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PubMed2002

Microarrays in ecology and evolution: a preview.

Microarray technology provides a new tool with which molecular ecologists and evolutionary biologists can survey genome-wide patterns of gene expression within and among species. New analytical approaches based on analysis of variance will allow quantification of the contributions of among individual variation, genotype, sex, microenvironment, population structure, and geography to variation in gene expression. Applications of this methodology are reviewed in relation to studies of mechanisms of adaptation and divergence; delineation of developmental and physiological pathways and networks; characterization of quantitative genetic parameters at the level of transcription ('quantitative genomics'); molecular dissection of parasitism and symbiosis; and studies of the diversification of gene content. Establishment of microarray resources is neither prohibitively expensive nor technologically demanding, and a commitment to development of gene expression profiling methods for nonmodel organisms could have a tremendous impact on molecular and genetic research at the interface of organismal and population biology.

Animals↗
PubMed2003

A study of susceptibility of primary human Kupffer cells to hepatitis C virus.

BACKGROUND/AIMS: Histological examination of a liver-biopsy from a patient with chronic hepatitis C shows activated Kupffer cells. In vitro infection of human Kupffer cells (KC) was performed to study their interaction with hepatitis C virus (HCV). METHODS: KC, isolated by collagenase perfusion and centrifugal elutriation, were infected with various HCV positive sera. The presence of the viral genome was followed, at different times, quantitatively by a branched-DNA assay and qualitatively by reverse transcriptase-nested polymerase chain reaction. A strand-specific assay performed with the thermostable enzyme rTth was used to detect the synthesis of a negative replicative intermediate. Cytopathic effect was examined by electron microscopy. Production of cytokines and inducible nitric oxide synthase was evaluated in the supernatants. RESULTS: Quantification of HCV-RNA showed that the level of viral RNA associated with KC after adsorption decreased rapidly. Genomic viral RNA disappeared within 5 days of infection. Negative-strand RNA was never detected in any of these experiments. No cytopathic effects could be detected at any time. KC did not produce inflammatory nor antiviral cytokines. CONCLUSIONS: Our results strongly suggest that primary cultures of KC are not permissive for HCV in vitro.

Disease Susceptibility↗
PubMed1989

JC virus genomes in progressive multifocal leukoencephalopathy: detection using a sensitive non-radioisotopic in situ hybridization method.

JC virus genomes have been localized in formalin-fixed, paraffin-embedded brain tissues of two cases of known progressive multifocal leukoencephalopathy by in situ hybridization utilizing a biotinylated JC virus DNA probe. A three-stage immunoperoxidase system with gold-silver amplification of the diaminobenzidine substrate was used to visualize biotinylated nucleic acid hybrids. Dot-blot quantification of this visualization system indicates that subpicogramme amounts of biotinylated DNA can be detected. Optimal detection of the virus genomes in the brain tissues required a microwave irradiation step prior to hybridization. JC virus genomes were observed in the nuclei of enlarged oligodendrocytes and of some bizarre astrocytes. No other cell types were found to harbour the genomes.

Brain↗
PubMed2006

No evidence for parental imprinting of mouse 22q11 gene orthologs.

Non-Mendelian factors may influence central nervous system (CNS) phenotypes in patients with 22q11 Deletion Syndrome (22q11DS, also known as DiGeorge or Velocardiofacial Syndrome), and similar mechanisms may operate in mice carrying a deletion of one or more 22q11 gene orthologs. Accordingly, we examined the influence of parent of origin on expression of 25 murine 22q11 orthologs in the developing and mature CNS using single nucleotide polymorphism (SNP)-based analysis in interspecific crosses and quantification of mRNA in a murine model of 22q11DS. We found no evidence for absolute genomic imprinting or silencing. All 25 genes are biallelically expressed in the developing and adult brains. Furthermore, if more subtle forms of allelic biasing are present, they are very small in magnitude and most likely beyond the resolution of currently available quantitative approaches. Given the high degree of similarity of human 22q11 and the orthologous region of mmChr16, genomic imprinting most likely cannot explain apparent parent-of-origin effects in 22q11DS.

Alleles↗
PubMed2006

Real-time PCR protocol for the detection of porcine parvovirus in field samples.

