22 results on '"Primer, T."'
Search Results
2. Overexpression of Map3k7 activates sinoatrial node-like differentiation in mouse ES-derived cardiomyocytes.
- Author
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Brown, Kemar, Legros, Stephanie, Ortega, Francis A., Dai, Yunkai, Doss, Michael Xavier, Christini, David J., Robinson, Richard B., and Foley, Ann C.
- Subjects
GENETIC overexpression ,SINOATRIAL node ,HEART cells ,PLURIPOTENT stem cells ,REGENERATIVE medicine - Abstract
In vivo, cardiomyocytes comprise a heterogeneous population of contractile cells defined by unique electrophysiologies, molecular markers and morphologies. The mechanisms directing myocardial cells to specific sub-lineages remain poorly understood. Here we report that overexpression of TGFβ-Activated Kinase (TAK1/Map3k7) in mouse embryonic stem (ES) cells faithfully directs myocardial differentiation of embryoid body (EB)-derived cardiac cells toward the sinoatrial node (SAN) lineage. Most cardiac cells in Map3k7-overexpressing EBs adopt markers, cellular morphologies, and electrophysiological behaviors characteristic of the SAN. These data, in addition to the fact that Map3k7 is upregulated in the sinus venous—the source of cells for the SAN—suggest that Map3k7 may be an endogenous regulator of the SAN fate. [ABSTRACT FROM AUTHOR]
- Published
- 2017
- Full Text
- View/download PDF
3. Linear-After-The-Exponential (LATE)-PCR: primer design criteria for high yields of specific single-stranded DNA and improved real-time detection
- Author
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Lawrence J. Wangh, John Rice, Kenneth Pierce, and J. Aquiles Sanchez
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Multidisciplinary ,Hybridization probe ,Linear amplification ,Cystic Fibrosis Transmembrane Conductance Regulator ,DNA, Single-Stranded ,Biology ,Amplicon ,Biological Sciences ,Molecular biology ,Polymerase Chain Reaction ,Exponential function ,chemistry.chemical_compound ,chemistry ,Yield (chemistry) ,Transition Temperature ,Primer (molecular biology) ,Gene ,DNA ,DNA Primers - Abstract
Traditional asymmetric PCR uses conventional PCR primers at unequal concentrations to generate single-stranded DNA. This method, however, is difficult to optimize, often inefficient, and tends to promote nonspecific amplification. An alternative approach, Linear-After-The-Exponential (LATE)-PCR, solves these problems by using primer pairs deliberately designed for use at unequal concentrations. The present report systematically examines the primer design parameters that affect the exponential and linear phases of LATE-PCR amplification. In particular, we investigated how altering the concentration-adjusted melting temperature ( T m ) of the limiting primer ( T m L ) relative to that of the excess primer ( T m X ) affects both amplification efficiency and specificity during the exponential phase of LATE-PCR. The highest reaction efficiency and specificity were observed when T m L - T m X ≥ 5°C. We also investigated how altering T m X relative to the higher T m of the double-stranded amplicon ( T m A ) affects the rate and extent of linear amplification. Excess primers with T m X closer to T m A yielded higher rates of linear amplification and stronger signals from a hybridization probe. These design criteria maximize the yield of specific single-stranded DNA products and make LATE-PCR more robust and easier to implement. The conclusions were validated by using primer pairs that amplify sequences within the cystic fibrosis transmembrane regulator ( CFTR ) gene, mutations of which are responsible for cystic fibrosis.
- Published
- 2005
4. A New Role for Carbonic Anhydrase 2 in the Response of Fish to Copper and Osmotic Stress: Implications for Multi-Stressor Studies.
- Author
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de Polo, Anna, Margiotta-Casaluci, Luigi, Lockyer, Anne E., and Scrimshaw, Mark D.
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CARBONIC anhydrase ,OSMOSIS ,AQUATIC organisms ,COPPER ,GENE expression in fishes - Abstract
The majority of ecotoxicological studies are performed under stable and optimal conditions, whereas in reality the complexity of the natural environment faces organisms with multiple stressors of different type and origin, which can activate pathways of response often difficult to interpret. In particular, aquatic organisms living in estuarine zones already impacted by metal contamination can be exposed to more severe salinity variations under a forecasted scenario of global change. In this context, the present study aimed to investigate the effect of copper exposure on the response of fish to osmotic stress by mimicking in laboratory conditions the salinity changes occurring in natural estuaries. We hypothesized that copper-exposed individuals are more sensitive to osmotic stresses, as copper affects their osmoregulatory system by acting on a number of osmotic effector proteins, among which the isoform two of the enzyme carbonic anhydrase (CA2) was identified as a novel factor linking the physiological responses to both copper and osmotic stress. To test this hypothesis, two in vivo studies were performed using the euryhaline fish sheepshead minnow (Cyprinodon variegatus) as test species and applying different rates of salinity transition as a controlled way of dosing osmotic stress. Measured endpoints included plasma ions concentrations and gene expression of CA2 and the α1a-subunit of the enzyme Na
+ /K+ ATPase. Results showed that plasma ions concentrations changed after the salinity transition, but notably the magnitude of change was greater in the copper-exposed groups, suggesting a sensitizing effect of copper on the responses to osmotic stress. Gene expression results demonstrated that CA2 is affected by copper at the transcriptional level and that this enzyme might play a role in the observed combined effects of copper and osmotic stress on ion homeostasis. [ABSTRACT FROM AUTHOR]- Published
- 2014
- Full Text
- View/download PDF
5. Template strand scrunching during DNA gap repair synthesis by human polymerase λ.
- Author
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Garcia-Diaz, Miguel, Bebenek, Katarzyna, Larrea, Andres A., Havener, Jody M., Perera, Lalith, Krahn, Joseph M., Pedersen, Lars C., Ramsden, Dale A., and Kunkel, Thomas A.
