RECQ5 is one of five members of the RecQ family of helicases in humans, which include RECQ1, Bloo... more RECQ5 is one of five members of the RecQ family of helicases in humans, which include RECQ1, Bloom (BLM), Werner (WRN), RECQ4, and RECQ5. Both WRN and BLM have been shown to resolve G-quadruplex (GQ) structures. Deficiencies in unfolding GQ are known to result in DNA breaks and genomic instability, which are prominent in Werner and Bloom syndromes. RECQ5 is significant in suppressing sister chromatid exchanges during homologous recombination but its GQ unfolding activity are not known. We performed single-molecule studies under different salt (50-150 mM KCl or NaCl) and ATP concentrations on different GQ constructs including human telomeric GQ (with different overhangs and polarities) and GQ formed by thrombin-binding aptamer to investigate this activity. These studies demonstrated a RECQ5-mediated GQ unfolding activity that was an order of magnitude weaker than BLM and WRN. On the other hand, BLM and RECQ5 demonstrated similar single-stranded DNA (ssDNA) reeling activities that wer...
Recent charge and thermal transport measurements on the electron- doped high-Tc superconductor Pr... more Recent charge and thermal transport measurements on the electron- doped high-Tc superconductor Pr2-xCexCuO4-y (PCCO) show unusual behavior in the superconducting dome at the doping level going from x = 0.15 (optimally doped) to 0.17 (overdoped), such as quantum phase transition and electron paring symmetry changes, suggesting a possible connection between the superconductivity and other competing phases like antiferromagnetism. We report the ^63,65Cu-NMR spectrum in PCCO single crystals with x = 0.17 in a high magnetic field B0, and contrast their properties with those of x = 0.15. The temperature dependence of the ^63,65Cu-NMR Knight shift is dominated by the negative isotropic hyperfine coupling to the field-induced Pr^3+ ion moment, and changes little between x = 0.15 and 0.17. The ^63,65Cu-NMR linewidths are propotional to the applied magnetic field B0, but affected by the doping, indicating the existence of other sources besides the Pr^3+ ion moment that contributes to the static local field distribution at the Cu nuclei. The work at UCLA is supported at UCLA by NSF Grants DMR-0334869 (WGC) and 0203806 (SEB).
We report measurements of the irreversibility field (Hirf) in single crystals of the electron-dop... more We report measurements of the irreversibility field (Hirf) in single crystals of the electron-doped high-Tc superconductor (HTSC) Pr2-xCexCuO4-y (x = 0.15 and 0.17) with an applied magnetic field (B0) up to 28 T, using the method of the shift in a nuclear magnetic resonance (NMR) probe circuit resonance frequency (f) caused by the susceptibility of the sample. It is observed that Hirf is highly anisotropic, and that as the temperature T -> 0 the upper critical field [Hc2, c(T -> 0)] at B0 c is far less than the Pauli limit and very different from that at B0 c. A phase diagram that involves the vortex solid and/or vortex liquid states depending on the alignment of B0 relative to the lattice c-axis is proposed, and the obtained anisotropic Hc2 character along with the evaluated zero T coherence length [ξab(c)(T -> 0)] and penetration depth [λab(c)( T -> 0)] at B0 ab(c) is compared with that of hole-doped HTSCs. This work is supported at UCLA by NSF Grants DMR-0334869 (WGC) and 0520552 (SEB), at U. Maryland by 0352735 (RLG), and NHMFL by 0084173 and the State of Florida.
We report hysteresis in flux lattice (FL) annealing by an rf magnetic field for the cuprate super... more We report hysteresis in flux lattice (FL) annealing by an rf magnetic field for the cuprate superconductor Pr1.85Ce0.15CuO4-y. A strained FL is formed by placing the sample in the coil (inductance L) of a tuned NMR probe circuit and rotating the coil and sample in a fixed B at low temperature. A series of rf pulses in the coil generates an alternating magnetic field that anneals the FL. It decreases the sample's rf skin depth, which reduces L and increases the resonant frequency (f) of the probe. Rotation in small steps about an axis parallel to the CuO planes and perpendicular to B with annealing after each step generates a peak in f whose shape is the same for rotation in both directions (no rotation hysteresis) and is a maximum for B parallel to the CuO planes. When rotation is done in both directions without annealing, the substantial hysteresis reported here occurs. This work was supported at UCLA by NSF Grants DMR-0334869 and DMR-0804625, and at U. Maryland by NSF Grant DMR- 0653535.
