[Z-form of intraphage DNA]. / Z-forma vnutrifagovoi DNK.

The bacteriophage lambda gt10 DNA containing an insertion of 20 pairs of GC-bases capable of being arranged as Z-form was cloned. Two independent methodological approaches based on the main properties of Z-form were used to study the shape of the insertion: formation of transition bridges composed of unpaired nucleotides between left-rotating Z-forms and right-rotating B-forms of helix (j-domain) and high immunogenic activity of Z-form. O-beta-diethylaminoethylhydroxyamine (OHA), an analogue of hydroxylamine, is capable of reacting specifically with unpaired cytidines. In this work this modification was used to inhibit the process of restriction at BamH1-site adjacent to (gc)10 insertion, that N-Methyl-bis(2-chloethyl) amine (MBCA) is capable of fixing the Z-form of the insertion in situ. Fixed Z-form is conserved even after DNA has been isolated from bacteriophage, thereby providing an opportunity of its identification by anti-Z-antibodies. It was shown that from 4 to 6% of the total number of insertions are in the Z-form. The hypothesis of significant role of Z-form in the process of packing of DNA molecules in capsid is put forward.

[DNA inside bacteriophage lambda forms a Z-form]. / DNK vnutri bakteriofaga lambda obrazuet Z-formu.

The site of the transition regions between the B- and Z-forms of the DNA duplex (B-Z junction) may serve as marker of the existence of Z-DNA in situ. The structure of (dC-dG)10 insert in the bacteriophage lambda gt10 has been studied in situ by modification of B-Z junction with O-beta-diethylaminoethylhydroxylamine (OHA). The latter is an analogue of hydroxylamine possessing specificity with respect to unpaired cytidine. This modification inhibited the process of restriction at BamHI site adjacent to the Z-insert. Judging by the extent of the inhibition about 5% of all inserts has been converted in Z-form. The certain role of Z-form at process of the packaging DNA into bacteriophage's capsid suggest.

[Comparison of the state of the area between B- and Z-segments of superhelical plasmids in vitro and in situ]. / Sopostavlenie sostoianiia oblasti mezhdu B- i Z-uchastkami superspiral'noi plazmidy in vitro i in situ.

The structure of B-Z junction in a cloned plasmid pGC20 containing a (dG-dC)10 insert at the SmaI site has been studied in vitro and in situ by modifying the DNA with O-beta-diethylaminoethylhydroxylamine (OHA). The latter is an analog of hydroxylamine possessing specificity with respect to unpaired cytidine. Experiments in vitro showed a complicated pattern of inhibiting the restriction hydrolysis of the OHA-modified DNA within the polylinker region of the plasmid. As the duration of the DNA reaction with OHA grows, a gradual increase in the inhibition of restriction is observed at the BamHI site neighboring the Z-insert and at the HindIII site at a distance of about 30 bp from the insert, while an intact segment (containing the SalGI site) is retained in the intermediate region. On passing to the cell level, only the region immediately adjacent to the Z-insert appears to be modified. According to estimates, about 30 to 40% of pGC20 molecules have the (dG-dC)10 insert in the Z-form when modified in situ in 1M OHA, pH 5.0.

[Nitrogen mustard fixes the Z-conformation in poly[d(G-C)]poly[d(G-C)] and DNA]. / Embikhin fiksiruet Z-formu v poli[d(G-C)]poli[d)G-C)] i DNK.

The reactions of poly(dG-dC).poly(dG-dC) and (dG-dC)10 insert in the plasmid pGC20 with N-methyl-bis(2-chloroethyl)-amine (nitrogen mustard, HN-2) have been studied. It is shown that nitrogen mustard does not induce the B----Z transition in poly(dG-dC).poly(dG-dC), but produces fixation of the polynucleotide Z-conformation once this exists. In the case of pGC20 plasmid DNA, nitrogen mustard also fixes Z-form of the (dG-dC)-insert. The rate constant of the reaction of nitrogen mustard with guanine in the polynucleotide (k = 9,0.10(-3) min-1) is about one-third of that for the fixation of Z-form of the (dG-dC)-insert in the plasmid (k1 = 2,8.10(-2) min-1) which is attributed to a greater rate of formation of diguanyl derivative in the opposite DNA chains. It is suggested that nitrogen mustard is capable of fixing the Z-form DNA not only in vitro, but also in vivo.

