Complete conformational family of 2',3'-didehydro-2',3'-dideoxyguanosine: quantum chemical and electron density topological study

Comprehensive conformational analysis of the biologically active nucleoside 2',3'-didehydro-2',3'-dideoxyaguanosine (d4G) has been performed at the MP2/6-311++G(d,p)//DFT B3LYP/6-31G(d,p) level of theory. The energetic, geometrical and polar characteristics of twenty d4G conformers as well as their conformational equilibrium were investigated. The electron density topological analysis allowed us to establish that the d4G molecule is stabilized by nine types of intramolecular interactions: O5'H...N3, O5'H...C8, C8H...O5', C2'H...N3, C5'H1...N3, C5'H2...N3, C8H...H1C5', C8H...H2'C5' and N2H1...O5'. The obtained results of conformational analysis permit us to think that d4G may be a terminator of the DNA chain synthesis in the 5'-3' direction. Thus it can be inferred that d4G competes with canonical 2'-deoxyaguanosine in binding an active site of the corresponding enzyme.     

Intramolecular hygrogen bonds in conformers of 2'-deoxycytidine: results of quantum-chemical analysis of electron density topology

As many as 13 types of intramolecular hygrogen bonds are determined in 89 conformers of 2'-deoxycytidine nucleoside by means of quantum-chemical analysis (at DFT B3LYP/6-31G(d,p) theory level) of electron density topology with Atoms-in-Molecules (AIM) theory. The total number of H-bonds is 168 and their types are C1'H...O2, C2'H2...O5', C2'H2...O2, C3'H...O2, C5'H1...O2, C5'H2...O2, C6H...O4', C6H...O5', C3'H...HC6, O3'H...O5', O5'H...O3', O5'H...O4' and O5'H...O2. Conformational, geometric and electron-topological properties of H-bonds are presented.     

Conformational capacity of 2',3'-didehydro-2',3'-dideoxyadenosine as a key to understanding its biological activity: results of quantum chemical modelling

Comprehensive conformational analysis of the biologically active nucleoside 2',3'-didehydro-2',3'-dideoxyadenosine (d4A) has been performed at the MP2/6-311++G(d,p)//DFT B3LYP/6-31G(d,p) level of theory. The energetic, geometrical and polar characteristics of twenty one d4A conformers as well as their conformational equilibrium were investigated. The electron density topological analysis allowed us to establish that the d4A molecule is stabilized by eight types of intramolecular interactions: O5'H...N3, O5'H...C8, C8H...O5', C2'H...N3, C5'H1...N3, C5'H2...N3 Ta C8H...H1/2C5'. The obtained results of conformational analysis lead us to think that d4A may be a terminator of the DNA chain sythesis in the 5'-3' direction. Thus it can be inferred that d4A competes with canonical 2'-deoxyadenosine in binding an active site of the corresponding enzyme.     

Selective protection of 5' ... GGCC ... 3' and 5' ... GCNGC ... 3' sequences by the hypermodified oxopyrimidine in Bacillus subtilis bacteriophage SP10 DNA.

The DNA of Bacillus subtilis bacteriophage SP10 is partially resistant to cleavage and methylation in vitro by restriction enzyme R . BsuRI and its cognate methylase even though greater than 20 copies of the target sequence, 5' ... GGCC ... 3', are present on the phage genome. YThy, a hypermodified oxopyrimidine that replaces a fraction of the thymine residues in SP10 DNA, was responsible for this protection, since YThy-free DNA was no longer resistant. Sites that were normally resistant could nevertheless be cleaved or methylated in vitro if the salt concentration was reduced or dimethyl sulfoxide was added to the reaction buffer. Analysis of the termini produced by cleavage suggested that resistant sites occurred in the sequence 5' ... GGCC-YThy ... 3', whereas sensitive sites, of which there were only two per genome, occurred in the sequence 5' ... GGCCG ... 3'. These in vitro results provide an explanation for the in vivo resistance of SP10 to restriction-modification by B. subtilis R. They also suggest ways in which the presence of the atypical base YThy in regions that flank the target might upset critical DNA-enzyme interactions necessary to locate and recognize the specific site of cleavage or methylation. YThy also strongly protected 5' ... GCNGC ... 3' (R . Fnu4HI) sequences on SP10 DNA, but the biological relevance of this protection is unclear.     