This report describes a real-time polymerase chain reaction assay with SYBR Green for detection of a broad range of porcine parvoviruses (PPV) and accurate virus quantification in porcine tissues. The assay targets the VP2 gene of PPV and the porcine genomic c-myc gene for normalization. The detection limit of the SYBR Green reaction was shown to be equivalent to 6 x 10(0) to 6 x 10(1) PPV copies/reaction and the overall detection limit equivalent to 0.1 TCID(50)/100 microl. The assay was linear over a 10(7) dilution range of template concentrations. Other porcine pathogens involved in reproductive disorders such as porcine circovirus 2 (PCV-2), porcine reproductive and respiratory virus (PRRSV), Aujeszky's disease virus (PRV) and other parvoviruses such as feline parvovirus (FPV), canine parvovirus (CPV), minute virus of canines (MVC) and a human parvovirus (B19) were not detected by this assay.

Animals↗
PubMed2003

Comparative analyses of Saccharomyces cerevisiae RNAs using Agilent RNA 6000 Nano Assay and agarose gel electrophoresis.

Precise quantification and quality characterisation of isolated RNAs are prerequisites for their further exploitation in genome-wide microarrays, Northern blots, cDNA library preparation and others. Our data indicate that RNA analyses using Agilent RNA Nano Assay exhibit several advantages when compared with those performed on ethidium bromide-stained agarose gel electrophoresis or on a spectrophotometer. The RNA Nano Assay makes it possible to estimate RNA concentrations in the range from 1000 ng microl(-1) to 17 ng microl(-1). The presence of impurities including traces of DNA within RNA samples does not influence the concentration measurements. Like agarose gel electrophoresis, RNA Nano Assay allows to analyse RNAs dissolved in formamide and therefore protected against RNase action. Moreover, it allows a clearer distinction of partially degraded samples. The limitation of RNA Nano Assay is the impossibility to detect and to analyse double-stranded RNAs.

Electrophoresis, Agar Gel↗
PubMed2001

Expression of the splice variants of the p85alpha regulatory subunit of phosphoinositide 3-kinase in muscle and adipose tissue of healthy subjects and type 2 diabetic patients.

The regulation by insulin of the expression of the p85alpha regulatory subunit of phosphoinositide 3-kinase (PI 3-kinase) is impaired in skeletal muscle and adipose tissue of type 2 diabetic patients. The gene encoding p85alpha (named grb-1) can generate several variants by alternative splicing, all being able to activate the p110 catalytic subunits of PI 3-kinase. Our aims were (i) to determine the mRNA expression profiles of these variants in human skeletal muscle and adipose tissue; (ii) to investigate the effect of insulin on their expression in vivo and in vitro in muscle and (iii) to verify whether this regulation is defective in type 2 diabetes. We determined the human genomic organization of grb-1 and set up reverse transcriptase competitive PCR assays for the quantification of each mRNA variant. In muscle, p85alpha and p50alpha mRNAs were the most abundant, and p55alpha represented less than 20% of all grb-1-derived mRNAs. In adipose tissue, p85alpha was expressed predominantly and p55alpha mRNA was not detectable. These expression profiles were not different in type 2 diabetics. During a 3 h hyperinsulinaemic clamp, insulin increased the mRNA expression of the three variants in muscle of control subjects. In diabetic patients, the effect of insulin on p85alpha and p50alpha mRNAs was blunted, and largely reduced on p55alpha transcripts. In cultured human myotubes, up-regulation of p85alpha, p55alpha and p50alpha mRNAs by insulin was abolished by LY294002 (10 microM) and by rapamycin (50 nM), suggesting that the PI 3-kinase/protein kinase B/p70 S6 kinase pathway could be involved in the stimulation of grb-1 gene expression by insulin in human muscle cells.

Adipose Tissue↗
PubMedDate not supplied

[Quantitative assay of 5-HT(1A) serotonin receptor gene expression in the brain].

Serotonin 5-HT(1A) receptors participate in the regulation of many kinds of behavior and are implicated in the mechanism of action of anxiolitics and antidepressants. The investigation of 5-HT(1A) receptor gene expression is complicated by low concentration of the receptor mRNA. Our method of quantification of the receptor gene expression in brain structures includes estimation of the concentration of genomic DNA contamination, the number of cDNA copies of glyceraldehyde-3-phosphate dehydrogenase (GAPDH)--one of the "housekeeping genes", and the number of cDNA copies of 5-HT(1A) receptor in the sample. To evaluate the number of cDNA copies of the receptor and GAPDH, the fluorescence intensity of PCR-product was calibrated using genomic DNA-standard of a known concentration. The intensity of 5-HT(1A) receptor gene expression was corrected by genomic DNA contamination and was evaluated as a number of copies of 5-HT(1A) receptor cDNA per 100 copies of GAPDH cDNA. Using this method an increase of 5-HT(1A) receptor gene expression in the frontal cortex and amygdala in monoamine oxidase A knockout mice was shown.

