[Effect of single-stranded DNA binding proteins on template/primer-independent DNA synthesis in the presence of nicking endonuclease Nt.BspD6I].

In the presence of the Nt.BspD6I nicking endonuclease DNA polymerase Bst stimulates intensive template/primer-independent DNA synthesis. Template/primer-independent DNA synthesis could be the reason for appearing nonspecific DNA products in many DNA amplification reactions particularly in the reactions with using nicking endonucleases. Search of the modes for inhibition template/primer-independent DNA synthesis becomes an urgent task because of broadening the DNA amplification methods with using nicking endonucleases. We report here that the E. coli single-stranded DNA binding protein has no effect on the template/primer-independent DNA synthesis. In the absence of the nicking endonuclease the single-stranded DNA binding protein encoded by bacteriophage T4 gene 32 completely inhibits template/primer-independent DNA synthesis. This protein does not inhibit synthesis of specific DNA product in the presence of nicking endonuclease but remarkably decreases the amount of nonspecific products.

[Oligomerization of site-specific nicking endonuclease BspD6I at high protein concentrations].

Ability of site-specific nickase BspD6I (Nt.BspD6I) to oligomerize at concentrations > or = 0.5 microM (> or = 0.035 mg/mL) is studied. Three states of Nt.BspD6I are registered via electrophoretic studies both in the presence and in the absence of DNA. Estimation of their molecular mass allows assigning them as a monomer, a dimer and a trimer. Both dimeric and monomeric Nt.BspD6I are shown to hydrolyze its DNA substrate with the identical specificity. Calculation of the electrostatic potential distribution on the Nt.BspD6I globule surface shows that the protein molecule is a dipole. The Nt. BspD6I oligomeric forms are likely to be the result of ionic protein interactions.

[Investigation of site-specific DNA binding with nicking endonuclease Nt.BspD6I at single molecule level by atomic force microscopy].

Nicking endonuclease Nt.BspD6I is a heterodimeric restriction endonuclease, one subunit of which exhibits specific nicking activity. It gets bound to double-stranded DNA and makes a break (nick) in one chain at a distance of 4 nucleotides from the binding site. In this work, for visualization of the specific binding and protein landing site an atomic force microscopy was used. In five minutes after incubation of DNA solution with nicking endonuclease, the DNA molecules with associated proteins which located at the expected binding site and "shared" DNA strand into two segments (approximately, 1/3 and 2/3 of length) were observed in the images. In addition, near the binding site DNA molecule had a height corresponding to a single-stranded DNA molecule, which was in good agreement with single-stranded cleavage by nickase in the course of complex formation.

[Influence of low-intensity laser radiation on the formation of liquid crystalline structures in a solution of glycoproteins]. / Vliianie nizkointensivnogo lazernogo izlucheniia na formiravanie zhidkokristallicheskikh struktur v rastvore glikoproteinov.

Liquid-crystalline structure formation in glycoprotein solutions irradiated by helium-neon laser in the presence of hydrogen peroxide was observed by both polarizing microscopy and spectrophotometry. High molecular weight (2.10(6) Da) and heavily glycosylated (about 80%) glycoprotein was isolated from the mucus layer of pig small intestine. Remarkable changes of both optic parameters of the solutions and the morphology of liquid-crystalline structures were detected in irradiated samples compared to the non-irradiated ones.

[Deletion of late genes of the prophage lambda]. / Deletsiia pozdnikh genov profaga lambda.

The deletions in tandem prophage lambda appear with high frequency (to 10%) in rec A- strain of Escherichia coli. The deletions were shown by marker rescue and hybridization of fragments of DNA on nitrocellulose filters with nick-translated phage lambda DNA localized only in prophage area. Right and left att sites are not involved. The majority of defective lysogens had all regulatory regions and deletions of late structural genes. These strains may be used for construction of the host-vector systems with the strongest promoter p'R of phage lambda.

[BspLS2I--a new site-specific endonuclease from the thermophilic bacteria Bacillus species LS2]. / BspLS2I--novaia sait-spetsificheskaia éndonukleaza iz termofil'noi bakterii Bacillus species LS2.

A new restriction endonuclease BspLS2I was isolated from the thermophilic bacterium Bacillus species LS2 and purified by blue sepharose and hydroxyapatite chromatographies. The enzyme is an isoschizomer of SduI from Streptococcus durans. BspLS2I recognizes the sequence 5' G(G/A/T)GC(C/T/A) decreases C 3' on double-stranded DNA and cleaves it is indicated by the arrow to yield sticky-ended DNA fragments. Maximum catalytic activity of endonuclease was found in 10 mM tris-HCl (pH 7.9) in the presence of 15-30 mM MgCl2 at 50 degrees C. The phage T4 glucosylated DNA is not cleaved by the enzyme.