A strained flux lattice (FL) in a superconductor (SC) can be annealed to a lower free energy by t... more A strained flux lattice (FL) in a superconductor (SC) can be annealed to a lower free energy by the RF-field used to generate an NMR spin-echo signal [W.G. Clark et al., J. Phys. IV Proceedings 9, Pr10-49-52 (1999)]. Here, we report the change in the rf magnetic susceptibility (chi) in the SC phase of a single crystal of Pr1.85Ce0.15CuO4-y with a strained FL by successive RIFLA pulses. The distorted FL is prepared by rotating the sample through a small angle in a magnetic field well below the SC transition temperature, leaving the FL pinned in a non-equilibrium configuration. RIFLA pulses then reduce chi, which is measured by the change in the inductance of a coil surrounding the sample using an NMR probe and spectrometer. The interpretation is that shaking the FL by the RF pulses progressively anneals it to a lower free energy configuration, which has a smaller chi. These measurements provide a very sensitive measure of the RIFLA effect. Work at UCLA was supported by NSF Grants DMR-0334869 (WGC) and DMR-0520552 (SEB).
G-Quadruplexes (GQs) found within the promoter regions of genes are known to mostly act as repres... more G-Quadruplexes (GQs) found within the promoter regions of genes are known to mostly act as repressors of transcription. Here we report a guanosine (G)-rich segment in the 3'-proximal promoter region of human tyrosine hydroxylase (TH), which acts as a necessary element for transcription. Tyrosine hydroxylase catalyzes the rate-limiting step in the catecholamine biosynthesis and is linked to several common neurological disorders such as Parkinson's and schizophrenia. A 45 nucleotide (nt) sequence (wtTH49) within the human TH promoter contains multiple G-stretches that are extremely well conserved among the primates but deviate in rodents, which raises the possibility of variation in the GQ structures formed in the two orders with the potential for a distinctive functional outcome. Biochemical and biophysical studies, including single-molecule Förster resonance energy transfer, indicate that the wtTH49 sequence can adopt multiple GQ structures by using different combinations of...
G-Quadruplexes (GQs) found within the promoter regions of genes are known to mostly act as repres... more G-Quadruplexes (GQs) found within the promoter regions of genes are known to mostly act as repressors of transcription. Here we report a guanosine (G)-rich segment in the 3'-proximal promoter region of human tyrosine hydroxylase (TH), which acts as a necessary element for transcription. Tyrosine hydroxylase catalyzes the rate-limiting step in the catecholamine biosynthesis and is linked to several common neurological disorders such as Parkinson's and schizophrenia. A 45 nucleotide (nt) sequence (wtTH49) within the human TH promoter contains multiple G-stretches that are extremely well conserved among the primates but deviate in rodents, which raises the possibility of variation in the GQ structures formed in the two orders with the potential for a distinctive functional outcome. Biochemical and biophysical studies, including single-molecule Förster resonance energy transfer, indicate that the wtTH49 sequence can adopt multiple GQ structures by using different combinations of G-stretches. A functional assay performed with 2.8 kb of the 3'-proximal end of the TH promoter and a mutated version (TH49fm; mutated wtTH49) that is unable to form any GQ structure indicates that overall the GQ-enabling wtTH49 sequence is functionally necessary and enhances human TH promoter activity by 5-fold compared to that of the mutant. Two additional mutants, each of which was designed to form distinct GQs, differentially affected reporter gene transcription. A cationic porphyrin TMPyP4 destabilizes the wtTH49 GQ and lowers the level of reporter gene expression, although its analogue, TMPyP2, fails to elicit any response. The 45 nt G-rich sequence within the human TH promoter can form multiple GQ structures, is a necessary element in transcription, and depending on the utilized combination of G-stretches affects transcription in different ways.
