EUROPEAN JOURNAL OF IMMUNOLOGY, Issue 5 2005
Daniel Lucas
Abstract Mammalian DNA polymerase,, (Pol,), preferentially expressed in secondary lymphoid organs, is shown here to be up-regulated in germinal centers after immunization. Alternative splicing appears to be part of Pol, regulation during an immune response. We generated Pol,-deficient mice that are viable and show no anatomical malformation or serious alteration in lymphoid populations, with the exception of an underrepresentation of the B,cell compartment. Young and aged homozygous Pol,,/, mice generated similar immune responses after immunization with the hapten (4-hydroxy-3-nitrophenyl)acetyl (NP) coupled to chicken gammaglobulin (CGG), compared with their wild-type littermates. Nonetheless, the kinetics of development of the centroblast population showed significant differences. Hypermutation analysis of the rearranged heavy chain intron region in centroblasts isolated from NP-CGG-immunized Pol,,/, mice showed a similar quantitative and qualitative somatic mutation spectrum, but a lower representation of heavily mutated clones. These results suggest that although it is not a critical partner, Pol, modulates the in vivo somatic hypermutation process. [source]

Urea Sensitization Caused by Separation of Helicobacter pylori RNA Polymerase , and ,, Subunits

HELICOBACTER, Issue 2 2007
Daiva Dailidiene
Abstract Background:, The , and ,, subunits of RNA polymerase are fused in all Helicobacters, but separate in most other taxa. Prior studies had shown that this fusion is not essential for viability in culture or in vivo, but had not tested it for potentially important quantitative effects on phenotype. Methods:, The effect of separating rpoB and rpoC sequences on Helicobacter pylori growth was tested in culture and during mouse infection. Results:, Derivatives of strains X47 and SS1 carrying this "rpoBCsplit" allele colonized mice less vigorously than their wild-type parents in competition tests. With X47 rpoBCsplit, this reduced vigor was evident in wild-type mice, whereas with SS1 rpoBCsplit it was seen only in cytokine IL-10- and IL-12,-deficient mice. In culture, the rpoBCsplit allele sensitized each of four strains tested (X47, SS1, 88-3887, and AM1) to urea, a metabolite that is secreted into the gastric mucosa; urea sensitization was more severe in X47 than in SS1 genetic backgrounds. The rpoBCsplit allele also caused poorer growth on Ham's F12 agar, a nutritionally limiting medium, but had little effect on sensitivity to mild acidity. Conclusions:,H. pylori's normal RNA polymerase ,-,, subunit fusion contributes quantitatively to fitness. We propose that urea, although important to H. pylori in vivo, also be considered inhibitory; and that H. pylori's natural ,-,, subunit fusion helps it cope with urea exposure. [source]

Deoxyribonucleic acid-based diagnostic techniques to detect Helicobacter pylori

ALIMENTARY PHARMACOLOGY & THERAPEUTICS, Issue 11 2004
A. Ruzsovics
Summary Helicobacter pylori is an important cause of many gastrointestinal disorders, ranging from chronic gastritis to gastric lymphoma and adenocarcinoma. The deoxyribonucleic acid-based assays have the potential to be a powerful diagnostic tool given its ability to specifically identify H. pylori deoxyribonucleic acid. Markers used to include general H. pylori structures and pathogenetic factors like ureaseA, cagA, vacA, iceA. Deoxyribonucleic acid or bacterial ribonucleic acid for polymerase chain reaction assays can be collected from gastric biopsy, gastric juice, stool, buccal specimens. Polymerase chain reaction can yield quantitative and genotyping results with sensitivity and specificity that approaches 100%. A clear trend in the direction of the determination of quantitative H. pylori infection by real-time polymerase chain reaction can be observed. Fluorescent in situ hybridization is suggested for routine antibiotic resistance determination. To identify the organism, deoxyribonucleic acid structure and its virulence factors may be feasible by using oligonucleotide microarray specifically recognizing and discriminating bacterial deoxyribonucleic acid and various virulence factors. Deoxyribonucleic acid-based H. pylori diagnosis yields higher sensitivity, however, specificity requires sophisticated labour environment and associated with higher costs. [source]

Titelbild: Kristallstruktur eines Cisplatin-(1,3-GTG)-Schadens im Komplex mit DNA-Polymerase , (Angew. Chem.

