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PubMed2001

Pre-clinical validation of a novel, highly sensitive assay to detect PML-RARalpha mRNA using real-time reverse-transcription polymerase chain reaction.

We have developed a sensitive and quantitative reverse-transcription polymerase chain reaction (RT-PCR) assay for detection of PML-RARalpha, the fusion oncogene present as a specific marker in >99% of cases of acute promyelocytic leukemia (APL). The assay is linear over at least 5 orders of magnitude of input DNA or RNA, and detects as few as 4 copies of PML-RARalpha plasmid DNA. PML-RARalpha transcripts could be detected in mixtures containing 2 to 5 pg of RNA from fusion-containing cells in a background of 1 microg of RNA from PML-RARalpha-negative cells. Using 1.0 to 2.5 microg of input RNA, the sensitivity of the assay was between 10(-5) and 10(-6). Furthermore, determination of GAPDH copy number in each reaction allowed an accurate assessment of sample-to-sample variation in RNA quality and reaction efficiency, with consequent definition of a detection limit for each sample assayed. Using an internal calibrator, assay precision was high, with coefficients of variation between 10 and 20%. An interlaboratory study using coded samples demonstrated excellent reproducibility and high concordance between laboratories. This assay will be used to test the hypothesis that sensitive and quantitative measurement of leukemic burden, during or after therapy of APL, can stratify patients into discrete risk groups, and thereby serve as a basis for risk-adapted therapy in APL.

Biomarkers, Tumor↗

PubMed2000

Transgenic zebrafish as sentinels for aquatic pollution.

Using the golden mutant zebrafish having a decrease in interfering pigmentation, we are developing transgenic lines in which DNA motifs that respond to selected environmental pollutants are capable of activating a reporter gene that can be easily assayed. We have begun with three response elements that recognize three important classes of foreign chemicals. Aromatic hydrocarbon response elements (AHREs) respond to numerous polycyclic hydrocarbons and halogenated coplanar molecules such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; dioxin) and polychlorinated biphenyls. Electrophile response elements (EPREs) respond to quinones and numerous other potent electrophilic oxidants. Metal response elements (MREs) respond to heavy metal cations such as mercury, copper, nickel, cadmium, and zinc. Soon, we will include estrogen response elements (EREs) to detect the effects of environmental endocrine disruptors, and retinoic acid response elements (RARE, RXRE) to detect the effects of retinoids in the environment. Each of these substances is known to be bioconcentrated in fish to varying degrees; for example, 10(-17) M TCDD in a body of water becomes concentrated to approximately 10(-12) M TCDD in a fish, where it would act upon the AHRE motif and turn on the luciferase (LUC) reporter gene. The living fish as a sentinel will not only be assayed intact in the luminometer, but--upon several days or weeks of depuration--would be usable again. To date, we have established that zebrafish transcription factors are able to recognize both mammalian and trout AHRE, EPRE, and MRE sequences in a dose-dependent and chemical-class-specific manner, and that expression of both the LUC and jellyfish green fluorescent protein (GFP) reporter genes is easily detected in zebrafish cell cultures and in the intact live zebrafish. Variations in sensitivity of this model system can be achieved by increasing the copy number of response elements and perhaps by altering the sequence of each core consensus response element and flanking regions. This transgenic technology should allow for a simple, exquisitely sensitive, and inexpensive assay for monitoring aquatic pollution. We have already initiated studies using sentinel zebrafish to monitor a public drinking water source.

Animals↗

PubMed1999

Abr1, a transposon-like element in the genome of the cultivated mushroom Agaricus bisporus (Lange) Imbach.

A 300-bp repetitive element was found in the genome of the white button mushroom, Agaricus bisporus, and designated Abr1. It is present in approximately 15 copies per haploid genome in the commercial strain Horst U1. Analysis of seven copies showed 89 to 97% sequence identity. The repeat has features typical of class II transposons (i.e., terminal inverted repeats, subterminal repeats, and a target site duplication of 7 bp). The latter shows a consensus sequence. When used as probe on Southern blots, Abr1 identifies relatively little variation within traditional and present-day commercial strains, indicating that most strains are identical or have a common origin. In contrast to these cultivars, high variation is found among field-collected strains. Furthermore, a remarkable difference in copy numbers of Abr1 was found between A. bisporus isolates with a secondarily homothallic life cycle and those with a heterothallic life cycle. Abr1 is a type II transposon not previously reported in basidiomycetes and appears to be useful for the identification of strains within the species A. bisporus.

Agaricus↗

PubMed2007

Gene copy number detection in animal studies.