A strained flux lattice (FL) in a superconductor (SC) can be annealed to a lower free energy by t... more A strained flux lattice (FL) in a superconductor (SC) can be annealed to a lower free energy by the RF-field used to generate an NMR spin-echo signal [W.G. Clark et al., J. Phys. IV Proceedings 9, Pr10-49-52 (1999)]. Here, this effect is reported for the SC phase of Pr1.85Ce0.15CuO4-y. The distorted FL is prepared by rotating the sample through a small angle in a magnetic field well below the transition temperature, which leaves the FL pinned in a non-equilibrium configuration. Then, the first spin echo has almost no amplitude. Subsequent echoes have a progressively larger amplitude until a final, steady value is reached. The interpretation is that shaking of the distorted vortex lattice by the RF pulses progressively anneals it to a lower free energy configuration. The corresponding change in the local field between the de-phasing and re-phasing pulse for each echo then reduces the height of the echo.
INTRODUCTIONFIONA, short for fluorescence imaging with one-nanometer accuracy, is a simple method... more INTRODUCTIONFIONA, short for fluorescence imaging with one-nanometer accuracy, is a simple method for achieving localization of single (or single groups of) fluorophores with nanometer accuracy in the xy plane. This protocol provides details on constructing an inexpensive sample chamber for use in single-molecule FIONA experiments and two methods for cleaning slides and coverslips.
INTRODUCTIONFIONA, short for fluorescence imaging with one-nanometer accuracy, is a simple method... more INTRODUCTIONFIONA, short for fluorescence imaging with one-nanometer accuracy, is a simple method for achieving localization of single (or single groups of) fluorophores with nanometer accuracy in the xy plane. Data analysis of a FIONA experiment requires the use of several software programs. Data must be acquired and exported into a proper format before analysis can take place. This article describes various options for data analysis.
INTRODUCTIONFIONA, short for fluorescence imaging with one-nanometer accuracy, is a simple method... more INTRODUCTIONFIONA, short for fluorescence imaging with one-nanometer accuracy, is a simple method for achieving localization of single (or single groups of) fluorophores with nanometer accuracy in the xy plane. This article describes procedures for setting up the equipment necessary for FIONA and achieving total internal reflection (TIR).
We have probed, for the first time, the density of states (DOS) near the Fermi level (E_F) in the... more We have probed, for the first time, the density of states (DOS) near the Fermi level (E_F) in the normal state of the electron-doped cuprate superconductor Pr_2-xCe_xCuO_4-delta (PCCO) (0.1 < x < 0.2), using tunneling spectroscopy. Such experiments were possible for PCCO since the critical field parallel to the c-axis is about 10 tesla at 1.5 K. A magnetic field was applied parallel to the c-axis to obtain the normal state and tunneling spectra were recorded using either point contact or vacuum tunnel junctions in a temperature range of 1.5 K to 30 K. The normal-state spectra for the overdoped samples show a relatively flat DOS with a small dip at zero bias. This feature deepens as the value of x is reduced and for the underdoped samples a gap-like feature is observed at E_F. These results will be discussed in the context of the varying effects of electron-electron interactions and localization in the normal state of PCCO. Supported by NSF DMR 97-32736
Recent studies of the electron-doped high-temperature superconductor Pr2-xCexCuO4-y (PCCO) show v... more Recent studies of the electron-doped high-temperature superconductor Pr2-xCexCuO4-y (PCCO) show very unusual physical properties at the doping level x=0.17. Transport experiments are consistent with the existence of a quantum critical point in the normal state. Within the superconducting state, specific heat measurements in a magnetic field are interpreted as evidence for a transition in superconducting order parameter symmetry. We report the ^ 63,65 Cu-NMR spectrum and spin dynamics of PCCO single crystals with x=0.17 and contrast their properties with crystals of x=0.15 . The anisotropic Knight shifts are dominated by the Pr^3+ moments and change little between the samples. The spin lattice relaxation rates are affected by the doping, as are the linewidths at low temperatures. ^*This work is supported at UCLA by NSF Grants DMR-0334869 (WGC) and 0203806 (SEB).