ANGEWANDTE CHEMIE, Issue 17 2010
17/2010)
Cisplatin-DNA-Schäden hemmen die DNA-Transkription und führen zum Zelltod. In ihrer Zuschrift auf S.,3142,ff. beschreiben T. Carell und Mitarbeiter die Kristallstruktur eines Cisplatin-(1,3-GTG)-Schadens im Komplex mit Polymerase,,. Die Bewegung dieses Enzyms entlang des DNA-Doppelstrangs wird verhindert, indem sich das zentrale Desoxythymidin des Schaden aus dem Doppelstrang herausdreht. [source]

A Population of Thermostable Reverse Transcriptases Evolved from Thermus aquaticus DNA Polymerase,I by Phage Display,

ANGEWANDTE CHEMIE, Issue 7 2010
Sophie Vichier-Guerre Dr.
No abstract is available for this article. [source]

Direct Polymerase Synthesis of Reactive Aldehyde-Functionalized DNA and Its Conjugation and Staining with Hydrazines,

ANGEWANDTE CHEMIE, Issue 6 2010
Veronika Raindlová
In zwei Stufen zu reaktiver Aldehyd-modifizierter DNA: durch Suzuki-Kreuzkupplung halogenierter Nucleosidtriphosphate (dNTPs) mit 4-Formylthiophen-2-boronsäure und Polymerase- vermittelten Einbau der modifizierten Nucleotide in DNA (siehe Schema; PEX=Primerverlängerung, PCR=Polymerasekettenreaktion). Die Bildung von Hydrazonen mit Arylhydrazinen unter wässrigen Bedingungen wurde zum Anfärben der DNA verwendet. [source]

A Light-Activated DNA Polymerase,

ANGEWANDTE CHEMIE, Issue 32 2009
Chungjung Chou Dr.
Wenn die Zeit reif ist: Die häufig genutzte Thermus-aquaticus -DNA-Polymerase wurde durch Einbau der photoaktivierbaren Aminosäure ortho -Nitrobenzyltyrosin anstelle eines Tyrosinrests im aktiven Zentrum lichtaktivierbar (siehe Bild). Da das modifizierte Enzym erst nach Bestrahlen mit UV-Licht aktiv ist, kann die Polymerase-Aktivität zeitlich reguliert werden. [source]

Stabilization of Taq DNA Polymerase at High Temperature by Protein Folding Pathways From a Hyperthermophilic Archaeon, Pyrococcus furiosus

BIOTECHNOLOGY & BIOENGINEERING, Issue 1 2006
Pongpan Laksanalamai
Abstract Pyrococcus furiosus, a hyperthermophilic archaeon growing optimally at 100°C, encodes three protein chaperones, a small heat shock protein (sHsp), a prefoldin (Pfd), and a chaperonin (Cpn). In this study, we report that the passive chaperones sHsp and Pfd from P. furiosus can boost the protein refolding activity of the ATP-dependent Cpn from the same hyperthermophile. The thermo-stability of Taq polymerase was significantly improved by combinations of P. furiosus chaperones, showing ongoing protein folding activity at elevated temperatures and during thermal cycling. Based on these results, we propose that the protein folding apparatus in the hyperthermophilic archaeon, P. furiosus can be utilized to enhance the durability and cost effectiveness of high temperature biocatalysts. © 2005 Wiley Periodicals, Inc. [source]

ChemInform Abstract: Discovery of Pentacyclic Compounds as Potent Inhibitors of Hepatitis C Virus NS5B RNA Polymerase.

CHEMINFORM, Issue 26 2009
Joerg Habermann
Abstract ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a "Full Text" option. The original article is trackable via the "References" option. [source]

ChemInform Abstract: Asymmetric Synthesis of an N-Acylpyrrolidine for Inhibition of HCV Polymerase.