Amygdala↗
PubMed2002

Compact disc with both numeric and genomic information as DNA microarray platform.

The compact disc (CD) is an ideal toolfor reading, writing, and storing numeric information. It was used in this work as a support for constructing DNA microarrays suited for genomic analysis. The CD was divided into two functional areas: the external ring of the CD was used for multiparametric DNA analysis on arrays, and the inner portion was usedfor storing numeric information. Because polycarbonate and CD resins autofluoresce, a colorimetric method for DNA microarray detection was used that is well adaptedfor the fast detection necessary when using a CD reader. A double-sided CD reader was developed for the simultaneous analysis of both array and numeric data. The numeric data are engraved as pits in the CD tracks and result in the succession of 0/1, which results from the modulation of the laser reflection when one reads the edges of the pits. Another diffraction-based laser was placed above the CD for the detection of the DNA targets on the microarrays. Both readersfit easily in a PC tower. Both numeric and genomic information data were simultaneously acquired, and each array was reconstituted, analyzed, and processed for quantification by the appropriate software.

Compact Disks↗
PubMed2001

[Proteomic analysis: why and how ?].

The proteome, first formalized in 1995, designs all the proteins expressed by the genome of a cell, tissu, or organ at a defined time. Proteomic analysis leads to a description of the regulation of gene expression by the study of proteins and of their post-translational modifications. Proteomic analysis is based on three technologies: 1) Two-dimensional electrophoresis allowing the separation of thousands of proteins from a single mixture; 2) mass spectrometry allowing the characterization of picoquantities of polypeptides and providing data on post-translational modifications; 3) Bioinformatic which is required for the quantification of protein level and for the constitution of databases of protein expression profiles. Complementing the methods of the genomics, the use of proteomic analysis is widely spreading in the fields of fundamental biology, biomedicine and pharmacology for the identification of new biological markers and therapeutic targets.

Animals↗
PubMed1995

Substrate preferences of Vsr DNA mismatch endonuclease and their consequences for the evolution of the Escherichia coli K-12 genome.

The substrate spectrum of Vsr DNA mismatch endonuclease of Escherichia coli K-12 was investigated using fluorescence-labelled oligonucleotide substrates and a DNA sequencer for detection and quantification of substrates and reaction products. Fourteen substrates were found to be processed by the enzyme, which differ in one or two positions from the canonical pentanucleotide sequence CTA/TGG (T mismatched to G). Relative second-order rate constants of these substrates were determined in groups of four by multiple substrate kinetics and compared to the underresentation of the corresponding pentanucleotides in the E. coli K-12 genome. The high quality of correlation further establishes active mutagenesis by VSP repair as a significant driving force of the evolution of the E. coli K-12 genome and provides clues to its possible selective value.

Base Sequence↗
PubMed2003

Quantification of UVR-induced DNA damage: global- versus gene-specific levels of thymine dimers.

The induction and repair of DNA damage has been shown to occur heterogeneously throughout the mammalian genome. As a consequence, analysis of these parameters at a global genome level may not reflect important gene-level events. Few techniques have been established to explore quantitatively gene-specific DNA damage and repair. Most of these are polymerase chain reaction (PCR)-based assays and are relatively insensitive, relying on decreased PCR amplification arising from damage in template DNA. We have developed a quantitative assay that combines specific immunocapture of damaged DNA by an antiserum specific for thymine dimers (IgG479), with PCR amplification of a 149 bp fragment of the human H-ras proto-oncogene. Quantification of DNA damage was based upon proportionality between the amount of the PCR product and the initial amount of damage. Detection of thymine dimers was possible with nanogram amounts of genomic DNA and increased in a linear, dose-responsive manner. Using this assay, gene-level induction of thymine dimers was shown to be directly proportional to levels induced in the global genome of ultraviolet radiation (UVR)-exposed, extracted DNA as measured by gas chromatography-mass spectrometry (GC-MS). This result suggests that global damage assessments do indeed reflect gene-level events although we predict that this relationship may not be maintained when applied to a cellular system. These findings demonstrate the suitability of this approach to the detection of UVR-induced DNA damage at the level of individual genes.