RECQ5 is one of five members of the RecQ family of helicases in humans, which include RECQ1, Bloo... more RECQ5 is one of five members of the RecQ family of helicases in humans, which include RECQ1, Bloom (BLM), Werner (WRN), RECQ4, and RECQ5. Both WRN and BLM have been shown to resolve G-quadruplex (GQ) structures. Deficiencies in unfolding GQ are known to result in DNA breaks and genomic instability, which are prominent in Werner and Bloom syndromes. RECQ5 is significant in suppressing sister chromatid exchanges during homologous recombination but its GQ unfolding activity are not known. We performed single-molecule studies under different salt (50-150 mM KCl or NaCl) and ATP concentrations on different GQ constructs including human telomeric GQ (with different overhangs and polarities) and GQ formed by thrombin-binding aptamer to investigate this activity. These studies demonstrated a RECQ5-mediated GQ unfolding activity that was an order of magnitude weaker than BLM and WRN. On the other hand, BLM and RECQ5 demonstrated similar single-stranded DNA (ssDNA) reeling activities that wer...
Recent charge and thermal transport measurements on the electron- doped high-Tc superconductor Pr... more Recent charge and thermal transport measurements on the electron- doped high-Tc superconductor Pr2-xCexCuO4-y (PCCO) show unusual behavior in the superconducting dome at the doping level going from x = 0.15 (optimally doped) to 0.17 (overdoped), such as quantum phase transition and electron paring symmetry changes, suggesting a possible connection between the superconductivity and other competing phases like antiferromagnetism. We report the ^63,65Cu-NMR spectrum in PCCO single crystals with x = 0.17 in a high magnetic field B0, and contrast their properties with those of x = 0.15. The temperature dependence of the ^63,65Cu-NMR Knight shift is dominated by the negative isotropic hyperfine coupling to the field-induced Pr^3+ ion moment, and changes little between x = 0.15 and 0.17. The ^63,65Cu-NMR linewidths are propotional to the applied magnetic field B0, but affected by the doping, indicating the existence of other sources besides the Pr^3+ ion moment that contributes to the static local field distribution at the Cu nuclei. The work at UCLA is supported at UCLA by NSF Grants DMR-0334869 (WGC) and 0203806 (SEB).
We report measurements of the irreversibility field (Hirf) in single crystals of the electron-dop... more We report measurements of the irreversibility field (Hirf) in single crystals of the electron-doped high-Tc superconductor (HTSC) Pr2-xCexCuO4-y (x = 0.15 and 0.17) with an applied magnetic field (B0) up to 28 T, using the method of the shift in a nuclear magnetic resonance (NMR) probe circuit resonance frequency (f) caused by the susceptibility of the sample. It is observed that Hirf is highly anisotropic, and that as the temperature T -> 0 the upper critical field [Hc2, c(T -> 0)] at B0 c is far less than the Pauli limit and very different from that at B0 c. A phase diagram that involves the vortex solid and/or vortex liquid states depending on the alignment of B0 relative to the lattice c-axis is proposed, and the obtained anisotropic Hc2 character along with the evaluated zero T coherence length [ξab(c)(T -> 0)] and penetration depth [λab(c)( T -> 0)] at B0 ab(c) is compared with that of hole-doped HTSCs. This work is supported at UCLA by NSF Grants DMR-0334869 (WGC) and 0520552 (SEB), at U. Maryland by 0352735 (RLG), and NHMFL by 0084173 and the State of Florida.
We report hysteresis in flux lattice (FL) annealing by an rf magnetic field for the cuprate super... more We report hysteresis in flux lattice (FL) annealing by an rf magnetic field for the cuprate superconductor Pr1.85Ce0.15CuO4-y. A strained FL is formed by placing the sample in the coil (inductance L) of a tuned NMR probe circuit and rotating the coil and sample in a fixed B at low temperature. A series of rf pulses in the coil generates an alternating magnetic field that anneals the FL. It decreases the sample's rf skin depth, which reduces L and increases the resonant frequency (f) of the probe. Rotation in small steps about an axis parallel to the CuO planes and perpendicular to B with annealing after each step generates a peak in f whose shape is the same for rotation in both directions (no rotation hysteresis) and is a maximum for B parallel to the CuO planes. When rotation is done in both directions without annealing, the substantial hysteresis reported here occurs. This work was supported at UCLA by NSF Grants DMR-0334869 and DMR-0804625, and at U. Maryland by NSF Grant DMR- 0653535.