[Isolation and properties of site-specific endonuclease BspTS514I from the thermophilic bacteria Bacillus species TS514]. / Vydelenie i svoistva sait-spetsificheskoi éndonukleazy BspTS514I iz termofil'noi bakterii Bacillus species TS514.

New site-specific endonucleases BspBS31I, BstBS32I, BspIS41, BstTS5I, BspTS514I were isolated from five thermophilic soil bacteria Bacillus sp. BS31, B. stearothermophilus BS32, Bacillus sp. IS4, B. stearothermophilus TS5, Bacillus sp. TS514. The enzymes are isoschizomers of the restriction endonuclease BbvII. Endonuclease BspTS514I was obtained pure from interfering contaminations by two consecutive chromatographies on blue agarose and hydroxyapatite. The enzyme exhibits a maximal activity at 55 degrees C in 10 mM tris-HCl (pH 9.2), 10 mM MgCl2 and 50 mM NaCl.

[Transactivation of p'R promoter of phage lambda]. / Transaktivatsiia promotora p'R faga lambda.

The host-vector system for efficient expression of the cloned genes under the control of transactivated promoter p'R of bacteriophage lambda has been elaborated. The Q protein activating p'R promoter is coded by the defective prophage constructed in vitro by means of excision of the late phage genes between the distant sites of the restriction endonuclease MluI and change of the central SalI fragment carrying the kill gene for the kanamycin resistance gene. The general recombination system is impaired during the change, thus the bacteriophage DNA can be obtained from the induced RecA cells as a plasmid DNA. The induction of the prophage results in a sharp increase of beta-lactamase synthesis (30% of soluble cell protein) under the control of p'R promoter in a plasmid derived of pBR322.

[Features of expression of cloned genes under the control of tandem promotors pL and pR of phage lambda]. / Ossobennosti ékspressii klonirovannykh genov pod kontrolem tandema promotorov pL and pR faga lambda.

The regulatory block P'R from the bacteriophage lambda has been inserted between the promoter and initial part of the gene into the plasmid pCJ55 carrying the gene for the Klenow fragment under the control of pL. As it should be predicted, at the inverted orientation the sharp decrease in the Klenow fragment quantity is registered. However, at the direct orientation there is some decrease in the synthesis of the protein, as compared with the synthesis of the Klenow fragment in the strain harbouring the plasmid pCJ55. A plausible explanation of the fact may be in the transcriptional interference of the promoters pL and p'R in artificially constructed structures.

[Isolation and properties of BstBSI restriction endonuclease from the thermophilic soil bacteria Bacillus stearothermophilus BS]. / Vydelenie i svoistva éndonukleazy restriktsii BstBSI iz termofil'noipochvennoi bakterii Bacillus stearothermophilus BS.

A new restriction endonuclease BstBSI was isolated and purified from the thermophilic soil bacterium Bacillus stearothermophilus BS by the blue sepharose and hydroxyapatite chromatographies. The enzyme is an isoschizomer of SnaI from Sphaerotilus natans C. It recognizes the hexanucleotide GTATAC and cleaves DNA in the center of the sequence. The maximal catalytic activity of the endonuclease is registered in 50 mM tris-HCl (pH 9.0) buffer with the high ionic strength (100 mM NaCl) in the presence of 10 mM MgCl2 at 45 degrees C. Glucosylated DNA of the phage T4 is not cleaved by the enzyme.

[X-ray diffraction study of adaptive changes in the structure of dense phase juice from the duodenum]. / Rentgenograficheskoe issledovanie adaptivnykh izmenenii struktury plotnoi fazy soka dvenadtsatiperstnoi kishki.

Low-angle X-ray diffraction patterns of canine duodenal juice precipitates taken at two stages of digestion, after 20 h fasting and after food stimulation have been obtained. Both diffraction patterns are due to glycoproteins. However, the glycoproteins contained in the precipitate after food stimulation occur in the native form while in fasting animals they are partially degraded.

[Experience in the use of synchrotron radiation for the x-ray study of biopolymers]. / Opyt ispol'zovaniia sinkhrotronnogo izlucheniia dlia rentgenograficheskogo issledovaniia biopolimerov

The results of methodical work, carried out on the sources of synchrotron radiation (SR) with the aim of using SR as a powerful source of X-rays for studying biopolymer structure, are presented. The questions of monochromatization are considered. The technique designed for photoregistration of diffraction patterns within the wide range of scattering angles is described. X-ray diffraction patterns of feather ceratin, collagen and striated muscle are obtained with exposure periods ten times less than those in the case of X-ray tubes. The high resolution of diffraction lines, the absence of parasitic phone and the presence of reflections within the wide range of scattering angles are characteristic of these patterns.

[Roentgenographic study of the duodenal juice components in the dog]. / Rentgenograficheskoe izuchenie komponentov soka dvenadtsatiperstnoi kishki sobaki.

