A genetic method for dissecting the mechanism of transcriptional activator synergy by identical activators

Pairs of transcriptional activators in prokaryotes have been shown to activate transcription synergistically from promoters with two activator binding sites. In some cases, such synergistic effects result from cooperative binding, but in other cases each DNA-bound activator plays a direct role in the activation process by interacting simultaneously with separate surfaces of RNA polymerase. In such cases, each DNA-bound activator must possess a functional activating region, the surface that mediates the interaction with RNA polymerase. When transcriptional activation depends on two or more identical activators, it is not straightforward to test the requirement of each activator for a functional activating region. Here we describe a method for directing a mutationally altered activator to either one or the other binding site, and we demonstrate the use of this method to examine the mechanism of transcriptional activator synergy by the Escherichia coli cyclic AMP receptor protein (CRP) working at an artificial promoter bearing two CRP-binding sites.

Electrostatic surface plasmon resonance: Direct electric field-induced hybridization and denaturation in monolayer nucleic acid films and label-free discrimination of base mismatches

We demonstrate that in situ optical surface plasmon resonance spectroscopy can be used to monitor hybridization kinetics for unlabeled DNA in tethered monolayer nucleic acid films on gold in the presence of an applied electrostatic field. The dc field can enhance or retard hybridization and can also denature surface-immobilized DNA duplexes. Discrimination between matched and mismatched hybrids is achieved by simple adjustment of the electrode potential. Although the electric field at the interface is extremely large, the tethered single-stranded DNA thiol probes remain bound and can be reused for subsequent hybridization reactions without loss of efficiency. Only capacitive charging currents are drawn; redox reactions are avoided by maintaining the gold electrode potential within the ideally polarizable region. Because of potential-induced changes in the shape of the surface plasmon resonance curve, we account for the full curve rather than simply the shift in the resonance minimum.

Binding of a hairpin polyamide in the minor groove of DNA: sequence-specific enthalpic discrimination.

Hairpin polyamides are synthetic ligands for sequence-specific recognition in the minor groove of double-helical DNA. A thermodynamic characterization of the DNA-binding properties exhibited by a six-ring hairpin polyamide, ImPyPy-gamma-PyPyPy-beta-Dp (where Im = imidazole, Py = pyrrole, gamma = gamma-aminobutyric acid, beta = beta-alanine, and Dp = dimethylaminopropylamide), reveals an approximately 1-2 kcal/mol greater affinity for the designated match site, 5'-TGTTA-3', relative to the single base pair mismatch sites, 5'-TGGTA-3' and 5'-TATTA-3'. The enthalpy and entropy data at 20 degrees C reveal this sequence specificity to be entirely enthalpic in origin. Correlations between the thermodynamic driving forces underlying the sequence specificity exhibited by ImPyPy-gamma-PyPyPy-beta-Dp and the structural properties of the heterodimeric complex of PyPyPy and ImPyPy bound to the minor groove of DNA provide insight into the molecular forces that govern the affinity and specificity of pyrrole-imidazole polyamides.

Real-time detection of DNA hybridization and melting on oligonucleotide arrays by using optical wave guides.

The challenge of the Human Genome Project is to increase the rate of DNA sequence acquisition by two orders of magnitude to complete sequencing of the human genome by the year 2000. The present work describes a rapid detection method using a two-dimensional optical wave guide that allows measurement of real-time binding or melting of a light-scattering label on a DNA array. A particulate label on the target DNA acts as a light-scattering source when illuminated by the evanescent wave of the wave guide and only the label bound to the surface generates a signal. Imaging/visual examination of the scattered light permits interrogation of the entire array simultaneously. Hybridization specificity is equivalent to that obtained with a conventional system using autoradiography. Wave guide melting curves are consistent with those obtained in the liquid phase and single-base discrimination is facile. Dilution experiments showed an apparent lower limit of detection at 0.4 nM oligonucleotide. This performance is comparable to the best currently known fluorescence-based systems. In addition, wave guide detection allows manipulation of hybridization stringency during detection and thereby reduces DNA chip complexity. It is anticipated that this methodology will provide a powerful tool for diagnostic applications that require rapid cost-effective detection of variations from known sequences.