- Subjects
DNA polymerases ,DNA repair ,PROTEIN binding ,NUCLEOTIDES ,AMINO acids ,BIOCHEMISTRY ,MOLECULAR biology - Abstract
Family X polymerases such as DNA polymerase λ (Pol λ) are well suited for filling short gaps during DNA repair because they simultaneously bind both the 5′ and 3′ ends of short gaps. DNA binding and gap filling are well characterized for 1-nucleotide (nt) gaps, but the location of yet-to-be-copied template nucleotides in longer gaps is unknown. Here we present crystal structures revealing that, when bound to a 2-nt gap, Pol λ scrunches the template strand and binds the additional uncopied template base in an extrahelical position within a binding pocket that comprises three conserved amino acids. Replacing these amino acids with alanine results in less processive gap filling and less efficient NHEJ when 2-nt gaps are involved. Thus, akin to scrunching by RNA polymerase during transcription initiation, scrunching occurs during gap filling DNA synthesis associated with DNA repair. [ABSTRACT FROM AUTHOR]
- Published
- 2009
- Full Text
- View/download PDF
6. A novel specificity for the primer-template pairing requirement in Tetrahymena telomerase.
- Author
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He Wang, Gilley, David, and Blackburn, Elizabeth H.
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TELOMERASE ,DNA polymerases ,REVERSE transcriptase ,TELOMERES ,BIOSYNTHESIS ,TETRAHYMENA ,MOLECULAR biology - Abstract
Telomerase is a specialized reverse transcriptase with a built-in RNA template. Base pairing between the templating domain of telomerase RNA and a telomeric DNA primer is normally a characteristic of elongation of telomeric DNA. Here we demonstrate the mechanism by which Tetrahymena telomerase bypasses a requirement for template—primer pairing in order to add telomeric DNA de novo to completely non-telomeric DNA primers. We show that this reaction initiates by copying the template residue at the 3 boundary of the telomerase RNA template sequence. Unexpectedly, as the RNA template moves through the telomerase catalytic center, the number of required potential Watson—Crick base pairs between RNA template and DNA primer increases from zero to five. We propose that this unprecedented position specificity of a base pairing potential requirement in a polymerase underlies the chromosome healing mechanism of telomerase, and reflects constraints inherent in an internal template. [ABSTRACT FROM AUTHOR]
- Published
- 1998
- Full Text
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7. Amino acid substitutions away from the RNase H catalytic site increase the thermal stability of Moloney murine leukemia virus reverse transcriptase through RNase H inactivation
- Author
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Kanta Yokokawa, Luis Menéndez-Arias, Kiyoshi Yasukawa, Atsushi Konishi, Tetsuro Hisayoshi, and Verónica Barrioluengo
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RNase P ,RNase H activity ,Thermostabilization ,Ribonuclease H ,Biophysics ,Template–primer ,Biochemistry ,chemistry.chemical_compound ,Catalytic Domain ,Murine leukemia virus ,Escherichia coli ,Reverse transcriptase ,RNase H ,Molecular Biology ,Base Sequence ,biology ,Protein Stability ,Mutagenesis ,Temperature ,RNA ,RNA-Directed DNA Polymerase ,DNA ,Cell Biology ,biology.organism_classification ,Molecular biology ,Recombinant Proteins ,Amino Acid Substitution ,chemistry ,Mutagenesis, Site-Directed ,biology.protein ,Nucleic acid ,Moloney murine leukemia virus - Abstract
We have previously used site-directed mutagenesis to introduce basic residues (i.e., Arg; Lys) in the nucleic acid binding cleft of the Moloney murine leukemia virus reverse transcriptase (MMLV RT) in order to increase its template-primer (T/P) binding affinity. Three stabilizing mutations (i.e., E286R, E302K, and L435R) were identified (Yasukawa et al., 2010). Now, we studied the mechanism by which those mutations increase the thermal stability of the RT. The three single-mutants (E286R, E302K, and L435R), an RNase H-deficient MMLV RT (carrying the RNase H-inactivating mutation D524A), a quadruple mutant (E286R/E302K/L435R/D524A, designated as MM4) and the wild-type enzyme (WT) were produced in Escherichia coli. All RTs exhibited similar dissociation constants (Kd) for heteropolymeric DNA/DNA (2.9-6.5 nM) and RNA/DNA complexes (1.2-2.9 nM). Unlike the WT, mutant enzymes (E286R, E302K, L435R, D524A, and MM4) were devoid of RNase H activity, and were not able to degrade RNA in RNA/DNA complexes. These results suggest that the mutations, E286R, E302K, and L435R increase the thermostability of MMLV RT not by increasing its affinity for T/P but by abolishing its RNase H activity.