CHEMINFORM, Issue 35 2008
Martin J. Slater
Abstract ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a "Full Text" option. The original article is trackable via the "References" option. [source]

Design and Synthesis of 2,3,4,9-Tetrahydro-1H-carbazole and 1,2,3,4-Tetrahydro-cyclopenta[b]indole Derivatives as Non-Nucleoside Inhibitors of Hepatitis C Virus NS5B RNA-Dependent RNA Polymerase.

CHEMINFORM, Issue 31 2006
Ariamala Gopalsamy
Abstract ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF. [source]

Tetrahydrobenzothiophene Inhibitors of Hepatitis C Virus NS5B Polymerase

CHEMINFORM, Issue 13 2006
M. G. LaPorte
Abstract ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF. [source]

Active Site Inhibitors of HCV NS5B Polymerase.

CHEMINFORM, Issue 4 2005
6-dihydroxypyrimidine-4-carboxylic Acid., Pharmacophore of 2-Thienyl-, The Development
Abstract For Abstract see ChemInform Abstract in Full Text. [source]

Structure of Microcin J25, a Peptide Inhibitor of Bacterial RNA Polymerase, is a Lassoed Tail.

CHEMINFORM, Issue 4 2004
Kelly-Anne Wilson
No abstract is available for this article. [source]

Optimization of Thienopyrrole-Based Finger-Loop Inhibitors of the Hepatitis,C Virus NS5B Polymerase

CHEMMEDCHEM, Issue 10 2009
Hernando Dr., Ignacio Martin
Abstract Infections caused by the hepatitis,C virus (HCV) are a significant world health problem for which novel therapies are in urgent demand. The NS5B polymerase of HCV is responsible for the replication of viral RNA and has been a prime target in the search for novel treatment options. We had discovered allosteric finger-loop inhibitors based on a thieno[3,2- b]pyrrole scaffold as an alternative to the related indole inhibitors. Optimization of the thienopyrrole series led to several N -acetamides with submicromolar potency in the cell-based replicon assay, but they lacked oral bioavailability in rats. By linking the N4-position to the ortho -position of the C5-aryl group, we were able to identify the tetracyclic thienopyrrole 40, which displayed a favorable pharmacokinetic profile in rats and dogs and is equipotent with recently disclosed finger-loop inhibitors based on an indole scaffold. [source]

Incorporation of Thymine Nucleotides by DNA Polymerases through T,HgII,T Base Pairing

ANGEWANDTE CHEMIE, Issue 37 2010
Hidehito Urata Prof.
DNA in schlechter Gesellschaft: In Gegenwart von HgII -Ionen führten DNA-Polymerasen Thymidin-5,-triphosphat (TTP) gegenüber einem Thyminrest im Templatstrang ein bildeten zur Verlängerung des Primerstrangs eine Phosphodiesterbindung. DNA-Polymerasen erkannten dieses ungewöhnliche, durch ein Metallion verknüpfte Basenpaar und synthetisierten darüber hinweg das Volllängenprodukt (siehe Bild). [source]

Directed Evolution of DNA Polymerases for Next-Generation Sequencing,

ANGEWANDTE CHEMIE, Issue 34 2010
Aaron
Evolution mit mehr Tempo: Mit einem aktivitätsbasierten Phagen-Display wurde die Titelaufgabe gelöst. Dabei wurde eine Mutante der Taq-Polymerase gefunden (siehe Schema), die fluorophormarkierte dA, dT, dC und dG 50- bis 400-fach effizienter in ,vernarbte" Primer einbaut und die zudem unter realen Sequenzierungsbedingungen ein deutlich verbessertes Verhalten zeigt. [source]

Structure,Activity Relationships of Untenone A and Its Derivatives for Inhibition of DNA Polymerases.