Biogas from MSW landfill: Composition and determination of chlorine content with the AOX (adsorbable organically bound halogens) technique

An exhaustive characterization of the biogas from some waste disposal facilities has been carried out. The analysis includes the main components (methane, carbon dioxide, nitrogen and oxygen) as well as trace components such as hydrogen sulphide, ammonia and VOCs (volatile organic compounds) including siloxanes and halogenated compounds. VOCs were measured by GC/MS (Gas Chromatography/Mass Spectrometry) using two different procedures: thermal desorption of the Tenax TA and Carbotrap 349 tubes and SPME (Solid Phase Micro-Extraction). A method has been established to measure the total halogen content of the biogas with the AOX (adsorbable organically bound halogens) technique. The equipment used to analyze the samples was a Total Organic Halogen Analyzer (TOX-100). Similar results were obtained when comparing the TOX (Total Organic Halogen) values with those obtained by GC/MS. The halogen content in all the samples was under 22 mg Cl/Nm3 which is below the limit of 150 mg/Nm3 proposed in the Spanish Regulations for any use of the biogas. The low chlorine content in the biogas studied, as well as the low content of other trace compounds, makes it suitable for use as a fuel for electricity generating engines.

A synopsis of naturall philosophy according to the method of the ancients, but improved & augmented with the notions of later philosophers : manuscript, 1677-1679

Possibly autograph, dated at end of volume: Finitu[m] mart: 14, 1678/9. Imperfect copy with title page missing; supplied from a MS copy, dated 29 March 1680, now in the Bodleian Library.

The improved practical system of educating the horse : ... Also a treatise on shoeing ... : A statement of their method of training cattle and dogs ... : The rules of Union Course /

Based on Dennis Magner's New system of training horses, Utica, 1863 (Cf. Libr. of Congr. Cat. of printed books)

Geography; or, A description of the world. In three parts. Part I.--Geographical orthography, divided and accented. Part II. A grammar of geography, to be committed to memory. Part III.--A description of the earth...Accompanied with an atlas. To which is added, an esay method of constructing maps,

Imperfect: Atlas lacking.

Observations on the Discourse of natural theology by Henry, lord Brougham; chiefly relating to His Lordship's doctrine of the immateriality of the human mind, as proved by psychological phenomena; the applicability of the inductive method of proof to natural theology, so far as relates to psychological facts; dreams ... and the bearing of the "Discourse" on divine revelation ...

With this is bound the author's Additional observations on the Discourse of natural theology by Henry, lord Brougham. 1835.

Converged quantum calculations of HO2 bound states and resonances for J=6 and 10

Bound and resonance states of HO2 are calculated quantum mechanically using both the Lanczos homogeneous filter diagonalization method and the real Chebyshev filter diagonalization method for nonzero total angular momentum J=6 and 10, using a parallel computing strategy. For bound states, agreement between the two methods is quite satisfactory; for resonances, while the energies are in good agreement, the widths are in general agreement. The quantum nonzero-J specific unimolecular dissociation rates for HO2 are also calculated. (C) 2004 American Institute of Physics.

Iterative quantum computations of HO2 bound states and resonances for J=4 and 5

Bound and resonance states of HO2 have been calculated by both the complex Lanczos homogeneous filter diagonalisation (LHFD) method(1,2) and the real Chebyshev filter diagonalisation method(3,4) for non-zero total angular momentum J = 4 and 5. For bound states, the agreement between the two methods is quite satisfactory; for resonances while the energies are in good agreement, the widths are only in general agreement. The relative performances of the two iterative FD methods have also been discussed in terms of efficiency as well as convergence behaviour for such a computationally challenging problem. A helicity quantum number Ohm assignment (within the helicity conserving approximation) is performed and the results indicate that Coriolis coupling becomes more important as J increases and the helicity conserving approximation is not a good one for the HO2 resonance states.

Unimolecular Rovibrational Bound and Resonance States for Large Angular Momentum: J=20 Calculations for HO2

We explore the calculation of unimolecular bound states and resonances for deep-well species at large angular momentum using a Chebychev filter diagonalization scheme incorporating doubling of the autocorrelation function as presented recently by Neumaier and Mandelshtam [Phys. Rev. Lett. 86, 5031 (2001)]. The method has been employed to compute the challenging J=20 bound and resonance states for the HO2 system. The methodology has firstly been tested for J=2 in comparison with previous calculations, and then extended to J=20 using a parallel computing strategy. The quantum J-specific unimolecular dissociation rates for HO2-> H+O-2 in the energy range from 2.114 to 2.596 eV have been reported for the first time, and comparisons with the results of Troe and co-workers [J. Chem. Phys. 113, 11019 (2000) Phys. Chem. Chem. Phys. 2, 631 (2000)] from statistical adiabatic channel method/classical trajectory calculations have been made. For most of the energies, the reported statistical adiabatic channel method/classical trajectory rate constants agree well with the average of the fluctuating quantum-mechanical rates. Near the dissociation threshold, quantum rates fluctuate more severely, but their average is still in agreement with the statistical adiabatic channel method/classical trajectory results.