- Published
- 2014
8. Genotyping strategy for the FMR1 gene: An alternative diagnostic method for the Fragile X syndrome and other trinucleotide expansion diseases
- Author
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Saúl Lindo-Samanamud, Olimpio Ortega, Mario Cornejo-Olivas, Victoria Marca, Keren Espinoza-Huertas, and Pilar Mazzetti
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Physics ,Cytosine ,Citosinas metilación ,Medicine ,uracil ,General Medicine ,methylation ,microsatellites repeats ,Molecular biology ,uracilo ,repetición de microsatélite - Abstract
Objetivos: Diseñar una estrategia alternativa por PCR para el genotipado de secuencias ricas en citosinas, basada en modificación nucleotídica. Material y métodos: Se modificó el gen FMR1 nativo de ocho individuos clínicamente no afectados por el Síndrome X frágil, cambiando las citosinas por uracilos, empleando bisulfito de sodio. El ADN modificado fue purificado y cuantificado por espectrofotometría. Las estructuras alternativas y potenciales islas CpG que adopta el microsatélite inestable fueron simuladas con los programas MFOLD y CpGplot. Se generaron cebadores específicos que hibriden tanto con el microsatélite modificado (Primer T) y con una secuencia modificada de las islas CpG (Primer M), utilizando el programa MethPrimer. Finalmente, ambas secuencias fueron amplificadas por PCR y los amplicones fueron separados por electroforesis en gel de poliacrilamida (PAGE por sus siglas en inglés) al 6% y visualizados con tinción de nitrato de plata. Resultados: La modificación del ADN fue evidenciada por espectrofotometría al uracilo. Las estructuras observadas en la simulación fueron las horquillas encontrándose dos potenciales islas CpG. La amplificación con los cebadores T, confirmó el diseño in silico desarrollado para abordar la estructura en horquillas. La amplificación con los cebadores M permitió detectar metilación de la primera isla CpG del gen FMR1.Conclusión: Se propone un diseño alternativo para amplificación de secuencias de microsatélite que contengan citosinas metiladas y no metiladas. Se requieren estudios posteriores con muestras de ADN que contengan microsatélites muy expandidos para validar su aplicación para diagnóstico molecular. Objectives: To design an alternative strategy for genotyping cytosine-rich sequences using PCR and nucleotide modification. Methods: The FMR1 gene wild type was modified in the DNA obtained from eight individuals clinically unaffected for Fragile X Syndrome; cytosines were replaced by uracils using sodium bisulfite. Modified DNA was purified and quantified by spectrophotometry. Alternative structures and potential CpG islands of the unstable microsatellite were simulated using MFOLD and CpGplot tools. Specific primers were generated to hybridize with both the modified microsatellite (Primer G) and a modified sequence of CpG islands (Primer M) using the MethPrimer software. Finally, both sequences were amplified by PCR and the amplicons were separated by electrophoresis in silver-stained PAGE 6% gels. Results: The DNA modification was evidenced by spectrophotometry to uracil. We found two potential CpG islands. The amplification with T primers confirmed the "in silico" design developed to engage hairpin structures. The amplification with M primers detected methylation of the first CpG island in the FMR1 gene. Conclusion: We propose an alternative design for amplifying microsatellite sequences that contain methylated and unmethylated cytosine bases. Further studies are required with DNA samples containing expanded microsatellites to validate its molecular diagnostic application
- Published
- 2013
9. Improving the thermal stability of avian myeloblastosis virus reverse transcriptase α-subunit by site-directed mutagenesis
- Author
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Atsushi Konishi, Kiyoshi Yasukawa, and Kuniyo Inouye
- Subjects
Hot Temperature ,Insecta ,Protein subunit ,Gene Expression ,cDNA synthesis ,Bioengineering ,Biology ,Applied Microbiology and Biotechnology ,Cell Line ,chemistry.chemical_compound ,Complementary DNA ,Enzyme Stability ,Reverse transcriptase ,Animals ,Site-directed mutagenesis ,Avian myeloblastosis virus ,Protein Stability ,Mutagenesis ,RNA ,RNA-Directed DNA Polymerase ,General Medicine ,Virology ,Molecular biology ,Protein Subunits ,chemistry ,Amino Acid Substitution ,Mutagenesis, Site-Directed ,Mutant Proteins ,Primer (molecular biology) ,Stability ,DNA ,Biotechnology - Abstract
Avian myeloblastosis virus reverse transcriptase (AMV RT) is a heterodimer consisting of a 63 kDa α-subunit and a 95 kDa β subunit. Moloney murine leukaemia virus reverse transcriptase (MMLV RT) is a 75 kDa monomer. These two RTs are the most extensively used for conversion of RNA to DNA. We previously developed several mutations that increase the thermostability of MMLV RT and generated a highly stable MMLV RT variant E286R/E302K/L435R/D524A by combining three of them (Glu286→Arg, Glu302→Lys, and Leu435→Arg) and the mutation to abolish RNase H activity (Asp524→Ala) [Yasukawa et al. (2010) J Biotechnol 150:299-306]. To generate a highly stable AMV RT variant, we have introduced the triple mutation of Val238→Arg, Leu388→Arg, and Asp450→Ala into AMV RT α-subunit and the resulted variant V238R/L388R/D450A, was expressed in insect cells and purified. The temperature decreasing the initial activity by 50 %, measured over 10 min, of the variant with or without template primer (T/P), poly(rA)-p(dT)(15), was 50 °C; for the wild-type AMV RT α-subunit (WT) this was 44 °C. The highest temperature at which the variant exhibited cDNA synthesis activity was 64 °C; the WT was 60 °C. A highly stable AMV RT α-subunit is therefore generated by the same mutation strategy as applied to MMLV RT and that positive charges are introduced into RT at positions that have been implicated to interact with T/P by site-directed mutagenesis.