Sensitive methods for the quantification of DNA fragments can be used to study an individual's genetic constitution for duplicated regions of the genome or to determine the relative proportion of different DNA fragments in heterogeneous samples such as pooled DNA from different individuals or in samples in which a fraction of the chromosomes carry a mutation. Here, we describe how we are using Pyrosequencing for this purpose. In Subheading 3., we describe a sensitive method that can be used to quantify the relative proportion of X- and Y-carrying sperm after sperm sorting in cattle. We also discuss our method for determining the copy numbers at a duplicated locus. This method has been applied to study genetic variation at the KIT locus in pigs, which have a major effect on coat color variation in this species.

Animals↗

PubMed2005

[Chromosomal aberration in human keloid analyzed by comparative genomic hybridization].

OBJECTIVE: To identify the genetic alteration in human keloid. METHODS: Comparative genomic hybridization was applied in 6 cases of keloid to investigate the genomic imbalance (the gain or loss of genetic material). RESULTS: The study showed that the loss of chromosome DNA copies included chromosome, 1,7,9,13,16,17,18,19,20,22. Among them, the frequently detected chromosome loss was chromosome 1 p(66.7%), 16 (83.3%), 20 (83.3%) and 22 (83.3%). The minimum overlapping regions were 1 pter-32.2,16p13.2p11.l,20q11.1-q13.2 and 16p13.2-p11.1. Frequent gain of DNA copy numbers was not found in the special regions. CONCLUSIONS: The mapping of DNA copy variation frequency in keloid showed that there may be inhibitory genes in chromosomes 1p,16,20,22. The loss of these genes may be involved in the development and progress of keloid.

Adult↗

PubMed1995

PCR amplification of intergenic spacers in the ribosomal DNA of Drosophila melanogaster reveals high levels of turnover in length and copy-number of spacers in geographically separated populations.

A recently described PCR-based method for the analysis of intergenic spacer (IGS) length variation in the ribosomal (r) DNA of Drosophila melanogaster was used to analyse the distribution of IGS length variants in the rDNA of a number of recently collected D. melanogaster populations. One group of populations, from Europe and North Africa, was shown to have low intrapopulation IGS length variation following maintenance of massed populations in the laboratory for an extended period. However, a greater degree of IGS profile variability was detected at a number of levels in the majority of laboratory-maintained isofemale lines from two of these populations plus a second group of populations which were collected more recently from the eastern coast of Australia; all of which were immediately divided into isofemale lines following collection. Interestingly, PCR analysis of pooled DNA extracts from 30 individuals of either sex showed almost identical PCR profiles from each of the Australian populations. These preliminary results are discussed with regard to the possible combinations of forces (natural selection, neutral drift and genomic molecular drive) on the patterns of IGS length variation.

Animals↗

PubMed1984

A mathematical model for lambda dv plasmid replication: analysis of wild-type plasmid.

A mathematical model for lambda dv plasmid replication in a growing single cell of Escherichia coli has been formulated and solved numerically. Quantitative description of the molecular control mechanism for initiation of lambda dv replication presumes regulatory functions of repressor and initiator proteins and transcriptional activation of the origin region. Random selection of a single plasmid for activation and replication is assumed, as is regular plasmid segregation to daughter cells. The model is capable of simulating the periodic changes in each regulatory element and the plasmid copy number during the cell cycle. The calculated average copy number, repressor concentration, and timing of plasmid replication agree well with experimental data. The simulated lambda dv plasmid replication rate is controlled primarily by transcription frequency. Initiation of plasmid replication is not related to variations in the levels of repressor or initiator proteins during the cell cycle. Simulation studies of perturbations in plasmid and repressor segregation indicate that replication regulation of the lambda dv plasmid compensates to readjust copy number to normal values in a few generations. Implications of these studies relative to the molecular mechanisms of replication control are discussed.

DNA Replication↗

PubMed1995

Segment selection during 'phase variation'-type regulation of gene replacement mediated by FLP recombinase in the yeast Saccharomyces cerevisiae.

FLP recombinase has recently been used as a tool to direct the exchange between invertible DNA segments, called 'Phase variation'-type regulation of gene replacement in eukaryotic cells. Using an appropriate selective medium, positive segment selection was shown to be efficient during the regulation of gene replacement. The efficiency was determined from the copy number ratio of invertible segments with the use of the neomycinphosphotransferase II (NPTII) gene bearing invertible segments located on the episomal yeast plasmid, and the resident 2-microns circle. Without the selection the segments copy number ratio was retained in growing cells. The results obtained are an evidence for the efficiency of positive segment selection during the 'Phase variation'-type regulation of gene replacement in eukaryotic cells.

