Binding of platinum-diaminonitroxyl complexes to animal DNA.

Reaction of PtII(DAPO)X2 complexes (where DAPO is trans-3,4-diamino-2,2,6,6-tetramethylpiperidine-1-oxyl, X2 = (NO3)2, oxalato (Ox) or 1,1-cyclobutanedicarboxylato (Cbdca)) with a bovine spleen DNA in 0.01 M NaHCO3 at 37 degrees C for 24 h gives rise to formation of platinated DNA. The [bound PtII(DAPO)]/[nucleotide] ratio (r) depends on the initial ratio of the reagents and on the nature of leaving ligands X. Nitroxyl-nitroxyl distances in platinated DNA determined by the ESR suggest that at r > or = 0.1 PtII(DAPO) fragments are uniformly attached to DNA. But at lower r, the thermal characteristics of modified DNA (melting temperature Tm, melting range width delta T) and the guanine-to-adenine platination degree ratios GPt/APt imply that the nature of leaving ligands X affect the selectivity of DNA platination. At r > or = 0.1, nitroxyl groups can approach each other so close that, in an acidic medium, the electron transfer from one nitroxyl group to another becomes possible, and the nitroxyls readily disproportionate to diamagnetic products. Correlation time of nitroxyl rotation in PtII(DAPO)-DNA adducts is approximately 10(-8) s, which is related to predominantly bifunctional bonding of PtII(DAPO) with DNA. Platination-induced distortion of DNA was evidenced by changes in Tm, delta T and degree of hyperchromicity H. The major part of adducts form the intrastrand cross-links which destabilize the structure of DNA duplex. The interstrand PtII(DAPO) cross-linking (approximately 1% of the adducts) facilitates renaturation of despiralized DNA molecules upon cooling. Two types of PtII(DAPO)-DNA adducts are revealed, which differ substantially in their rates of deplatination with NaCN. ESR, electron spin resonance; r, degree of modification; cisplatin, cis-diamminedichloroplatinum(II); Tm, melting temperature; delta T, melting range width; H, degree of hyperchromicity; R, degree of renaturation; AAS, atomic absorption spectroscopy; HPLC, high performance liquid chromatography.

[Dibromoethylacetate - a new agent for fixing the unwound regions of DNA. Denaturation maps of mitochondrial DNA from the rat liver]. / Dibrométilatsetat - novyi agent dlia fiksatsii despiralizovannykh uchastkov DNK. Denaturatsionnaia karta mitokhondrial'noi DNK pecheni krysy.

On the basis of the reaction of dibromoethylacetate with adenine and cytosine at the uncoiled regions of DNA it was possible to fix these molten regions (the degree of denaturation was about 16%) for rat liver mitochondrial DNA. Modification of all accessible adenine and cytosine residues in 0.14 M Na-acetate, pH 5.65, was completed at 70-80 degrees C in 15-20 min. Using the computer orientation for the set of fixed BamHI-fragments and the linear molecules of full length the denaturation map of mtDNA was constructed, the GC-content of the molten regions was about 28%. The dibromoethylacetate is a perspective agent for screening DNA with a low content of destabilizing regions (for instance, DNA of malignant cells).

[Functions of bacteriophage T4 rII genes. Preparatory isolation and several properties of rIIB protein]. / Funktsii genov rII bakteriofaga T4. Preparativnoe vydelenie i nekotorye svoistva belka rIIB

A method of preparative isolation of membrane rIIB protein from bacteriophage T4 is worked out. Conditions are found to maximal rIIB protein accumulation in membranes of E. coli cells infected with bacteriophage T4. The membrane isolation by ultracentrifugation is substituted with their sedimentation from polyethyleneglycol solutions by low-speed centrifugation. A fraction, enriched with rIIB protein, is obtained using the treatment of cell walls with detergents. Preparative polyacrylamide gel electrophoresis in the presence of sodium dodecylsulphate was used for further rIIB protein purification. The method described can be applied for the purification and preparative isolation of memorane and poor-soluble proteins. The problem on location and ufunctioning of rIIB protein is discussed.