- Published
- 2012
10. Homology Modeling and Analysis of Structure Predictions of the Bovine Rhinitis B Virus RNA Dependent RNA Polymerase (RdRp)
- Author
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Elizabeth Rieder and Devendra K. Rai
- Subjects
Picornavirus ,Rhinovirus ,viruses ,homology modeling ,RNA-dependent RNA polymerase ,Sequence alignment ,Catalysis ,Article ,Protein Structure, Secondary ,Inorganic Chemistry ,lcsh:Chemistry ,Viral Proteins ,Aphthovirus ,Animals ,Humans ,3Dpol structure predictions ,Homology modeling ,Physical and Theoretical Chemistry ,BRBV ,Molecular Biology ,lcsh:QH301-705.5 ,Spectroscopy ,Polymerase ,Phylogeny ,Genetics ,biology ,Organic Chemistry ,Respiratory infection ,RNA-Directed DNA Polymerase ,General Medicine ,biology.organism_classification ,Virology ,Computer Science Applications ,Protein Structure, Tertiary ,lcsh:Biology (General) ,lcsh:QD1-999 ,Structural Homology, Protein ,biology.protein ,Cattle ,Rabbits ,Foot-and-mouth disease virus - Abstract
Bovine Rhinitis B Virus (BRBV) is a picornavirus responsible for mild respiratory infection of cattle. It is probably the least characterized among the aphthoviruses. BRBV is the closest relative known to Foot and Mouth Disease virus (FMDV) with a ~43% identical polyprotein sequence and as much as 67% identical sequence for the RNA dependent RNA polymerase (RdRp), which is also known as 3D polymerase (3Dpol). In the present study we carried out phylogenetic analysis, structure based sequence alignment and prediction of three-dimensional structure of BRBV 3Dpol using a combination of different computational tools. Model structures of BRBV 3Dpol were verified for their stereochemical quality and accuracy. The BRBV 3Dpol structure predicted by SWISS-MODEL exhibited highest scores in terms of stereochemical quality and accuracy, which were in the range of 2Å resolution crystal structures. The active site, nucleic acid binding site and overall structure were observed to be in agreement with the crystal structure of unliganded as well as template/primer (T/P), nucleotide tri-phosphate (NTP) and pyrophosphate (PPi) bound FMDV 3Dpol (PDB, 1U09 and 2E9Z). The closest proximity of BRBV and FMDV 3Dpol as compared to human rhinovirus type 16 (HRV-16) and rabbit hemorrhagic disease virus (RHDV) 3Dpols is also substantiated by phylogeny analysis and root-mean square deviation (RMSD) between C-α traces of the polymerase structures. The absence of positively charged α-helix at C terminal, significant differences in non-covalent interactions especially salt bridges and CH-pi interactions around T/P channel of BRBV 3Dpol compared to FMDV 3Dpol, indicate that despite a very high homology to FMDV 3Dpol, BRBV 3Dpol may adopt a different mechanism for handling its substrates and adapting to physiological requirements. Our findings will be valuable in the design of structure-function interventions and identification of molecular targets for drug design applicable to Aphthovirus RdRps.
- Published
- 2012
11. Increase in thermal stability of Moloney murine leukaemia virus reverse transcriptase by site-directed mutagenesis
- Author
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Atsushi Konishi, Masaki Mizuno, Kiyoshi Yasukawa, and Kuniyo Inouye
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Models, Molecular ,DNA, Complementary ,Hot Temperature ,cDNA synthesis ,Bioengineering ,medicine.disease_cause ,Applied Microbiology and Biotechnology ,Virus ,Moloney murine leukaemia virus ,Complementary DNA ,medicine ,Reverse transcriptase ,Site-directed mutagenesis ,Gammaretrovirus ,Mutation ,biology ,Protein Stability ,Mutagenesis ,RNA-Directed DNA Polymerase ,General Medicine ,Nucleotidyltransferase ,biology.organism_classification ,Virology ,Molecular biology ,Protein Structure, Tertiary ,Kinetics ,Mutagenesis, Site-Directed ,Electrophoresis, Polyacrylamide Gel ,Moloney murine leukemia virus ,Stability ,Biotechnology - Abstract
We hypothesized that the thermal stability of Moloney murine leukaemia virus reverse transcriptase (MMLV RT) will increase with increases in its ability to bind with a template-primer (T/P). To test this hypothesis, we introduced positive charges into MMLV RT by site-directed mutagenesis at positions that have been implicated in the interaction with T/P. Thirty-six variants were constructed in which one of the twelve residues (Glu69, Gln84, Asp108, Asp114, Glu117, Glu123, Asp124, Glu286, Glu302, Trp313, Leu435, and Asn454) was replaced with Lys, Arg, or Ala, and these were expressed in Escherichia coli. In about half of these 36 variants, thermal inactivation at 50°C was reduced in the presence of the T/P, which suggested that this strategy was effective at stabilizing MMLV RT. We next combined three of the 36 mutations, Glu286→Ala, Glu302→Lys, and Leu435→Arg, and the mutation, Asp524→Ala, which is known to abolish the RNase H activity and increase the stability. Temperatures of 54 and 56°C reduced the initial reverse transcription activity by 50% over a 10-min incubation in the triple variant E286R/E302K/L435R and quadruple variant E286R/E302K/L435R/D524A, respectively. These temperatures were higher than that observed for WT (45°C). The highest temperature at which the triple and quadruple variants exhibited cDNA synthesis activity was 60°C, which was again higher than for WT (54°C). Thus, highly stable MMLV RT variants were generated by this mutation strategy.
