosome 11 are among the most common chromosome abnormalities in lymphoproliferative disorders (LPD). Translocations involving BCL1 at 11q13 are strongly associated with mantle cell lymphoma. other nonrandom aberrations, especially deletions and, less frequently, translocations, involving bands 11q21-923 have been identified by chromosome banding analysis. To date, the critical genomic segment and candidate genes involved in these deletions have not been identified. In the present study, we have analyzed tumors from 43 patients with LPD (B-cell chronic lymphocytic leukemia, n = 40; mantle cell lymphoma, n = 3) showing aberrations of bands 11q21-923 by fluorescence in situ hybridization. As probes we used Alu-PCR products from 17 yeast artificial chromosome clones spanning chromosome bands 11q14.3-923.3, including a panel of yeast artificial chromosome clones recognizing a contiguous genomic DNA fragment of approximately 9-10 Mb in bands 11q22.3-923.3. In the 41 tumors exhibiting deletions, we identified a commonly deleted segment in band 11q22.3-923.1; this region is approximately 2-3 Mb in size and contains the genes coding for ATM (ataxia telangiectasia mutated), RDX (radixin), and FDX1 (ferredoxin 1). Furthermore, two translocation break-points were localized to a 1.8-Mb genomic fragment contained within the commonly deleted segment. Thus, we have identified a single critical region of 2-3 Mb in size in which 11q14-923 aberrations in LPD cluster. This provides the basis for the identification of the gene(s) at 11q22.3-923.1 that are involved in the pathogenesis of LPD.