A strained flux lattice (FL) in a superconductor (SC) can be annealed to a lower free energy by t... more A strained flux lattice (FL) in a superconductor (SC) can be annealed to a lower free energy by the RF-field used to generate an NMR spin-echo signal [W.G. Clark et al., J. Phys. IV Proceedings 9, Pr10-49-52 (1999)]. Here, we report the change in the rf magnetic susceptibility (chi) in the SC phase of a single crystal of Pr1.85Ce0.15CuO4-y with a strained FL by successive RIFLA pulses. The distorted FL is prepared by rotating the sample through a small angle in a magnetic field well below the SC transition temperature, leaving the FL pinned in a non-equilibrium configuration. RIFLA pulses then reduce chi, which is measured by the change in the inductance of a coil surrounding the sample using an NMR probe and spectrometer. The interpretation is that shaking the FL by the RF pulses progressively anneals it to a lower free energy configuration, which has a smaller chi. These measurements provide a very sensitive measure of the RIFLA effect. Work at UCLA was supported by NSF Grants DMR-0334869 (WGC) and DMR-0520552 (SEB).
G-Quadruplexes (GQs) found within the promoter regions of genes are known to mostly act as repres... more G-Quadruplexes (GQs) found within the promoter regions of genes are known to mostly act as repressors of transcription. Here we report a guanosine (G)-rich segment in the 3'-proximal promoter region of human tyrosine hydroxylase (TH), which acts as a necessary element for transcription. Tyrosine hydroxylase catalyzes the rate-limiting step in the catecholamine biosynthesis and is linked to several common neurological disorders such as Parkinson's and schizophrenia. A 45 nucleotide (nt) sequence (wtTH49) within the human TH promoter contains multiple G-stretches that are extremely well conserved among the primates but deviate in rodents, which raises the possibility of variation in the GQ structures formed in the two orders with the potential for a distinctive functional outcome. Biochemical and biophysical studies, including single-molecule Förster resonance energy transfer, indicate that the wtTH49 sequence can adopt multiple GQ structures by using different combinations of...
G-Quadruplexes (GQs) found within the promoter regions of genes are known to mostly act as repres... more G-Quadruplexes (GQs) found within the promoter regions of genes are known to mostly act as repressors of transcription. Here we report a guanosine (G)-rich segment in the 3&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;-proximal promoter region of human tyrosine hydroxylase (TH), which acts as a necessary element for transcription. Tyrosine hydroxylase catalyzes the rate-limiting step in the catecholamine biosynthesis and is linked to several common neurological disorders such as Parkinson&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s and schizophrenia. A 45 nucleotide (nt) sequence (wtTH49) within the human TH promoter contains multiple G-stretches that are extremely well conserved among the primates but deviate in rodents, which raises the possibility of variation in the GQ structures formed in the two orders with the potential for a distinctive functional outcome. Biochemical and biophysical studies, including single-molecule Förster resonance energy transfer, indicate that the wtTH49 sequence can adopt multiple GQ structures by using different combinations of G-stretches. A functional assay performed with 2.8 kb of the 3&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;-proximal end of the TH promoter and a mutated version (TH49fm; mutated wtTH49) that is unable to form any GQ structure indicates that overall the GQ-enabling wtTH49 sequence is functionally necessary and enhances human TH promoter activity by 5-fold compared to that of the mutant. Two additional mutants, each of which was designed to form distinct GQs, differentially affected reporter gene transcription. A cationic porphyrin TMPyP4 destabilizes the wtTH49 GQ and lowers the level of reporter gene expression, although its analogue, TMPyP2, fails to elicit any response. The 45 nt G-rich sequence within the human TH promoter can form multiple GQ structures, is a necessary element in transcription, and depending on the utilized combination of G-stretches affects transcription in different ways.