- Published
- 2010
12. Study of MMLV RT- Binding with DNA using Surface Plasmon Resonance Biosensor
- Author
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Jianlong Zhao, Minghui Huang, Lei Wu, and Mengsu Yang
- Subjects
biology ,Chemistry ,DNA damage ,RNA-Directed DNA Polymerase ,Biophysics ,General Medicine ,Biochemistry ,Molecular biology ,Reverse transcriptase ,Receptor–ligand kinetics ,chemistry.chemical_compound ,biology.protein ,Surface plasmon resonance ,RNase H ,DNA ,Polymerase - Abstract
Surface plasmon resonance biosensor technique was used to study the binding of Moloney murine leukemia virus reverse transcriptase without RNase H domain (MMLV RT-) with DNA in the absence and in the presence of inhibitors. Different DNA substrates, including single-stranded DNA (ssDNA), DNA template-primer (T-P) duplex and gapped DNA, were immobilized on the biosensor chip surface using streptavidin-biotin, and MMLV RT(-)-DNA binding kinetics were analyzed by different models. MMLV RT-; could bind with ssDNA and the binding was involved in conformation change. MMLV RT-; binding DNA T-P duplex and gapped DNA could be analyzed using the simple 1:1 Langmuir model. The lack of RNase H domain reduced the affinity between MMLV RT-; and T-P duplex. The effects of RT inhibitors, including efavirenz, nevirapine and quercetin, on the interaction between MMLV RT-; and gapped DNA were analyzed according to recovered kinetics parameters. Efavirenz slightly interfered with the binding between RT and DNA and the affinity constant in the presence of the inhibitor (K(A) = 1.21 x 10(6) M(-1)) was lower than in the absence of the inhibitor (KA = 4.61 x 10(6) M(-1)). Nevirapine induced relatively tight binding between RT and DNA and the affinity constant in the presence of the inhibitor (K(A) = 1.47 x 10(7) M(-1)) was approximately three folds higher than without nevirapine, mainly due to rapid association and slow dissociation. Quercetin, a flavonoid originating from plant which has previously shown strong inhibition of the activity of RT, was found to have minimal effect on the RT-DNA binding.
- Published
- 2005
13. A Gripping New Mechanism of Drug Resistance in HIV-1 Reverse Transcriptase
- Author
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Grant D. Schauer, Nic Sluis-Cremer, and Sanford H. Leuba
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Molecular model ,Chemistry ,Mechanism (biology) ,Human immunodeficiency virus (HIV) ,Biophysics ,virus diseases ,Drug resistance ,medicine.disease_cause ,Molecular biology ,Reverse transcriptase ,Intramolecular force ,medicine ,Human genome ,Primer (molecular biology) - Abstract
HIV-1 Reverse Transcriptase (RT) converts viral RNA into dsDNA that is integrated into the human genome. Nonnucleoside RT inhibitors (NNRTIs) are highly effective in the treatment and prevention of HIV. NNRTI resistance mutations arise from therapy, yet the mechanism(s) of how these mutations inhibit polymerization by RT is unclear. We examine the role of NNRTI on the dynamics between RT and its Template/Primer (T/P) substrate using a combination of molecular modeling, single-molecule and bulk fluorescence techniques and provide an unprecedented glimpse into the dynamics of RT-T/P interaction as well as the intramolecular conformation of RT itself while bound to its substrate. The data suggests a unique mechanism of resistance that is mediated by interplay between intramolecular conformational changes in RT and intermolecular dynamics of the RT-template/primer-dNTP complex.
- Published
- 2014
- Full Text
- View/download PDF
14. The fidelity of reverse transcription differs in reactions primed with RNA versus DNA primers
- Author
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Ben Berkhout and Belinda B. Oude Essink
- Subjects
Transcription, Genetic ,DNA polymerase ,Base pair ,Endocrinology, Diabetes and Metabolism ,Clinical Biochemistry ,Transcription (biology) ,Pharmacology (medical) ,Molecular Biology ,Polymerase ,DNA Primers ,Transcription bubble ,Base Sequence ,biology ,Chemistry ,Biochemistry (medical) ,Templates, Genetic ,Cell Biology ,General Medicine ,Molecular biology ,Reverse transcriptase ,RNA, Ribosomal ,HIV-2 ,biology.protein ,RNA ,RNA, Transfer, Lys ,RNA, Viral ,Primase ,Primer (molecular biology) - Abstract
Reverse transcriptase enzymes (RT) convert single-stranded retroviral RNA genomes into double-stranded DNA. The RT enzyme can use both RNA and DNA primers, the former being used exclusively during initiation of minus- and plus-strand synthesis. Initiation of minus-strand DNA synthesis occurs by extension of a tRNA primer that is associated with the viral genome, and plus-strand DNA synthesis is initiated from an RNase H- resistant polypurine tract of the genomic RNA that remains bound to the newly synthesized minus-strand DNA. All other phases of reverse transcription represent elongation of a DNA primer. We demonstrate that the polymerase fidelity of RT enzymes is significantly higher in tRNA-primed reverse transcription compared with DNA-primed reactions. Two mechanistic explanations can be proposed. First, the type of template-primer (T- P) duplex (RNA-RNA versus RNA-DNA) may affect the RT enzyme conformation such that the discrimination against incorrect nucleotides is affected. Second, the tRNA primer may act as a fidelity co-factor through specific association with the RT enzyme. According to the latter hypothesis, the increased fidelity observed for an RNA-RNA T-P should persist at a distance from the initiation site, where the enzyme-bound nucleic acid duplex will consist of RNA-cDNA. However, we measured that the effect of tRNA on the fidelity is detectable only at a short distance from the initiation site. These results indicate that the type of T-P duplex influences the fidelity of reverse transcription, suggesting that two small segments of the viral genome downstream of the initiation sites for minus- and plus-strand DNA synthesis are copied with a fidelity that is greater than average.