Spatial and ontogenetic variation in growth of nursery-bound juvenile lemon sharks, Negaprion brevirostris: a comparison of two age-assigning techniques

We compared growth rates of the lemon shark, Negaprion brevirostris, from Bimini, Bahamas and the Marquesas Keys (MK), Florida using data obtained in a multi-year annual census. We marked new neonate and juvenile sharks with unique electronic identity tags in Bimini and in the MK we tagged neonate and juvenile sharks. Sharks were tagged with tiny, subcutaneous transponders, a type of tagging thought to cause little, if any disruption to normal growth patterns when compared to conventional external tagging. Within the first 2 years of this project, no age data were recorded for sharks caught for the first time in Bimini. Therefore, we applied and tested two methods of age analysis: ( 1) a modified 'minimum convex polygon' method and ( 2) a new age-assigning method, the 'cut-off technique'. The cut-off technique proved to be the more suitable one, enabling us to identify the age of 134 of the 642 previously unknown aged sharks. This maximised the usable growth data included in our analysis. Annual absolute growth rates of juvenile, nursery-bound lemon sharks were almost constant for the two Bimini nurseries and can be best described by a simple linear model ( growth data was only available for age-0 sharks in the MK). Annual absolute growth for age-0 sharks was much greater in the MK than in either the North Sound (NS) and Shark Land (SL) at Bimini. Growth of SL sharks was significantly faster during the first 2 years of life than of the sharks in the NS population. However, in MK, only growth in the first year was considered to be reliably estimated due to low recapture rates. Analyses indicated no significant differences in growth rates between males and females for any area.

Development of an oligonucleotide-based SNP detection method on lateral flow strips using hexapet tages

Detection of point mutations or single nucleotide polymorphisms (SNPs) is important in relation to disease susceptibility or detection in pathogens of mutations determining drug resistance or host range. There is an emergent need for rapid detection methods amenable to point-of-care applications. The purpose of this study was to reduce to practice a novel method for SNP detection and to demonstrate that this technology can be used downstream of nucleic acid amplification. The authors used a model system to develop an oligonucleotide-based SNP detection system on nitrocellulose lateral flow strips. To optimize the assay they used cloned sequences of the herpes simplex virus-1 (HSV-1) DNA polymerase gene into which they introduced a point mutation. The assay system uses chimeric polymerase chain reaction (PCR) primers that incorporate hexameric repeat tags ("hexapet tags"). The chimeric sequences allow capture of amplified products to predefined positions on a lateral flow strip. These "hexapet" sequences have minimal cross-reactivity and allow specific hybridization-based capture of the PCR products at room temperature onto lateral flow strips that have been striped with complementary hexapet tags. The allele-specific amplification was carried out with both mutant and wild-type primer sets present in the PCR mix ("competitive" format). The resulting PCR products carried a hexapet tag that corresponded with either a wild-type or mutant sequence. The lateral flow strips are dropped into the PCR reaction tube, and mutant sequence and wild-type sequences diffuse along the strip and are captured at the corresponding position on the strip. A red line indicative of a positive reaction is visible after 1 minute. Unlike other systems that require separate reactions and strips for each target sequence, this system allows multiplex PCR reactions and multiplex detection on a single strip or other suitable substrates. Unambiguous visual discrimination of a point mutation under room temperature hybridization conditions was achieved with this model system in 10 minutes after PCR. The authors have developed a capture-based hybridization method for the detection and discrimination of HSV-1 DNA polymerase genes that contain a single nucleotide change. It has been demonstrated that the hexapet oligonucleotides can be adapted for hybridization on the lateral flow strip platform for discrimination of SNPs. This is the first step in demonstrating SNP detection on lateral flow using the hexapet oligonucleotide capture system. It is anticipated that this novel system can be widely used in point-of-care settings.

Determination of the orientational distribution and orientation factor for transfer between membrane-bound fluorophores using a confocal microscope

Orientational fluorophores have been a useful tool in physical chemistry, biochemistry, and more recently structural biology due to the polarized nature of the light they emit and that fact that energy can be transferred between them. We present a practical scheme in which measurements of the intensity of emitted fluorescence can be used to determine limits on the mean and distribution of orientation of the absorption transition moment of membrane-bound. uorophores. We demonstrate how information about the orientation of. uorophores can be used to calculate the orientation factor k(2) required for use in FRET spectroscopy. We illustrate the method using images of AlexaFluor probes bound to MscL mechanosensitive transmembrane channel proteins in spherical liposomes.