CHEMINFORM, Issue 31 2004
Fumiyo Saito
Abstract For Abstract see ChemInform Abstract in Full Text. [source]

Polymerase-Catalysed Incorporation of Glucose Nucleotides into a DNA Duplex

CHEMISTRY - A EUROPEAN JOURNAL, Issue 22 2009
Marleen Renders
Abstract Active but unselective: Nucleoside triphosphates possessing glucose moieties (such as those depicted) instead of the natural furanose rings are recognised by the active sites of polymerases. Polymerases therefore seem to be very unspecific in their recognition patterns. The enzymatic recognition of six-membered ring nucleoside triphosphates,in particular the 6,-triphosphates of (,- D -glucopyranosyl)thymine, (2,,3,-dideoxy-,- D -glucopyranosyl)thymine, (3,,4,-dideoxy-,- D -glucopyranosyl)thymine and (2,,3,-dideoxy-,- D -glucopyranosyl)adenine,was investigated. Despite the facts that the pyranose nucleic acids obtained by polymerisation of these monomers do not hybridise in solution with DNA and that the geometry of a DNA strand in a natural duplex differs from that of a pyranose nucleic acid, elongation of the DNA duplex with all four nucleotide analogues by Vent,(exo,) polymerase was observed. Modelling experiments showed that hydrogen bonds are formed when 2,,3,-dideoxy-,-homo-T building blocks or ,- D - gluco -T building blocks are incorporated opposite adenosine residues in the template but not when they are incorporated opposite thymine residues in the template. The model shows a near perfect alignment of a secondary hydroxy group at the end of the primer and the ,-phosphate group of the incoming triphosphate. The results of these experiments provide new information on the role of the active site of the enzyme in the polymerisation reaction. [source]

Induction of G2/M phase arrest and apoptosis by a novel indoloquinoline derivative, IQDMA, in K562 cells

DRUG DEVELOPMENT RESEARCH, Issue 9 2006
Yi-Hsiung Lin
Abstract The indoloquinoline, IQDMA (N,-(11H-indolo[3,2-c]quinolin-6-yl)-N,N-dimethylethane-1,2-diamine), was identified as a novel antineoplastic agent with broad spectrum of antitumor activities against several human cancer cells. IQDMA-induced G2/M arrest was accompanied by up-regulation of the cyclin-dependent kinase inhibitors (CDKIs), p21 and p27, and down-regulation of Cdk1and Cdk2. IQDMA had no effect on the levels of cyclin A, cyclin B1, cyclin D3, or Cdc25C. IQDMA also increased apoptosis, as characterized by apoptotic body formation, increase of the sub G1 population and poly (ADP-ribose) polymerase (PARP) cleavage. Further mechanistic analysis demonstrated that IQDMA upregulated FasL protein expression, and kinetic studies showed the sequential activation of caspases-8, -3, and -9. Both caspase-8 and caspase-3 inhibitors, but not a caspase-9-specific inhibitor, suppressed IQDMA-induced cell death. These molecular alterations provide an insight into IQDMA-caused growth inhibition, G2/M arrest, and apoptotic death of K562 cells. Drug Dev. Res. 67:743,751, 2006. © 2006 Wiley-Liss, Inc. [source]

Separation of nuclear protein complexes by blue native polyacrylamide gel electrophoresis

ELECTROPHORESIS, Issue 7 2006
Zora Nováková
Abstract The nucleus is a highly structured organelle with distinct compartmentalization of specific functions. To understand the functions of these nuclear compartments, detailed description of protein complexes which form these structures is of crucial importance. We explored therefore the potential of blue native PAGE (BN-PAGE) method for the separation of nuclear protein complexes. We focused on (i),solubility and stability of nuclear complexes under conditions prerequisite for the separation by BN-PAGE, (ii),improved separation of native nuclear protein complexes using 2-D colorless native/blue native PAGE (CN-/BN-PAGE), and (iii),mass spectrometric analysis of protein complexes which were isolated directly from native 1-D or from 2-D CN/BN-PAGE gels. The suitability of BN-PAGE for nuclear proteomic research is demonstrated by the successful separation of polymerase,I and polymerase,II complexes, and by mass spectrometric determination of U1 small nuclear ribonucleoprotein particle composition. Moreover, practical advice for sample preparation is provided. [source]

