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H Lederer

Publications and source records attributed to H Lederer.

15 recordsLinked to original sources
PubMed1992

Domain structure of the human immunodeficiency virus reverse transcriptase.

The spatial arrangement of subunits p51 and p66 of the HIV-1 reverse transcriptase and the position of the RNase H containing domain, p15, have been determined by means of neutron small-angle scattering. The reverse transcriptase (p66/p51) is a flat molecule, which can be approximated by an ellipsoid with the half axes of 5.2 nm, 4.8 nm and 1.4 nm. The two subunits p51 and p66 having a centre-to-centre distance of 3.3 +/- 0.3 nm are attached at their flat sides, slightly shifted sideways. The p15 domain is located at the long axis of the ellipsoidal reverse transcriptase having a distance of 5.0 +/- 0.5 nm to the centre of the p51d domain, which is part of the p66 subunit, and a distance of 5.3 +/- 1.2 nm to the centre of the neighbouring p51s subunit.

Chromatography, Affinity↗
PubMed1991

Spatial arrangement of sigma-factor and core enzyme of Escherichia coli RNA polymerase. A neutron solution scattering study.

By means of neutron solution scattering we determined the position and orientation of core enzyme and sigma-factor within the Escherichia coli RNA polymerase holoenzyme with the aim of improving existing models. The individual components, core enzyme (E) and sigma-factor (sigma), were highlighted by deuterium labeling and their center-to-center distances determined in the monomeric and the dimeric holoenzyme. The following distance parameters were obtained: dE1-sigma 1 = 8.6(+/- 1) nm, dE1-E2 = 11.5(+/- 1) nm, d sigma 1-sigma 2 = 12.0(+/- 0.7) nm, dE1-sigma 2 = 9(+/- 3) nm. Using a triangulation procedure the position of the sigma-factors, sigma 1 and sigma 2, were determined with respect to the mass center of the core enzyme molecules, E1 and E2, assuming a symmetrical arrangement of the holoenzyme molecules in the dimer (C2 symmetry). In addition, the orientation of the sigma-factor with respect to core enzyme was estimated by means of model calculations. The obtained model of holoenzyme depicts the sigma-factor as buried in a groove of core enzyme, probably between the large subunits beta' and beta.

DNA-Directed RNA Polymerases↗
PubMed1990

Topography of intermediates in transcription initiation of E.coli.

Three characteristic footprinting patterns resulted from probing the Escherichia coli RNA polymerase T7 A1 promoter complex by hydroxyl radicals in the temperature range between 4 degrees C and 37 degrees C. These were attributed to the closed complex, the intermediate complex and the open complex. In the closed complex, the RNA polymerase protects the DNA only at one side over five helical turns. In the intermediate complex, the range of the protected area is extended further downstream by two helical turns. This region of the DNA helix is fully protected, indicating that the RNA polymerase wraps around the DNA between base positions -13 and +20. In the open complex, a stretch between base positions -7 and +2, which was fully protected in the intermediate complex, becomes accessible towards hydroxyl radicals but only in the codogenic strand, indicating that the DNA strands are unwound. Our data suggest that only the DNA downstream of the promoter is involved in this unwinding process.

DNA, Viral↗
PubMed1989

The quaternary structure of Tet repressors bound to the Tn10-encoded tet gene control region determined by neutron solution scattering.

The spatial arrangement of Tet repressor dimer, both free and in complex with an 80 bp DNA fragment spanning the wild-type Tn10-encoded tet transcriptional control sequence containing a tandem repeat of two operators, has been determined by neutron small-angle scattering. The active, free Tet repressor dimer is an elongated and flat molecule with a maximum dimension of 11 +/- 1.5 mm which can be approximated by an ellipsoid with the half-axes 6 nm, 2.5 nm and 1 nm. The overall conformation undergoes no detectable change when the repressor dimer is bound to a DNA fragment containing a single tet operator. The normal distance between the centre of gravity of the protein and the DNA axis is 3.0 +/- 0.1 nm, indicating that the repressor dimer is mainly located on one side of the DNA. When bound to the wild type tet control DNA, the two repressor dimers have a centre-to-centre distance of 11.0 +/- 0.5 nm. Their minimal distance is 5 +/- 2 nm. Protein-protein contacts via loop formation of the DNA by repressor binding is excluded. The repressors are well separated and have no direct contact. A model is proposed where the two repressor dimers are located on opposite sides of the DNA and the DNA is not strongly bent in the complex.

Base Sequence↗
PubMed1988

Spatial arrangement of DNA-dependent RNA polymerase of Escherichia coli and DNA in the specific complex. A neutron small angle scattering study.

In this paper we demonstrate that neutron small angle scattering is a suitable method to study the spatial arrangement of large specific protein-DNA complexes. We studied the complex of DNA-dependent RNA polymerase of Escherichia coli and a 130 base-pair DNA fragment containing the strong promoter A1 of bacteriophage T7. Contrast variation of the complex with deuterium allowed us to "visualize" either RNA polymerase, or DNA, or both components in situ. From the corresponding scattering curves information was derived about: (1) Conformational changes of RNA polymerase and DNA by complex formation: comparison of the scattering profiles of the isolated and complexed components showed that by specific complex formation the cross-section of RNA polymerase decreases, while the DNA fragment does not undergo a gross conformational change. (2) The spatial arrangement of RNA polymerase and DNA in the specific complex from the cross-sectional radii of gyration of the complex the normal distance dn between the centre of gravity of the RNA polymerase and the axis of the DNA fragment was derived as 5.0 (+/- 0.3) nm. On the basis of these and footprinting data a low resolution model of the RNA polymerase-promoter complex is proposed. The main feature of this model is the positioning of RNA polymerase to only one side of the DNA.