- Published
- 1999
15. Stabilization of human immunodeficiency virus type 1 reverse transcriptase by site-directed mutagenesis
- Author
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Kosaku Nishimura, Kiyoshi Yasukawa, Atsushi Konishi, and Mayu Shinomura
- Subjects
Hot Temperature ,Mutant ,Molecular Sequence Data ,Human immunodeficiency virus type 1 ,Bioengineering ,Applied Microbiology and Biotechnology ,Complementary DNA ,Murine leukemia virus ,Enzyme Stability ,Reverse transcriptase ,Amino Acid Sequence ,Site-directed mutagenesis ,Thermostability ,chemistry.chemical_classification ,biology ,Base Sequence ,Mutagenesis ,HIV ,virus diseases ,General Medicine ,Sequence Analysis, DNA ,biology.organism_classification ,Virology ,Molecular biology ,HIV Reverse Transcriptase ,Kinetics ,Enzyme ,chemistry ,Mutation ,Mutagenesis, Site-Directed ,RNA, Viral ,Electrophoresis, Polyacrylamide Gel ,Biotechnology - Abstract
Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is a heterodimer containing 66 kDa p66 and 51 kDa p51 subunits. We previously showed that HIV-1 group M (HIV-1 M) RT and HIV-1 group O (HIV-1 O) RT have higher affinities for dTTP and template-primer (T/P) than Moloney murine leukemia virus RT, which is currently used for cDNA synthesis, suggesting that they might also be useful for cDNA synthesis (Konishi et al. Appl Biochem Biotechnol 2013, 169:77-87). Here, we have increased the thermostability of both HIV-1 M RT and HIV-1 O RT by site-directed mutagenesis. The Asp443 → Ala mutation, which abolishes RNase H activity, was introduced into the p66 subunits of HIV-1 M RT and HIV-1 O RT. The temperatures that reduced the initial activity by 50 % of the resulting mutants, HIV-1 M p66D443A/p51 and HIV-1 O p66D443A/p51, were 44 and 52 °C, respectively, which were higher than those of wild-type HIV-1 M p66/p51 (42 °C) and HIV-1 O p66/p51 (48 °C). The highest temperature at which both HIV-1 M p66D443A/p51 and HIV-1 O p66D443A/p51 exhibited cDNA synthesis activity was 68 °C, which was higher than for the wild-type enzymes (62 and 66 °C, respectively).
- Published
- 2013
16. The K65R Mutation Confers Increased DNA Polymerase Processivity to HIV-1 Reverse Transcriptase
- Author
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Michael A. Parniak, Gadi Borkow, Zhengian Gu, Dominique Arion, and Mark A. Wainberg
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biology ,DNA synthesis ,DNA polymerase ,viruses ,RNA-Directed DNA Polymerase ,Point mutation ,Mutant ,Drug Resistance ,DNA ,Templates, Genetic ,Cell Biology ,Processivity ,biochemical phenomena, metabolism, and nutrition ,Biochemistry ,Molecular biology ,HIV Reverse Transcriptase ,Recombinant Proteins ,Reverse transcriptase ,Structure-Activity Relationship ,biology.protein ,Point Mutation ,Primer (molecular biology) ,Molecular Biology - Abstract
The K65R mutation in HIV-1 reverse transcriptase (RT) is associated with viral cross-resistance to 2',3'-dideoxyinosine, 2',3'-dideoxycytidine, and 2',3'-dideoxy-3'-thiacytidine. We have found that in vitro DNA synthesis by K65R RT is significantly more processive than that of wild type (wt) RT. Depending on the template/primer (T/P) used, the total incorporation of nucleotides under single processive cycle conditions was 20-50% higher with K65R RT than with wt RT. With heteropolymeric T/P, the total incorporation of dNMP by K65R and wt RT was similar under continuous DNA synthesis reaction conditions. However, under single processive cycle conditions, the rate of full-length polymerization product synthesis by K65R RT was about 2-fold higher than that by wt RT. We also found a decreased rate of T/P dissociation during K65R RT DNA synthesis, which is consistent with the increased processivity of the enzyme. We postulate that the increased processivity of the K65R RT may be a compensatory response to the decreased affinity of this mutant for certain dNTP substrates, allowing normal viral replication kinetics.
- Published
- 1996
17. Human Immunodeficiency Virus Type 1 Reverse Transcriptase
- Author
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Samuel H. Wilson, Madhuri Jaju, and William A. Beard
- Subjects
chemistry.chemical_classification ,Conformational change ,Stereochemistry ,Chemistry ,Substrate (chemistry) ,Context (language use) ,Cell Biology ,Biochemistry ,Dissociation constant ,Enzyme ,Reaction rate constant ,Nucleotide ,Enzyme kinetics ,Molecular Biology - Abstract
Human immunodeficiency virus type-1 (HIV-1) reverse transcriptase (RT) catalyzes DNA synthesis by an ordered sequential mechanism. After template-primer (T·P) binds to free enzyme, the deoxynucleoside triphosphate to be incorporated binds to the RT and T·P binary complex (RTT·P). After incorporation of the bound nucleotide, catalytic cycling is limited either by a conformational change (for processive synthesis) or release of the enzyme from the extended T·P (for single-nucleotide incorporation). To explore cycling through these alternate rate-limiting steps, we determined kinetic parameters for single-nucleotide incorporation by HXB2R HIV-1 RT with chain-terminating nucleotide substrates 3′-azido-3′-deoxythymidine triphosphate (AZTTP) and dideoxythymidine triphosphate on a homopolymeric T·P system, poly(rA)-oligo(dT)16. Inhibition of processive deoxythymidine monophosphate incorporation by these chain-terminating substrates was also examined. Because AZTTP is a substrate, its Kmshould be equivalent to Ki, and since Kmfor AZTTP should be influenced by the dissociation rate constant for RTT·P, we examined the effect of altering RTT·Pdissociation on AZTTP kinetic parameters. The dissociation rate constant was modulated by making use of different T·P substrates, viral sources of RT, and a mutant RT altered at a residue that perturbs T·P binding. As expected from earlier work, the time course of AZTMP incorporation on poly(rA)-oligo(dT)16was biphasic, with a burst followed by a slower steady-state phase representing kcat(0.42 min−1) which was similar to the rate constant for RTT·Pdissociation. Additionally, Kmfor AZTTP (110 nM) was lower than its equilibrium dissociation constant (1200 nM). AZTTP inhibition (KiAZTTP) of processive dTMP incorporation and incorporation of a single nucleotide were similar. However, a simple correlation between the RTT·Pdissociation rate constant and KiAZTTPwas not observed. These results indicate that a simple ordered model for single-nucleotide incorporation is inadequate and that different forms of RTT·Pexist which can limit catalysis. The results are discussed in the context of a two-step binding reaction for T·P where the binary RTT·Pcomplex undergoes an isomerization before binding of the deoxynucleotide substrate.