Mutagenic repair of DNA interstrand crosslinks

ENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 6 2010
Xi Shen
Abstract Formation of DNA interstrand crosslinks (ICLs) in chromosomal DNA imposes acute obstruction of all essential DNA functions. For over 70 years bifunctional alkylators, also known as DNA crosslinkers, have been an important class of cancer chemotherapeutic regimens. The mechanisms of ICL repair remains largely elusive. Here, we review a eukaryotic mutagenic ICL repair pathway discovered by work from several laboratories. This repair pathway, alternatively termed recombination-independent ICL repair, involves the incision activities of the nucleotide excision repair (NER) mechanism and lesion bypass polymerase(s). Repair of the ICL is initiated by dual incisions flanking the ICL on one strand of the double helix; the resulting gap is filled in by lesion bypass polymerases. The remaining lesion is subsequently removed by a second round of NER reaction. The mutagenic repair of ICL likely interacts with other cellular mechanisms such as the Fanconi anemia pathway and recombinational repair of ICLs. These aspects will also be discussed. Environ. Mol. Mutagen., 2010. © 2010 Wiley-Liss, Inc. [source]

Effect of deletion of SOS-induced polymerases, pol II, IV, and V, on spontaneous mutagenesis in Escherichia coli mutD5

ENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 4 2004
Anetta Nowosielska
Abstract The E. coli dnaQ gene encodes the , subunit of DNA polymerase III (pol III) responsible for the proofreading activity of this polymerase. The mutD5 mutant of dnaQ chronically expresses the SOS response and exhibits a mutator phenotype. In this study we have constructed a set of E. coli AB1157 mutD5 derivatives deleted in genes encoding SOS-induced DNA polymerases, pol II, pol IV, and pol V, and estimated the frequency and specificity of spontaneous argE3,Arg+ reversion in exponentially growing and stationary-phase cells of these strains. We found that pol II exerts a profound effect on the specificity of spontaneous mutation in exponentially growing cells. Analysis of growth-dependent Arg+ revertants in mutD5 polB+ strains revealed that Arg+ revertants were due to tRNA suppressor formation, whereas those in mutD5 ,polB strains arose by back mutation at the argE3 ochre site. In stationary-phase bacteria, Arg+revertants arose mainly by back mutation, regardless of whether they were proficient or deficient in pol II. Our results also indicate that in a mutD5 background, the absence of pol II led to increased frequency of Arg+ growth-dependent revertants, whereas the lack of pol V caused its dramatic decrease, especially in mutD5 ,umuDC and mutD5 ,umuDC ,polB strains. In contrast, the rate of stationary-phase Arg+revertants increased in the absence of pol IV in the mutD5 ,dinB strain. We postulate that the proofreading activity of pol II excises DNA lesions in exponentially growing cells, whereas pol V and pol IV are more active in stationary-phase cultures. Environ. Mol. Mutagen. 43:226,234, 2004. © 2004 Wiley-Liss, Inc. [source]

At the birth of molecular radiation biology ,

ENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 2-3 2001
Raymond Devoret
Abstract Rational thinking builds on feelings, too. This article starts with a tribute to Richard Setlow, an eminent scientist; it retraces as well some studies in molecular genetics that helped to understand basic questions of radiation biology. In the mid-1950s, the induction of a dormant virus (prophage) by irradiation of its host was an intriguing phenomenon. Soon, it was found that prophage induction results from the inactivation of the prophage repressor. Similarly, a score of induced cellular SOS functions were found to be induced when the LexA repressor is inactivated. Repressor inactivation involves the formation of a newly formed distinctive structure: a RecA-polymer wrapped around single-stranded DNA left by the arrest of replication at damaged sites. By touching this RecA nucleofilament, the LexA repressor is inactivated, triggering the sequential expression of SOS functions. The RecA nucleofilament acts as a chaperone, allowing recombinational repair to occur after nucleotide excision repair is over. The UmuD,C complex, synthesized slowly and parsimoniously, peaks at the end of recombinational repair, ready to be positioned at the tip of a RecA nucleofilament, placing the UmuD,C complex right at a lesion. At this location, UmuD,C prevents recombinational repair, and now acts as an error-prone paucimerase that fills the discontinuity opposite the damaged DNA. Finally, the elimination of lesions from the path of DNA polymerase, allows the resumption of DNA replication, and the SOS repair cycle switches to a normal cell cycle. Environ. Mol. Mutagen. 38:135,143, 2001. © 2001 Wiley-Liss, Inc. [source]