DNA Nucleotidyltransferases↗

PubMed1990

Generation of chromosome size polymorphism during in vivo mitotic multiplication of Plasmodium berghei involves both loss and addition of subtelomeric repeat sequences.

Extensive chromosome size polymorphism arises in Plasmodium berghei during in vivo mitotic multiplication. Size differences between homologous chromosomes involve rearrangements occurring in the subtelomeric portions while internal chromosomal regions do not contribute significantly to chromosome size polymorphism. Differences in the copy number of a 2.3-kb subtelomeric repeated unit are shown to correlate with size variations, and in at least one case to account completely for the size difference between two variants of the same chromosome.

Animals↗

PubMed2006

Comparison of real-time PCR and hemagglutination assay for quantitation of human polyomavirus JC.

Human polyomavirus JC (JCV), the etiological agent of the disease progressive multifocal leukoencephalopathy (PML) affects immunocompromised patients particularly patients with AIDS. In vitro studies of JCV infection are hampered by the lack of sensitive JCV quantitation tests. Although the hemagglutination (HA) assay has been routinely employed for in vitro quantitation of JCV, its sensitivity is severely limited. We have employed a real-time PCR assay which compares favorably with the HA assay for the in vitro quantitation of JCV. JCV(Mad1), propagated in primary human fetal glial (PHFG) cells in two independent laboratories, was purified and quantitated by the HA assay. Both batches of purified JCV(Mad1) were then serially diluted in Dulbecco's Modified Eagle's Medium to obtain HA titers ranging from 64 to 0.001 HA units (HAU) per 100 microL of virus suspension. DNA was extracted from 100 microL of virus suspension and eluted in 50 microL of buffer, and DNA amplification and quantitation were performed in the Bio-Rad iCycler iQ Multicolor Real-Time PCR Detection System using T-antigen as the target gene. Real-time PCR for quantitation of JCV was sensitive and consistently detected 1.8 x 10(1) copies of JCV DNA, and as low as 0.001 HAU equivalent of JCV. Moreover, there was a strong linear correlation between the HA assay and the DNA copy number of JCV(Mad1). The intra-run and inter-run coefficients of variation for the JCV standard curve were 0.06% to 4.8% and 2.6% to 5.2%, respectively. Based on these data, real-time PCR can replace the less-sensitive HA assay for the reliable detection, quantitation and monitoring of in vitro JCV replication.

DNA, Viral↗

PubMed2004

Quantification of multiple gene expression in individual cells.

Quantitative gene expression analysis aims to define the gene expression patterns determining cell behavior. So far, these assessments can only be performed at the population level. Therefore, they determine the average gene expression within a population, overlooking possible cell-to-cell heterogeneity that could lead to different cell behaviors/cell fates. Understanding individual cell behavior requires multiple gene expression analyses of single cells, and may be fundamental for the understanding of all types of biological events and/or differentiation processes. We here describe a new reverse transcription-polymerase chain reaction (RT-PCR) approach allowing the simultaneous quantification of the expression of 20 genes in the same single cell. This method has broad application, in different species and any type of gene combination. RT efficiency is evaluated. Uniform and maximized amplification conditions for all genes are provided. Abundance relationships are maintained, allowing the precise quantification of the absolute number of mRNA molecules per cell, ranging from 2 to 1.28 x 10(9) for each individual gene. We evaluated the impact of this approach on functional genetic read-outs by studying an apparently homogeneous population (monoclonal T cells recovered 4 d after antigen stimulation), using either this method or conventional real-time RT-PCR. Single-cell studies revealed considerable cell-to-cell variation: All T cells did not express all individual genes. Gene coexpression patterns were very heterogeneous. mRNA copy numbers varied between different transcripts and in different cells. As a consequence, this single-cell assay introduces new and fundamental information regarding functional genomic read-outs. By comparison, we also show that conventional quantitative assays determining population averages supply insufficient information, and may even be highly misleading.

Base Sequence↗

PubMed1985

Multiple duplications of complement C4 gene correlate with H-2-controlled testosterone-independent expression of its sex-limited isoform, C4-Slp.

Mouse liver cDNA clones related to the C4 and C4-Slp isoforms of the fourth component of complement differ by few nucleotide changes within a region of substantial divergence from human C4. It is suggested that the mouse C4 gene duplication is an evolutionarily recent event with respect to the time of mammalian radiation. This conclusion is reinforced by the presence of a single C4 gene in the Syrian hamster. Most H-2 haplotypes, including those characterized by an undetectable C4-Slp protein, possess two C4 gene copies which, in contrast to the neighboring factor B, show a marked restriction site polymorphism. The genetic variation of this region is emphasized by the presence in the mouse of a rare "polymorphism" for C4 gene number. Multiple C4-related gene copies characterize those exceptional wild-derived H-2 haplotypes, H-2w7, H-2w16, and H-2w19, that determine the expression of the C4-Slp protein in female animals.