- Published
- 1995
18. Extension of base mispairs byTaqDNA polymerase: implications for single nucleotide discrimination in PCR
- Author
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Norman Arnheim, Myron F. Goodman, and Mei mei Huang
- Subjects
Genetics ,Base Composition ,Base Sequence ,biology ,Thermus aquaticus ,Nucleotides ,DNA polymerase ,Base pair ,RNA-Directed DNA Polymerase ,Molecular Sequence Data ,Temperature ,DNA-Directed DNA Polymerase ,biology.organism_classification ,Polymerase Chain Reaction ,Molecular biology ,Reverse transcriptase ,chemistry.chemical_compound ,Oligodeoxyribonucleotides ,chemistry ,biology.protein ,Taq Polymerase ,Primer (molecular biology) ,Transversion ,Taq polymerase - Abstract
Thermus aquaticus (Taq) DNA polymerase was used to measure the extension efficiency for all configurations of matched and mismatched base pairs at template-primer 3'-termini. The transition mispairs, A(primer).C, C.A, G.T, and T.G were extended 10(-3) to 10(-4)-fold less efficiently than their correctly paired counterparts. Relative efficiencies for extending transversion mispairs were 10(-4) to 10(-5) for T.C and T.T, about 10(-6) for A.A, and less than 10(-6) for G.A, A.G, G.G and C.C. The transversion mispair C(primer).T was extended with high efficiency, about 10(-2) compared to a correct A.T basepair. The unexpected ease of extending the C.T mismatch was not likely to have been caused by primer-template misalignment. Taq polymerase was observed to bind with similar affinities to each of the correctly paired and mispaired primer-template 3'-ends. Thus, the failure of Taq polymerase to extend mismatches efficiently appears to be an intrinsic property of the enzyme and not due to an inability to bind to 3'-terminal mispairs. For almost all of the mispairs, C.T being the exception, Taq polymerase exhibits about 100 to 1000-fold greater discrimination against mismatch extension compared to avian myeloblastosis reverse transcriptase and HIV-1 reverse transcriptase which extend most mismatched basepairs permissively. Relative mismatch extension efficiencies for Taq polymerase were measured at 45 degrees C, 55 degrees C and 70 degrees C and found to be independent of temperature. The mispair extension data should be important in designing experiments using PCR to distinguish between sequences that vary by a single nucleotide.
- Published
- 1992
19. Base mispair extension kinetics. Comparison of DNA polymerase alpha and reverse transcriptase
- Author
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L V Mendelman, John Petruska, and Myron F. Goodman
- Subjects
Transition (genetics) ,biology ,Base pair ,DNA polymerase ,fungi ,Cell Biology ,Biochemistry ,Molecular biology ,Reverse transcriptase ,Deoxyribonucleotide ,chemistry.chemical_compound ,chemistry ,biology.protein ,Primer (molecular biology) ,Transversion ,Molecular Biology ,Polymerase ,Mathematics - Abstract
A polyacrylamide gel assay is used to measure the kinetics of adding a single deoxyribonucleotide onto either a correctly matched or mismatched primer 3' terminus (on M13 template) for all possible DNA base pairs and mispairs using Drosophila melanogaster DNA polymerase alpha (Pol alpha) and avian myeloblastosis virus reverse transcriptase. The reverse transcriptase catalyzes chain extension from transition mispairs (Pur.Pyr and Pyr.Pur, where Pur is purine and Pyr is pyrimidine) more efficiently than polymerase alpha. Reverse transcriptase extends G(primer).T almost 20% as efficiently as it extends A.T, while Pol alpha's G.T extension efficiency is less than 1%. For transversion mispairs (Pur.Pur and Pyr.Pyr), reverse transcriptase extends C.T and T.T with greater efficiency than polymerase alpha, while polymerase alpha is more efficient at extending A.G and G.G mispairs. Reverse transcriptase and polymerase alpha extend the G.G mispair at an efficiency of only 10(-6) and 10(-5), respectively, compared with G.C extension. The extension data for the two polymerases are compared with previously reported nucleotide misinsertion data for the same enzymes (Mendelman, L. V., Boosalis, M. S., Petruska, J., and Goodman, M. F. (1989) J. Biol. Chem. 264, 14415-14423). While the results obtained with reverse transcriptase and Pol alpha differ in detail, some general rules are indicated: (a) Pur.Pyr and Pyr.Pur mispairs, especially G.T and T.G, are easy to insert and even easier to extend; (b) Pyr.Pyr mispairs, especially C.C, are difficult to insert and slightly easier to extend; (c) Pur.Pur mispairs, notably G.G, are harder to extend than to insert. The comparison also shows that reverse transcriptase extends almost all mismatches more efficiently than it forms them, G.G being the only mismatch having a significantly lower efficiency of extension than insertion. Polymerase alpha inserts A.A mismatches most efficiently, but extends them inefficiently, thereby reducing the probability that such transversion mutations will occur in vivo. We show theoretically that when mispaired primers compete with properly matched primers for extension by polymerase, the relative velocities of extension depend on the concentration of the next correct dNTP substrate. The extension velocities depart from Michaelis-Menten kinetics by exhibiting positive cooperativity with respect to substrate concentration.