Prevalence of highly host-specific cyanophages in the estuarine environment

ENVIRONMENTAL MICROBIOLOGY, Issue 2 2008
Kui Wang
Summary Cyanophages that infect coastal and oceanic Synechococcus have been studied extensively. However, no cyanophages infecting estuarine Synechococcus have been reported. In this study, seven cyanophages (three podoviruses, three siphoviruses and one myovirus) isolated from four estuarine Synechococcus strains were characterized in terms of their morphology, host range, growth and genetic features. All the podoviruses and siphoviruses were highly host specific. For the first time, the photosynthesis gene (psbA) was found in two podoviruses infecting estuarine Synechococcus. However, the psbA gene was not detected in the three siphoviruses. The psbA sequences from the two Synechococcus podoviruses clustered with some environmental psbA sequences, forming a unique cluster distantly related to previous known psbA clusters. Our results suggest that the psbA among Synechococcus podoviruses may evolve independently from the psbA of Synechococcus myoviruses. All three estuarine Synechococcus podoviruses contained the DNA polymerase (pol) gene, and clustered with other podoviruses that infect oceanic Synechococcus and Prochlorococcus, suggesting that the DNA pol is conserved among marine picocyanobacterial podoviruses. Prevalence of host-specific cyanophages in the estuary suggests that Synechococcus and their phages in the estuarine ecosystem may develop a host,phage relationship different from what have been found in the open ocean. [source]

Environmental tuning of mutation rates

ENVIRONMENTAL MICROBIOLOGY, Issue 2 2006
Claude Saint-Ruf
Summary Through their life cycles, bacteria experience many different environments in which the relationship between available energy resources and the frequency and the nature of various stresses is highly variable. In order to survive in such changeable environments, bacteria must balance the need for nutritional competence with stress resistance. In Escherichia coli natural populations, this is most frequently achieved by changing the regulation of the RpoS sigma factor-dependent general stress response. One important secondary consequence of altered regulation of the RpoS regulon is the modification of mutation rates. For example, under nutrient limitation during stationary phase, the high intracellular concentration of RpoS diminishes nutritional competence, increases stress resistance, and, by downregulating the mismatch repair system and downregulating the expression of the dinB gene (coding for PolIV translesion synthesis polymerase) increases mutation rates. The reduction of the intracellular concentration of RpoS has exactly opposite effects on nutritional competence, stress resistance and mutation rates. Therefore, the natural selection that favours variants having the highest fitness under different environmental conditions results in high variability of stress-associated mutation rates in those variants. [source]

Multiple displacement amplification as a pre-polymerase chain reaction (pre-PCR) to process difficult to amplify samples and low copy number sequences from natural environments

ENVIRONMENTAL MICROBIOLOGY, Issue 7 2005
Juan M. Gonzalez
Summary Microbial assessment of natural biodiversity is usually achieved through polymerase chain reaction (PCR) amplification. Deoxyribonucleic acid (DNA) sequences from natural samples are often difficult to amplify because of the presence of PCR inhibitors or to the low number of copies of specific sequences. In this study, we propose a non-specific preamplification procedure to overcome the presence of inhibitors and to increase the number of copies prior to carrying out standard amplification by PCR. The pre-PCR step is carried out through a multiple displacement amplification (MDA) technique using random hexamers as priming oligonucleotides and ,29 DNA polymerase in an isothermal, whole-genome amplification reaction. Polymerase chain reaction amplification using specific priming oligonucleotides allows the selection of the sequences of interest after a preamplification reaction from complex environmental samples. The procedure (MDA-PCR) has been tested on a natural microbial community from a hypogean environment and laboratory assemblages of known bacterial species, in both cases targeting the small subunit ribosomal RNA gene sequences. Results from the natural community showed successful amplifications using the two steps protocol proposed in this study while standard, direct PCR amplification resulted in no amplification product. Amplifications from a laboratory assemblage by the two-step proposed protocol were successful at bacterial concentrations ,,10-fold lower than standard PCR. Amplifications carried out in the presence of different concentrations of fulvic acids (a soil humic fraction) by the MDA-PCR protocol generated PCR products at concentrations of fulvic acids over 10-fold higher than standard PCR amplifications. The proposed procedure (MDA-PCR) opens the possibility of detecting sequences represented at very low copy numbers, to work with minute samples, as well as to reduce the negative effects on PCR amplifications of some inhibitory substances commonly found in environmental samples. [source]