Animals↗

PubMed1992

16S rrn gene copy number in Helicobacter pylori and its application to molecular typing.

The copy number of the genes encoding 16S ribosomal RNA was analysed for the genomes of geographically diverse strains of Helicobacter pylori, and restriction site variation within and around the genes was characterized. A DNA probe of 550 bp was amplified by the polymerase chain reaction from genomic DNA of the type strain NCTC 11637. This probe constituted a sequence internal to the 3' end of the 16S rrn gene. Homology profiles were compared for genomic Southern blots made with four restriction enzymes cutting within and outside the probe sequence. A copy number of two was established for all 12 strains analysed. This approach yielded significantly simpler data than does conventional 'ribotyping ' of H. pylori. It was equally discriminatory, however, and provided strain-specific 16S rrn gene 'signatures'. These represent both fundamental physical-genetic information and a novel approach to typing this gastric pathogen.

Bacterial Typing Techniques↗

PubMed1995

Analysis of pfmdr1 and drug susceptibility in fresh isolates of Plasmodium falciparum from subsaharan Africa.

Resistance of Plasmodium falciparum to many therapeutic agents is an increasing problem in most endemic areas. The role of the mdr-like gene products of P. falciparum in resistance to quinoline-containing compounds is not clear. The purpose of this study was to further examine the role of pfmdr1 in drug resistance in fresh clinical isolates originating from Africa. Drug susceptibility testing (chloroquine, mefloquine, halofantrine and quinine) and a molecular analysis of pfmdr1 was completed for 51 fresh clinical isolates. A statistical association between the chloroquine sensitivity phenotype and an intragenic allele of pfmdr1 was noted at a position, amino acid 86, which was previously associated with chloroquine resistance. There was little variation in the other intragenic alleles previously associated with chloroquine resistance. No correlation between pfmdr1 intragenic allelic variation and susceptibility to mefloquine, halofantrine or quinine was found. There was no association between gene copy number of pfmdr1 and any drug resistant phenotype in an analysis of selected isolates. This, along with other data, suggests that mefloquine resistance may have arisen by two different mechanisms in African and Southeast Asian isolates. Much more variability in the polyasparaginated region of the pfmdr1 gene was noted in this study than previously reported. In addition, fingerprint analysis using multiplex PCR revealed considerable genetic variability among these isolates.

Africa South of the Sahara↗

PubMed1988

The rabbit alpha-like globin gene cluster is polymorphic both in the sizes of BamHI fragments and in the numbers of duplicated sets of genes.

The alpha-like globin gene cluster in rabbits contains embryonic zeta-globin genes, an adult alpha-globin gene, and theta-globin genes of undetermined function. The basic arrangement of genes, deduced from analysis of cloned DNA fragments, is 5'-zeta 0-zeta 1-alpha 1-theta 1-zeta 2-zeta 3-theta 2-3'. However, the pattern of restriction fragments containing zeta- and theta-globin genes varies among individual rabbits. Analysis of BamHI fragments of genomic DNA from 24 New Zealand white rabbits revealed eight different patterns of fragments containing zeta-globin genes. The large BamHI fragments containing genes zeta 0 and zeta 1 are polymorphic in length, whereas a 1.9-kb fragment containing the zeta 2 gene and the 3.5-kb fragment containing the zeta 3 gene do not vary in size. In contrast to this constancy in the size of the restriction fragments, the copy number of the zeta 2 and zeta 3 genes does vary among different rabbits. No length polymorphism was detected in the BamHI fragments containing the theta-globin genes, but again the copy number varies for restriction fragments containing the theta 2 gene. The alpha 1- and theta 1-globin genes are located in a nonpolymorphic 7.2-kb BamHI fragment. The combined data from hybridization with both zeta and theta probes shows that the BamHI cleavage pattern does not vary within the region 5'-alpha 1-theta 1-zeta 2-zeta 3-theta 2-3', but the pattern genomic blot-hybridization patterns for the progeny of parental rabbits with different zeta-globin gene patterns shows that the polymorphic patterns are inherited in a Mendelian fashion. Two different haplotypes have been mapped based on the genomic blot-hybridization data. The variation in the alpha-like globin gene cluster in the rabbit population results both from differences in the copy number of the duplication block containing the zeta-zeta-theta gene set and from the presence or absence of polymorphic BamHI sites.