[The Z-form of bacteriophage lambda DNA, modified in situ]. / Z-forma modifitsirovannoi in situ DNK bakteriofaga lambda.

Using the methods of chemical modification, restriction analysis and immune-electron microscopy it has been shown that the definite regions of the bacteriophage lambda DNA contain unpaired bases in situ. The distribution map of such sites along the genome has been constructed. The correlation of the in situ modification and the reaction with anti-Z-DNA antibodies is shown for the 44972 bp site of bacteriophage DNA. The possibility of the existence of Z-form DNA in situ is discussed.

[Unpaired bases in the B--Z junction of the supercoiled plasmid]. / Nesparennye osnovaniia v B--Z perekhodnoi oblasti superspiral'noi plazmidy.

The restriction analysis has been used to establish that O-beta-diethylaminoethylhydroxylamine (OHA) produces modification of unpaired cytidines in the polylinker region adjacent to the Z-insert (dG-dC)10. (dG-dC)10 in the negatively supercoiled plasmid pGC20. The length of the transition region between B- and Z-portions of DNA is not less than 36 bps. The reaction of OHA with the unpaired cytidines in the B-Z junction is a fixing one and produces no secondary despiralling of the neighboring regions. The reaction with DNA proceeds much slower than the one with monomers and single-strand polynucleotides. The structural nonuniformity has been observed, which is manifested in the alternating B and "non-B" form DNA in the B-Z junction. It is suggested that these junctions may contain nucleotide sequences which are stable to violation of the B structure during the change in superhelical density of DNA.

[Study of local changes in the structure of phage DNA by electron microscopy]. / Issledovanie lokal'nykh izmenenii struktury fagovoi DNK metodom élektronnoi mikroskopii.

DNA of defective bacteriophage PBSX was studied by electron microscopy. The presence and distribution of sites containing local structure changes were revealed using antibodies specific for the DNA molecules modified in situ. These structural changes are related to the capsid geometry, but not to the DNA primary sequence.

[Modification of the DNA of bacteriophage Sd in situ by O-beta-diethylaminoethylhydroxylamine]. / Modifikatsiia a DNK bakteriofaga Sd in situ O-beta-diétilaminoétilgidroksilaminom.

The interaction of a O-derivated analog of hydroxylamine-O-beta-diethylaminoethyl-hydroxylamine (OHA) with bacteriophage SD DNA in situ was studied. It was established that OHA modifies the cytidine of phage DNA up to 2% from the total cytidine quantity. The terminal ratio products of modification cytidine corresponds to the ration of modification products for completely denaturated DNA. The results obtained are interpreted in terms of locally denaturated regions spaced in the whole genome of SD bacteriophage.

[Double modification of cytidine residues in DNA]. / Dvoinaia modifikatsiia tsitidina v DNK.

The reaction of O-beta-diethylaminoethylhydroxylamine (O-beta-HA) with cytidine was studied and its mechanism was shown to be analogous to that of the reaction of hydroxylamine of O-methylhydroxylamine with cytidine. In experiments involving reaction of denatured DNA with O-beta-HA., Sephadex G-15 columns were used for the quantitative separation of normal and modified nucleosides after enzymatic hydrolysis of modified DNA by exonuclease A5 followed by alkaline phosphatase treatment. DNA cytidine residues of free cytidine with O-beta-HA. Modified cytidines can form complex with phosphotungstic acid (PTA). It was shown that one mole of PTA was bound per one mole of modified cytidine either in DNA or in free state. Electron microscopic examination of denatured DNA molecules modified by O-beta-HA and reacted with PTA revealed linear arrays of electron-scattering spots which presumably correspond to PTA molecules complexed with modified cytidine in DNA chains.