It is shown that X-ray pattern rich in reflections that we have received earlier from concentrated canine duodenal juice is the sum of the patterns arising from both mucin macromolecules and from unknown component of the juice. The functional role of the duodenal juice regularity observed is discussed.

[High time resolution x-ray study of the dynamics of a single muscle contraction]. / Rentgenograficheskoe issledovanie dinamiki odinochnogo myshechnogo sokrashcheniia s vysokim vremennym razresheniem.

A method of the diffraction cinema which enables to study the time-course of structural changes during twitch contraction is described. The method is based on using synchrotron radiation, position-sensitive counter and small-angle focusing X-ray camera. Only 0.1 s is required to record a good muscle X-ray diagram: meridional diagram contains all layer-lines beginning with the 429 A; the equatorial diagram contains 5 reflections including very weak alpha-reflection. The method allows to record 64 sequent diffraction patterns with different duration (1--2000 ms). The experiment is handled by a computer. Some tens of the films of isometric twitch contraction with time resolution of 3--20 ms have been obtained. During isometric contraction considerable changes in the intensity of both meridional and equatorial reflections were found. The changes were interpreted as indicating movement of cross-bridges toward the thin filaments. During the latent phase there are no visible changes in the intensity of the reflections; the result indicates that during this phase there are no structural changes in position and configuration of cross-bridges.

[Effect of thiol reagents on the structure of mucus glycoproteins (mucins)]. / Vliianie tiolovykh reagentov na strukturu glikoproteinov slizi (mutsinov).

The structure of both native mucins and subunits obtained by thiol reduction was studied by small-angle X-ray diffraction. X-ray pattern from the subunits significantly differs from that of the native mucins in the number, spacings and width of the reflections observed. Decrease in the number of the reflections and increase of their width pointed out that isolated subunits are less regular structure than the whole mucin molecule. This may be due to the two reasons: 1) conformation of the subunits in native molecule differs from that of in isolate state; 2) native mucin molecule is not a simple polymer, formed by the subunits.

[Two site-specific endonucleases from the thermophilic strain Bacillus species LU11]. / Dve sait-spetsificheskie éndonukleazy iz termofil'nogo shtamma Bacillus species LU11.

Upon screening of natural strains of thermophilic bacteria, a strain has been found which contains two restriction endonucleases. One of those, BspLU11II, is an isoschizomer of XbaI, while the other one, BspLU11I, recognizes the new palindromic sequence 5'-A decreases CATGT-3' and cleaves it as indicated by the arrow. Functionally pure enzymes were obtained by stepwise chromatography with blueagarose, hydroxyapatite and heparin-Sepharose. The restriction endonuclease BspLU11I produces sticky ends identical to those produced by the restriction endonuclease NcoI; hence a combination of BspLU11I and NcoI can be used for enzymatic selection of recombinant DNA. The recognition sequence of BspLU11I contains the ATG codon and can be used to construct expression vectors for chemically synthesized genes.

[A new site-specific endonuclease and methylase from a thermophilic strain of Bacillus species KT6]. / Novye sait-spetsificheskie éndonukleaza i metilaza iz termofil'nogo shtamma Bacillus species KT6.

The site-specific endonuclease R.BspKT6I and the cognate site-specific methylase M.BspKT6I have been isolated from the thermophilic strain of Bacillus species KT6 using gel-filtration on Sephadex G100 followed by chromatography on heparin-Sepharose and hydroxyapatite. Endonuclease BspKT6I is an isomer but not an isoschizomer of Sau3AI and MboI. It recognized on the DNA molecule the GAT decreases C sequence and cleaves it; however, unlike Sau3AI and MboI it produces 3'-protruding dinucleotides. The site cleavage is inhibited by dam-methylation. The sticky ends resulting from the BspKT6I cleavage are identical and complementary to the ends formed after the PvuI cleavage. The isolated from the B. species KT6 methylase protects the DNA from subsequent cleavage by BspKT6I. Adenine is a methylated base.

[Site-specific endonuclease BspKT8 from the thermophilic strain KT8 of Bacillus species]. / Sait-spetsificheskaia éndonukleaza BspKT8 iz termofil'nogo shtamma Bacillus species KT8.

A site-specific endonuclease capable of recognizing the sequence 5'-AAGCTT-3' was detected and purified to homogeneity from the thermophilic strain of Bacillus species KT8. The endonuclease has a molecular mass of 34 kDa and is found in solution in a monomeric form. The activity of BspKT8 does not depend on ATP and is not stimulated by S-adenosyl-L-methionine. The enzyme displays the highest activity with a broad range of temperatures (37 degrees-48 degrees C). Since DNA cleavage occurs in accordance with the scheme: [formula: see text] the enzyme can be assigned to the class-II of restriction endonucleases and represents an isoschizomer of HindIII.