Animals↗

PubMed1992

Mutations in bglY, the structural gene for the DNA-binding protein H1 of Escherichia coli, increase the expression of the kanamycin resistance gene carried by plasmid pGR71.

bglY mutants of Escherichia coli K12 which show higher levels of kanamycin resistance (Kmr) in the presence of plasmid pGR71 have been previously described. In this work, we show that this increased resistance to an aminoglycoside antibiotic is not due either to low drug uptake or to alteration of its target, the ribosome. The copy number of plasmid pGR71 is not modified. The fact that increased antibiotic resistance is observed with only some of the Kmr determinants used in this study suggests a specific role for the bglY gene product. Moreover, for one such determinant, a higher level of resistance was observed when it was inserted in the chromosome but not when harbored by a plasmid. This discrepancy can be explained by the twin transcriptional-loop model, which proposes that transcription can lead to local variation in topology. A kan-lacZ fusion was constructed from the Kmr gene of plasmid pGR71 and inserted into a low copy number vector. Assay of beta-galactosidase in wild-type and mutant strains showed that expression of the antibiotic resistance gene was directly affected by H1 protein, the bglY gene product.

Aminoglycosides↗

PubMed2003

Detection of beta-lactamase reporter gene expression by flow cytometry.

BACKGROUND: Flow cytometry of gene expression in living cells requires accurate, sensitive, nontoxic fluorescent indicators capable of detecting transcription of specific genes. This is typically achieved by using genes that encode fluorescent proteins or enzymes coupled to promoters of interest. The most commonly used reporters are green fluorescent protein and beta-galactosidase (lacZ). In this study, we characterized the performance of a cell-permeant, ratiometric, beta-lactamase substrate, coumarin cephalosporin fluorescein (CCF2/AM). We compared its characteristics with that of the beta-galactosidase/fluorescein di-beta-D-galactopyranoside reporter system. METHODS: Jurkat cell lines were generated for beta-lactamase and beta-galactosidase reporters with the use of similar plasmid constructs. Rare event flow cytometric detection for the beta-galactosidase and beta-lactamase reporters were assayed by using mixed populations of negative (WT) and positive (constitutively expressing) cells for each reporter. To determine sensitivity at low reporter copy number, we measured the activity of an unstimulated inducible promoter and detected positive events as a function of substrate incubation time. Technical issues related to data processing and optical configuration are also presented. RESULTS: The low population coefficients of variation afforded by ratiometric detection of the beta-lactamase system improved the statistical performance of the assay in comparison with a single-dye, intensity-based assay, leading to markedly improved detection for low copy number and rare events. At low levels of gene expression, beta-lactamase was detected with approximately 10-fold higher confidence than was beta-galactosidase. In rare event detection experiments, cells expressing high levels of beta-lactamase proteins were reliably detected at frequencies of 1:10(6) compared with about 1:10(4) for beta-galactosidase. CONCLUSION: The ratiometric fluorescence readout of the beta-lactamase system based on fluorescence resonance energy transfer allowed more sensitive and accurate detection of gene expression than the currently available beta-galactosidase substrates. Further, the cell-permeant nature of the substrate improved experimental convenience. These properties facilitated cell engineering and enabled a variety of applications including selection of rare cells from large populations and measurement of low-expressing or downregulated genes.

Artifacts↗

PubMed1992

Heterogeneity in state and expression of viral DNA in polyoma virus-induced tumors of the mouse.

We have examined the state and expression of polyoma viral DNA in representative epithelial and mesenchymal tumors, using a combination of biochemical and in situ methods. Results showed wide variations among tumor types and also in different regions within individual tumors, with respect to copy number of viral DNA, presence or absence of deletions, and expression of early and late viral proteins. Epithelial tumors showed the greatest heterogeneity. High copy free viral DNA, frequently with deletions, was found in all such tumors. A portion of free viral DNA was recoverable as transcriptionally active minichromosomes. Three distinct subpopulations of cells were distinguished by in situ analyses. Type 1 cells showed high copy free viral DNA and expressed the major viral capsid protein VP1; these cells appeared to be at various stages of productive (lytic) viral infection. Some productively infected cells were able to undergo mitosis; in a portion of these cells, VP1 was found in close association with the mitotic spindle. Type 2 cells contained high copy free DNA but did not express VP1; by some unknown mechanism, these cells manifest a post-replication block to late gene expression and lytic infection. Type 3 cells contained only low copy, presumably integrated, viral DNA and expressed no VP1; they thus resemble cells transformed in vitro by the virus. Epithelial tumors contained variable mixtures of these subpopulations, while mesenchymal tumors were composed of Type 3 cells only. Differences in virus-cell interactions are discussed in terms of their possible implications in tumor development.

Animals↗