- Published
- 1990
20. Mechanistic differences in RNA-dependent DNA polymerization and fidelity between murine leukemia virus and HIV-1 reverse transcriptases
- Author
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Mark Skasko, Baek Kim, Holly M. Reynolds, Varuni K. Jamburuthugoda, Kellie K. Weiss, and Kwi Y. Lee
- Subjects
Time Factors ,Base Pair Mismatch ,viruses ,DNA Mutational Analysis ,medicine.disease_cause ,Biochemistry ,Article ,chemistry.chemical_compound ,Mice ,Murine leukemia virus ,medicine ,Animals ,Humans ,Molecular Biology ,DNA Primers ,Mutation ,Binding Sites ,biology ,Dose-Response Relationship, Drug ,Mutagenesis ,Active site ,RNA ,RNA-Directed DNA Polymerase ,Cell Biology ,DNA ,biology.organism_classification ,Molecular biology ,HIV Reverse Transcriptase ,Leukemia Virus, Murine ,Kinetics ,Phenotype ,Polymerization ,chemistry ,biology.protein ,HIV-1 ,Primer (molecular biology) ,Protein Binding - Abstract
We compared the mechanistic and kinetic properties of murine leukemia virus (MuLV) and human immunodeficiency virus type 1 (HIV-1) reverse transcriptases (RTs) during RNA-dependent DNA polymerization and mutation synthesis using pre-steady-state kinetic analysis. First, MuLV RT showed 6.5-121.6-fold lower binding affinity (K(d)) to deoxynucleotide triphosphate (dNTP) substrates than HIV-1 RT, although the two RTs have similar incorporation rates (k(pol)). Second, compared with HIV-1 RT, MuLV RT showed dramatic reduction during multiple dNTP incorporations at low dNTP concentrations. Presumably, due to its low dNTP binding affinity, the dNTP binding step becomes rate-limiting in the multiple rounds of the dNTP incorporation by MuLV RT, especially at low dNTP concentrations. Third, similar fold differences between MuLV and HIV-1 RTs in the K(d) and k(pol) values to correct and incorrect dNTPs were observed. This indicates that these two RT proteins have similar misinsertion fidelities. Fourth, these two RT proteins have different mechanistic capabilities regarding mismatch extension. MuLV RT has a 3.1-fold lower mismatch extension fidelity, compared with HIV-1 RT. Finally, MuLV RT has a 3.8-fold lower binding affinity to mismatched template/primer (T/P) substrate compared with HIV-1 RT. Our data suggest that the active site of MuLV RT has an intrinsically low dNTP binding affinity, compared with HIV-1 RT. In addition, instead of the misinsertion step, the mismatch extension step, which varies between MuLV and HIV-1 RTs, contributes to their fidelity differences. The implications of these kinetic differences between MuLV and HIV-1 RTs on viral cell type specificity and mutagenesis are discussed.
- Published
- 2005
21. Use of arbitrarily primed polymerase chain reaction to differentiate Trichophyton dermatophytes
- Author
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J. Pedersen, D. Liu, S. Coloe, and Robert W. Baird
- Subjects
Biology ,medicine.disease_cause ,Microbiology ,Polymerase Chain Reaction ,law.invention ,chemistry.chemical_compound ,Trichophyton ,law ,Genetics ,medicine ,Dermatomycoses ,Humans ,DNA, Fungal ,Molecular Biology ,Polymerase chain reaction ,DNA Primers ,Fungal genetics ,biology.organism_classification ,Molecular biology ,genomic DNA ,chemistry ,Dermatophyte ,Primer (molecular biology) ,Arthrodermataceae ,DNA - Abstract
Dermatophytes such as Trichophyton species are common human pathogens, the infection of which results in dermatophytosis (also known as ringworm). Several laboratory tests are used routinely for the diagnosis of dermatophytosis, but they are either slow or lacking specificity. Through examination of genomic DNA from Trichophyton dermatophytes and other fungi in arbitrarily primed PCR, it was shown that a random primer 5'-ACCCGACCTG-3' produced bands of 4.3 kb, 1.9 kb, 1.7 kb and 0.7 kb in T. rubrum DNA, bands of 2.5 kb, 1.9 kb and 0.8 kb in T. mentagrophytes var. interdigitale and T. mentagrophytes var. mentagrophytes DNA, and bands of 2.5 kb, 1.9 kb, 1.5 kb and 0.9 kb in T. tonsurans DNA. This primer amplified bands of different sizes in other fungal DNA. Therefore, based on the distinct band patterns observed in arbitrarily primed PCR using this primer, T. rubrum, T. mentagrophytes and T. tonsurans dermatophytes could be rapidly differentiated.
- Published
- 1996
22. Direct Measurement of HIV-1 Reverse Transcriptase Binding Kinetics, One Complex at a Time
- Author
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Nicolas Sluis-Cremer, Sanford H. Leuba, and Grant D. Schauer
- Subjects
Human immunodeficiency virus (HIV) ,Biophysics ,Biology ,medicine.disease_cause ,Genome ,Molecular biology ,Receptor–ligand kinetics ,Reverse transcriptase ,chemistry.chemical_compound ,Viral replication ,chemistry ,Biochemistry ,Drug development ,medicine ,Viral rna ,DNA - Abstract
HIV-1 reverse transcriptase (RT) is entirely responsible for the conversion of the viral RNA genome into double stranded DNA. Due to its essential role in virus replication, it is a primary target for drug development. In order to rationally modulate RT binding to its Template/Primer (T/P) substrate using novel inhibitory compounds, an accurate description of DNA-protein binding kinetics in the absence of ensemble averaging is crucial. We are therefore developing a robust single-molecule assay to directly measure the association/dissociation rates of protein-nucleic acid complexes at equilibrium. Utilizing this method in comparison with bulk methods, we describe in detail the effects of various known RT inhibitors (RTIs) as well as novel RTIs under development in our laboratory on the binding kinetics of individual RT-T/P complexes.
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