Antibody Specificity↗
PubMed2006

Real-time PCR quantification of human complement C4A and C4B genes.

BACKGROUND: The fourth component of human complement (C4), an essential factor of the innate immunity, is represented as two isoforms (C4A and C4B) in the genome. Although these genes differ only in 5 nucleotides, the encoded C4A and C4B proteins are functionally different. Based on phenotypic determination, unbalanced production of C4A and C4B is associated with several diseases, such as systemic lupus erythematosus, type 1 diabetes, several autoimmune diseases, moreover with higher morbidity and mortality of myocardial infarction and increased susceptibility for bacterial infections. Despite of this major clinical relevance, only low throughput, time and labor intensive methods have been used so far for the quantification of C4A and C4B genes. RESULTS: A novel quantitative real-time PCR (qPCR) technique was developed for rapid and accurate quantification of the C4A and C4B genes applying a duplex, TaqMan based methodology. The reliable, single-step analysis provides the determination of the copy number of the C4A and C4B genes applying a wide range of DNA template concentration (0.3-300 ng genomic DNA). The developed qPCR was applied to determine C4A and C4B gene dosages in a healthy Hungarian population (N = 118). The obtained data were compared to the results of an earlier study of the same population. Moreover a set of 33 samples were analyzed by two independent methods. No significant difference was observed between the gene dosages determined by the employed techniques demonstrating the reliability of the novel qPCR methodology. A Microsoft Excel worksheet and a DOS executable are also provided for simple and automated evaluation of the measured data. CONCLUSION: This report describes a novel real-time PCR method for single-step quantification of C4A and C4B genes. The developed technique could facilitate studies investigating disease association of different C4 isotypes.

Adult↗
PubMed2026

Engineering bubble structures as Cas12a activators for highly sensitive monitoring of WRN helicase function.

The Werner syndrome helicase (WRN) is a critical synthetic lethal target in microsatellite instability cancers, essential for resolving complex genomic structures like replication bubbles and R-loops. However, strategies to simultaneously discriminate WRN activity on DNA versus DNA-RNA substrates in living cells are lacking. Here, we developed a structure-specific CRISPR/Cas12a biosensing strategy to visualize WRN functional activity by engineering bubble-structure probes. These probes were rationally designed to structurally mimic DNA replication bubbles and R-loop associated DNA-RNA hybrids. Upon specific unwinding by WRN, the probes release a sequestered activator strand that triggers Cas12a trans-cleavage, effectively converting the unwinding event into an amplified fluorescent signal. This assay achieves low picomolar sensitivity (LODs: 5.6-6.0 pM) and exceptional selectivity against homologous RecQ helicases. Uniquely, this strategy enables the parallel quantification of WRN activity on both substrate types, providing insights into distinct WRN-mediated pathways for resolving genomic stress. We further demonstrated the strategy's utility by visualizing endogenous WRN dynamics in living cells and profiling the efficacy of small-molecule inhibitors. This work offers a powerful molecular toolkit for dissecting WRN biology and facilitating high-throughput drug screening in targeted cancer therapy.

Werner Syndrome Helicase↗
PubMed2006

[EBV quantification in children after allogeneic hematopoietic stem cell transplantation].

BACKGROUND: Patients undergoing allogeneic hematopoietic stem cell transplantation (AHSCT) are endangered by developing Epstein-Barr virus-related post-transplant lymfoprolipherative disease (EBV-LPD). The aims of the study were to retrospectively characterise the viral loads in four patients who died of this complication, and to test possible risk factors for EBV reactivation in a prospectively observed cohort of children after AHSCT. METHODS AND RESULTS: Serial DNA samples extracted from whole blood from four patients who died of post-transplant EBV-LPD in year 2000 were retrospectively analysed for EBV load using quantitative real-time PCR. First detection of EBV activation preceded death by 24-91 days. All four patients exceeded a viral load of one million EBV copies per 100,000 human genome equivalents. A cohort of 72 children undergoing AHSCT between 2001-2004 was prospectively followed-on using the same quantification method from regularly obtained samples of whole blood, and clinical and laboratory data were recorded on a weekly basis, totalling at 3,896 person-weeks of observation. Approximately one half of the cohort experienced at least one episode of EBV reactivation during the first 100 days after AHSCT, four of the episodes being accompanied with viral loads higher than our provisional threshold of 10,000 copies per 100,000 human genome equivalents. Three of the four patients developed EBV-LPD and were successfully treated by intravenous administration of anti-CD20 antibody. Testing of possible clinical and laboratory predictors of EBV reactivation did not reveal any clinically useful association. CONCLUSIONS: The cornerstone of predicting EBV-LPD in AHSCT is a regular monitoring of EBV viral load using quantitative methods. Using this strategy with a threshold of 10,000 EBV copies per 100,000 human genome equivalents was proved to be effective, as shown by no death of EBV for the study period, compared to four cases in the year before the quantitative monitoring.