Fluorescence in situ hybridization of 16S rRNA gene clones (Clone-FISH) for probe validation and screening of clone libraries

ENVIRONMENTAL MICROBIOLOGY, Issue 11 2002
Andreas Schramm
Summary A method is presented for fluorescence in situ hybridization (FISH) of 16S rRNA gene clones targeting in vivo transcribed plasmid inserts (Clone-FISH). Several different cloning approaches and treatments to generate target-rRNA in the clones were compared. Highest signal intensities of Clone-FISH were obtained using plasmids with a T7 RNA polymerase promoter and host cells with an IPTG-inducible T7 RNA polymerase. Combined IPTG-induction and chloramphenicol treatment of those clones resulted in FISH signals up to 2.8-fold higher than signals of FISH with probe EUB338 to cells of Escherichia coli. Probe dissociation curves for three oligonucleotide probes were compared for reference cells containing native (FISH) or cloned (Clone-FISH) target sequences. Melting behaviour and calculated Td values were virtually identical for clones and cells, providing a format to use 16S rRNA gene clones instead of pure cultures for probe validation and optimization of hybridization conditions. The optimized Clone-FISH protocol was also used to screen an environmental clone library for insert sequences of interest. In this application format, 13 out of 82 clones examined were identified to contain sulphate-reducing bacterial rRNA genes. In summary, Clone-FISH is a simple and fast technique, compatible with a wide variety of cloning vectors and hosts, that should have general utility for probe validation and screening of clone libraries. [source]

Polymerase,, is up-regulated during the T,cell-dependent immune response and its deficiency alters developmental dynamics of spleen centroblasts

Role of GSK-3, activity in motor neuronal cell death induced by G93A or A4V mutant hSOD1 gene

EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 2 2005
Seong-Ho Koh
Abstract Point mutations such as G93A and A4V in the human Cu/Zn-superoxide dismutase gene (hSOD1) cause familial amyotrophic lateral sclerosis (fALS). In spite of several theories to explain the pathogenic mechanisms, the mechanism remains largely unclear. Increased activity of glycogen synthase kinase-3 (GSK-3) has recently been emphasized as an important pathogenic mechanism of neurodegenerative diseases, including Alzheimer's disease and ALS. To investigate the effects of G93A or A4V mutations on the phosphatidylinositol-3-kinase (PI3-K)/Akt and GSK-3 pathway as well as the caspase-3 pathway, VSC4.1 motoneuron cells were transfected with G93A- or A4V-mutant types of hSOD1 (G93A and A4V cells, respectively) and, 24 h after neuronal differentiation, their viability and intracellular signals, including PI3-K/Akt, GSK-3, heat shock transcription factor-1 (HSTF-1), cytochrome c, caspase-3 and poly(ADP-ribose) polymerase (PARP), were compared with those of wild type (wild cells). Furthermore, to elucidate the role of the GSK-3,-mediated cell death mechanism, alterations of viability and intracellular signals in those mutant motoneurons were investigated after treating the cells with GSK-3, inhibitor. Compared with wild cells, viability was greatly reduced in the G93A and A4V cells. However, the treatment of G93A and A4V cells with GSK-3, inhibitor increased their viability by activating HSTF-1 and by reducing cytochrome c release, caspase-3 activation and PARP cleavage. However, the treatment did not affect the expression of PI3-K/Akt and GSK-3,. These results suggest that the G93A or A4V mutations inhibit PI3-K/Akt and activate GSK-3, and caspase-3, thus becoming vulnerable to oxidative stress, and that the GSK-3,-mediated cell death mechanism is important in G93A and A4V cell death. [source]