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.     

On the nature of the iron sulfur cluster in a deuterated algal ferredoxin.

A protonated and a completely deuterated two-iron algal ferredoxin from Synechococcus lividus have been studied by optical, electron paramagnetic resonance, electron-nuclear double resonance, proton magnetic resonance and Mossbauer spectroscopies; temperature dependent magnetic susceptibility measurements are reported as well. These studies have confirmed the electron localized model of the active center in the two-iron ferredoxins, as previously deduced from studies of spinach ferredoxin, have yielded much more precise spectroscopic parameters for this center, and have thus greatly increased the confidence in this model.     

Ionic base radicals in gamma-irradiated oriented non-deuterated and fully deuterated DNA.

The free radicals induced by gamma-irradiation at 77 K in non-deuterated and fully deuterated oriented samples of moist algal DNA have been studied by electron paramagnetic resonance (e.p.r.). The e.p.r. spectra from the non-deuterated sample were found to be similar to those from calf-thymus DNA studied previously. Numerical spectra simulations were performed based on the previously proposed interpretation of the DNA spectra at 77 K in terms of a mixture of two spectral components arising from anionic and cationic base radicals (probably on thymine and guanine, respectively). The simulations were found to account satisfactorily for the main characteristics of the e.p.r. spectra from both the non-deuterated and the fully deuterated algal DNA sample.     

The induction of aneuploidy by nitrogen mustard in a human lymphoblastoid cell line

We report that the presence of an extra Y chromosome can be used as a marker for the induction of aneuploidy (mitotic non-disjunction) in a human lymphoblastoid cell line. This endpoint is easily visualized in metaphase chromosome preparations after staining with quinacrine mustard. The induction of cells with two Y chromosomes by nitrogen mustard (NM) was examined. Exposure to 150 ng/ml nitrogen mustard induced a 6-fold increase in aneuploid frequency relative to untreated control levels; maximal induction of aneuploidy was observed 2 days after treatment. Lower concentrations of nitrogen mustard (36 and 75 ng/ml) induced smaller increases in aneuploid frequency, with maximal induction observed 1 day after treatment. This system has the potential to be used as an assay for the induction of aneuploidy in cultured human cells.     

Resonance raman spectroscopy of chemically modified and isotopically labelled purple membranes. I. A critical examination of the carbon-nitrogen vibrational modes

Resonance Raman spectra of bacteriorhodopsin are compared to the spectra of this protein modified in the following ways: (1) selective deuteration at the C-15 carbon atom of retinal, (2) full deuteration of the retinal, (3) the addition of a conjugated double bond in the β-ionone ring (3-dehydroretinal), (4) full deuteration of the protein and lipid components, (5) ¹⁵N enrichment of the entire membrane and (6) deuteration of the entire membrane (including the retinal). A detailed comparison of the ¹⁵N-enriched membrane and naturally occurring purple membrane from 800 cm^?1 to 1700 cm^?1 reveals that ¹⁵N enrichment affects the frequency of only two vibrational modes. These occur at 1642 cm^?1 and 1620 cm^?1 in naturally occurring purple membrane and at 1628 cm^?1 and 1615 cm^?1 in the ¹⁵N-enriched samples. Therefore, this pair of bands reflects the states of protonation of the Schiff base. However, our data also indicate that neither of these modes are simple, localized $\text{C}\text{=}\textcolor{red}{}\text{?}\text{H}$ or C=N stretching vibrations. In the case of the 1642 cm^?1 band motions of the retinal chain beyond C-15 are not significantly involved. On the other hand, in the 1620 cm^?1 band atomic motions in the isoprenoid chain beyond C-15 are involved.     

Sucrose Synthase Expression during Cold Acclimation in Wheat

When wheat (Triticum aestivum) seedlings are exposed to a cold temperature (2-4°C) above 0°C, sucrose accumulates and sucrose synthase activity increases. The effect of a cold period on the level of sucrose synthase (SS) was investigated. Using antibodies against wheat germ SS, Western blots studies showed that the amount of the SS peptide increased during 14 days in the cold, when plants were moved from 23°C to 4°C. The level of SS diminished when plants were moved back to 23°C. Northern blots of poly(A)⁺ RNA, confirmed a five- to sixfold induction of SS in wheat leaves during cold acclimation. These results indicate that SS is involved in the plant response to a chilling stress.     

Tomato yellow leaf curl virus from Sardinia is a whitefly-transmitted monopartite geminivirus.

The genome of an isolate of tomato yellow leaf curl virus from Sardinia, Italy (TYLCV-S), a geminivirus transmitted by the whitefly Bemisia tabaci, has been cloned and sequenced. The single circular DNA molecule comprises 2770 nucleotides. Genome organisation closely resembles that of the DNA A component of the whitefly-transmitted geminiviruses with a bipartite genome. A 1.8 mer of the TYLCV-S genome in a binary vector of Agrobacterium tumefaciens is infectious upon agroinoculation of tomato plants. Typical tomato yellow leaf curl disease symptoms developed about three weeks after inoculation. The disease was transmitted by the natural vector B.tabaci from agroinfected plants to test plants, reproducing in this way the full biological cycle and proving that the genome of TYLCV-S consists of only one circular single-stranded DNA molecule. Contrary to the other whitefly-transmitted geminiviruses described so far, there is no evidence for the existence nor the necessity of a second component (B DNA) in the TYLCV-S genome.     

Assay for gene mutation in a human lymphoblast line, AHH-1, competent for xenobiotic metabolism

A novel quantitative gene-locus mutation assay has been developed using a line of human lymphoblast cells, designated AHH-1, competent in oxidative xenobiotic metabolism. AHH-1 cells are sensitive to the mutagenic action of both chemically reactive mutagens and mutagens which require oxidative metabolism to exert their mutagenicity. These cells are readily mutated by direct exposure to ethyl methanesulfonate, ICR-191, 2-acetoaminofluorene, aflatoxin B1, benzo[a]pyrene (BP), cyclopenta[c, d]pyrene, dimethylnitrosamine, lasiocarpine, and 1-methylphenanthrene.