Child↗
PubMed1998

Genetic stability of Sabin 1 strain of poliovirus: implications for quality control of oral poliovirus vaccine.

The Sabin vaccine strains of poliovirus, like all RNA viruses, exist as a quasispecies of genomic sequences whose composition can be altered during virus propagation. Since changes in vaccine virus during manufacture can enhance the neurovirulent potential of the vaccine, each monovalent lot of oral poliovirus vaccine (OPV) undergoes several tests to ensure consistency of manufacture, including the monkey neurovirulence test (MNVT). Recently, we proposed a new molecular approach for direct quantification of vaccine variants with neurovirulent potential as an alternative way to monitor consistency of OPV production. Analysis of the Sabin 1 genome allowed us to identify a limited number of specific loci that exhibit significant change during viral propagation in vitro and in vivo. Here we explore the possible roles of these changes and show that 7427-U-->C and 7441-G-->A alterations in the 3'-UTR of the Sabin 1 virus do not increase monkey neurovirulence. These, as well as our previous results, suggest that only mutations in the 5'-UTR play a significant role in the limited increase in Sabin 1 monkey neurovirulence observed after extended propagation of the virus beyond the passage level used in vaccine production. Our studies with high-passage batches of the Sabin 1 strain confirmed the stability of this strain, which retains acceptable levels of monkey neurovirulence even after serial passages at elevated temperature. Compared to the MNVT, molecular analysis of the genetic composition of Sabin 1 poliovirus provides a more sensitive analytical approach to monitor consistency of vaccine production.

Animals↗
PubMed2003

Aspirin-like molecules that inhibit human immunodeficiency virus 1 replication.

Some anti-inflammatory molecules are also known to possess anti-human immunodeficiency virus (HIV) activity. We found that o-(acetoxyphenyl)hept-2-ynyl sulfide (APHS), a recently synthesized non-steroidal anti-inflammatory molecule can inhibit HIV-1 replication. The aim of this study was to clarify the mechanism of action of APHS. When administered during the first steps of the infection, APHS was capable of inhibiting the replication of several HIV-1 strains (macrophage-tropic and/or lymphocytotropic) in a dose-dependent manner in both peripheral blood mononuclear cells (PBMC), monocyte-derived macrophages and peripheral blood lymphocytes with 50% inhibitory concentration values of approximately 10 microM. The 50% toxic concentration of APHS varied between 100 and 200 microM in the different primary cells tested. APHS did not affect HIV-1 replication once the provirus was already inserted into the cellular genome. APHS also did not inhibit HIV-1 entry into the host cells as determined by quantification of gag RNA inside PBMC 2h after infection. However, APHS did inhibit gag DNA synthesis during reverse transcription in primary cells, which indicates that APHS may target the reverse transcription process.

Acetylene↗
PubMed2005

Seed-specific expression of the lysine-rich protein gene sb401 significantly increases both lysine and total protein content in maize seeds.

The sb401 gene from potato (Solanum berthaultii) encoding a pollen-specific protein with high lysine content was successfully integrated into the genome of maize plants, and its expression was correlated with increased levels of lysine and total protein content in maize seeds. A plasmid vector containing the sb401 gene under the control of a maize seed-specific expression storage protein promoter (P19z) was constructed and introduced into maize calli by microprojectile bombardment. The integration of the sb401 gene into the maize genome was confirmed by Southern blot analysis, and its expression was confirmed by Western blot analysis. Quantification of the lysine and protein contents in R1 maize seeds showed that, compared with the nontransgenic maize control, the lysine content increased by 16.1% to 54.8% and the total protein content increased by 11.6% to 39.0%. There were no visible morphological changes in the vegetative parts and seeds of the transgenic maize plants. Lysine and protein analysis of the transgenic maize grains showed that the levels of lysine and total protein remained high for six continuous generations, indicating that the elevated lysine and total protein levels were heritable. These results indicate that the sb401 gene could be successfully employed in breeding programs aimed at improving the nutritional value of maize.

Consumer Product Safety↗