A strained flux lattice (FL) in a superconductor (SC) can be annealed to a lower free energy by t... more A strained flux lattice (FL) in a superconductor (SC) can be annealed to a lower free energy by the RF-field used to generate an NMR spin-echo signal [W.G. Clark et al., J. Phys. IV Proceedings 9, Pr10-49-52 (1999)]. Here, this effect is reported for the SC phase of Pr1.85Ce0.15CuO4-y. The distorted FL is prepared by rotating the sample through a small angle in a magnetic field well below the transition temperature, which leaves the FL pinned in a non-equilibrium configuration. Then, the first spin echo has almost no amplitude. Subsequent echoes have a progressively larger amplitude until a final, steady value is reached. The interpretation is that shaking of the distorted vortex lattice by the RF pulses progressively anneals it to a lower free energy configuration. The corresponding change in the local field between the de-phasing and re-phasing pulse for each echo then reduces the height of the echo.
INTRODUCTIONFIONA, short for fluorescence imaging with one-nanometer accuracy, is a simple method... more INTRODUCTIONFIONA, short for fluorescence imaging with one-nanometer accuracy, is a simple method for achieving localization of single (or single groups of) fluorophores with nanometer accuracy in the xy plane. This protocol provides details on constructing an inexpensive sample chamber for use in single-molecule FIONA experiments and two methods for cleaning slides and coverslips.
INTRODUCTIONFIONA, short for fluorescence imaging with one-nanometer accuracy, is a simple method... more INTRODUCTIONFIONA, short for fluorescence imaging with one-nanometer accuracy, is a simple method for achieving localization of single (or single groups of) fluorophores with nanometer accuracy in the xy plane. Data analysis of a FIONA experiment requires the use of several software programs. Data must be acquired and exported into a proper format before analysis can take place. This article describes various options for data analysis.
INTRODUCTIONFIONA, short for fluorescence imaging with one-nanometer accuracy, is a simple method... more INTRODUCTIONFIONA, short for fluorescence imaging with one-nanometer accuracy, is a simple method for achieving localization of single (or single groups of) fluorophores with nanometer accuracy in the xy plane. This article describes procedures for setting up the equipment necessary for FIONA and achieving total internal reflection (TIR).
We have probed, for the first time, the density of states (DOS) near the Fermi level (E_F) in the... more We have probed, for the first time, the density of states (DOS) near the Fermi level (E_F) in the normal state of the electron-doped cuprate superconductor Pr_2-xCe_xCuO_4-delta (PCCO) (0.1 < x < 0.2), using tunneling spectroscopy. Such experiments were possible for PCCO since the critical field parallel to the c-axis is about 10 tesla at 1.5 K. A magnetic field was applied parallel to the c-axis to obtain the normal state and tunneling spectra were recorded using either point contact or vacuum tunnel junctions in a temperature range of 1.5 K to 30 K. The normal-state spectra for the overdoped samples show a relatively flat DOS with a small dip at zero bias. This feature deepens as the value of x is reduced and for the underdoped samples a gap-like feature is observed at E_F. These results will be discussed in the context of the varying effects of electron-electron interactions and localization in the normal state of PCCO. Supported by NSF DMR 97-32736
Recent studies of the electron-doped high-temperature superconductor Pr2-xCexCuO4-y (PCCO) show v... more Recent studies of the electron-doped high-temperature superconductor Pr2-xCexCuO4-y (PCCO) show very unusual physical properties at the doping level x=0.17. Transport experiments are consistent with the existence of a quantum critical point in the normal state. Within the superconducting state, specific heat measurements in a magnetic field are interpreted as evidence for a transition in superconducting order parameter symmetry. We report the ^ 63,65 Cu-NMR spectrum and spin dynamics of PCCO single crystals with x=0.17 and contrast their properties with crystals of x=0.15 . The anisotropic Knight shifts are dominated by the Pr^3+ moments and change little between the samples. The spin lattice relaxation rates are affected by the doping, as are the linewidths at low temperatures. ^*This work is supported at UCLA by NSF Grants DMR-0334869 (WGC) and 0203806 (SEB).
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