DNA, Bacterial↗
PubMed1987

Large-scale preparation of a DNA fragment containing the strong promoter A1 of the phage T7.

A procedure has been developed to isolate DNA fragments on a large scale. A DNA fragment of 130 base-pairs containing the strong promoter A1 of the phage T7 was purified to homogeneity in amounts of 10 mg. The procedure includes the rapid purification of gram amounts of plasmid DNA, a new, simple method to separate small DNA fragments from the vector by a phenol/water partitioning system, and a liquid-liquid PEG-dextran partition chromatography for the final purification of the fragment. The fragment was cloned in two vector systems: The vector pDS1, to1+ (1), containing an efficient terminator downstream from the promoter integration site, gives high yields, 3-4 mg plasmid DNA per liter medium. In the plasmid pWH802 (2), which is not specially designed for the amplification of a strong promoter, the integration of the promoter was possible but the yield decreased by a factor of about 50. The stability of the inserts was tested in both systems. Monomeric inserts were stable in both plasmids, multimeric inserts up to a tetramer were only stable in pWH802. Only one orientation of the fragment was found.

Biotechnology↗
PubMed1986

Solution structure of a short DNA fragment studied by neutron scattering.

The solution structure of a DNA fragment of 130 base pairs and known sequence has been investigated by neutron small-angle scattering. In 0.1 M NaCl, the overall structure of the DNA fragment which contains the strong promoter A1 of the Escherichia coli phage T7 agrees with that expected for B-DNA. The neutron scattering curve is well fitted by that of a rigid rod with a length of 44 nm and a diameter of 2 nm. The results were confirmed by quasi-elastic light scattering and analytical centrifugation. The neutron measurements in H2O and D2O buffer reveal a cross-sectional inhomogeneity not detected by X-ray small-angle scattering. This inhomogeneity is caused by the hydration layer around the DNA core and not by the helical structure. The primary solvent shell has a density increased by at least 4-9% compared to bulk water.

Base Composition↗
PubMed1986

Deuterium incorporation into Escherichia coli proteins. A neutron-scattering study of DNA-dependent RNA polymerase.

Neutron small-angle scattering studies of single protein subunits in a protein-DNA complex require the adjustment of the neutron scattering-length densities of protein and DNA, which is attainable by specific deuteration of the protein. The neutron scattering densities of unlabelled DNA and DNA-dependent RNA polymerase of Escherichia coli match when RNA polymerase is isolated from cells grown in a medium containing 46% D2O and unlabelled glucose as carbon source. Their contrasts vanish simultaneously in a dialysis buffer containing 65% D2O. An expression was evaluated which allows the calculation of the degree of deuteration and match point of any E. coli protein from the D2O content of the growth medium, taking the 2H incorporation into RNA polymerase amino acids to be representative for all amino acids in E. coli proteins. The small-angle scattering results, on which the calculation of the degree of deuteration is based, were confirmed by mass spectrometric measurements.

DNA-Directed RNA Polymerases↗
PubMed1977

[EEG changes in the course of progressive cerebral disorders in children (author's transl)].

This survey presents EEG-data on the following disorders: Globoid-cell leucodystrophy (Krabbe), Metachromatic Leukodystrophy, GM2 Gangliosidosis I (Tay-Sachs), Neuronal Ceroidlipofuscinosis. During Krabbe's disease the background activity of the EEG slows down, multifocal and generalized hypersynchroneous activity (HSA) appears, in the final stage hypsarrythmia is found occasionally. In contrast HSA during Sulfatide lipidosis appears only in the final stages. The EEG changes in Tay Sachs disease are minimal in the beginning; later only high amplitude slowing and especially focal abnormalities with spikes and sharp-waves will appear and in advanced stages there is generalized depression of amplitudes. The Elektroretinogramm (ERG) remains intact but VEP disappear. In contrast, in Neuronal Ceroidlipofuscinosis irregular spikes and sharp-waves can be found shortly after onset of the disease. Later on there is a generalized depression of amplitudes; photic driving and differences between sleeping and waking diminish from the 3rd year of life on; thereafter, the EEG becomes isoelectric. The ERG is extinguished from the very beginning in infantile, lateinfantile and juvenile type of Ceroid-Lipofuscinosis.

Brain Diseases, Metabolic↗
PubMed1973

Effects of observer variation in population screening for cervical carcinoma.

A stratified random sample of 100 cervical smears was taken from routine population screening material and graded by three cytological laboratories. The sensitivity of the smear test for detecting cervical malignancies varied from 72 to 88%-a difference of 16%; for all serve epithelial abnormalities the sensitivity varied from 52 to 71% (a difference of 19%). The histological material pertaining to these smears was re-examined ;blindly' and then sent for diagnosis to a second pathologist. Using each set of histodiagnoses as reference, the sensitivity of the test for detecting cervical malignancies varied from 77 to 85%-a difference of 8%; the sensitivity for all epithelial abnormalities varied from 62 to 76% (a difference of 14%). The significance of these observer variations on the efficacy of screening for cervical carcinoma is discussed.

Carcinoma↗