Site-specificity of DNA methylases from Shigella sonnei 47 cells

A complex approach involving isoplith analysis, enzymatic treatment of methylated isopliths and a computer analysis of experimental data has been used for determining site specificity of six methylases from Shigella sonnei 47 cells termed according to their specificity for a nitrous base and pI as MC4.2, MC5.3, MC6.2, MC7.4, MC8.4 and MA9.5. It has been found that the recognition site of MA9.5 is a palyndrome six-member structure of the 5'...GAATTC...3' type and that this enzyme is an isometimer with respect to MEcoRI. It has been demonstrated for the first time for methylases that the recognition site of MC4.2 is represented by a non-symmetrical four-member sequence, 5'...NCCCCN...3' characterized by unique blocking of cytosines. MC8.4 possesses a broad specificity of substrate recognition and methylates the cytosine residue within the composition of the non-symmetrical unique sequence 5'...N (C/Pu) CCN...3', whose 5'-terminal base is depleted in three nucleotides. MC5.3 methylates the 3'-terminal cytosine residue within the composition of the pentanucleotide palindrome recognition site, 5'...CCNGG...3'. MC6.2 and MC7.4 possess identical pentanucleotide recognition sites of 5'...(Py)CNG(Pu)...3', but are distinguished in pI. The latter finding has been shown for the first time for different methylases within one strain.     

Long- and short-patch gene conversions in Streptococcus pneumoniae transformation

In pneumococcal transformation some point mutations are integrated by an excision-repair pathway which switches the heteroduplex DNA into homoduplex. This transfer of information is a gene conversion. We have reviewed some of the properties of this system especially those relating to heteroduplex specificity and given evidence that this extends over several kilobases of DNA. We then describe a new process of conversion in pneumococcal transformation which occurs over a very short distance (5 to 27 base-pairs) and is triggered by a single site mutation resulting from the transversion 5'-ATTCAT...to 5'...ATTAAT... Only one of the two heteroduplexes 5'...A...3'/3'...G...5', is converted.     

A phagemid vector library for cloning DNA with four-nucleotide 5' or 3' overhangs

A phagemid vector library for cloning DNA with four nucleotide 5' or 3' overhangs has been constructed. This library is based on the pT7T3 vector (Pharmacia) which is a modification of the phagemid pTZ18U vector. We have chosen pT7T3 as the parent vector because it can be used for Sanger's dideoxy sequencing and for the generation of RNA probes with either the T7 or T3 promoter. Each member of the cloning vector series pBM has recognition sites for both of the restriction enzymes BspM1 and BstX1 in addition to the basic multiple cloning sites. BspM1 recognizes the sequence 5'...ACCTGC NNNN/NNNN...3' whereas BstX1 recognizes the sequence 5'...CCAN NNNN/NTGG...3'. Thus these two sites can be overlapped, so that only 256 vectors (instead of 512 vectors) need be constructed to cover all the theoretical possible combinations of sites which give complementary cohesive ends for cloning DNA with four nucleotide 5' or 3' overhangs. This vector library can be used for amplification cloning of DNA in a tandem array by choosing appropriate vectors which have nonpalindromic sequences. We have obtained approximately 200 members of the 256 possible clones and have organized the vectors using a MacIntosh HyperCard program for easy retrieval.     

利用美国SMV株系初步鉴定中国大豆鉴别寄主的抗性基因

参照美国的大豆花叶病毒(SMV)株系鉴别系统,利用G1、G2、G3、G5和G7 5个株系对24个中国鉴别寄主进行接种鉴定,初步对中国SMV大豆鉴别寄主的抗性基因进行了推断.结果表明:'Davis'、'文丰5号'、'齐黄1号'、'齐黄10号'、'徐豆1号'、'徐豆2号'、'吉林26'(PI 612720A,B)、'铁丰25'和'诱变30'的表现型与'York'相同,可能携带相同的抗性基因Rsv1-y;'合丰25'可能携带一个新的Rsv1-n基因;'吉林26'(PI 597411A)和'1138-2'(PI 592914)可能携带Rsv3基因;'吉林26'(PI 597411B)、'早18'、'早熟18'、'吉林21'、'鲁豆4号'抗所有鉴定的SMV株系,可能携带Rsv1-h、Rsv4、Rsv1+3、Rsv1+4或Rsv3+4基因.利用SMV接种鉴定的反应型是对大豆抗性基因进行初步筛选的有效方法,本研究结果对构建中国SMV株系通用鉴别系统有一定借鉴意义.     

Determination of the recognition sites of cytosine DNA-methylases from Escherichia coli SK.

Two different cytosine DNA-methylases, NI and GII, are present in Escherichia coli SK. The GII methylase recognizes the five-member symmetric sequence: 5'...NpCpCpApGpGpN...3'. This sequence is identical with the recognition site of the hsp II type determined by RII plasmid but, in contrast to RII methylase, the GII enzyme methylates cytosine located on the 5' side of the site. By analogy with the isoshizomery of the restricting endonucleases, RII and GII DNA methylaeses may be called isomethymers which recognize the same site but methylate different bases. Since the phage of the SK and hsp II phenotypes is effectively restricted in respective cells it may be assumed that the isomethymeric modification does not provide any protection against the corresponding restrictases. NI methylase recognizes the five-member symmetric site which represents an inverted sequence of the GII site: 5'...NpGpGpApCpCpN...3'. In this case cytosine at the 3'-end of the recognition site is methylated.     

A 1H-NMR and MD study of intramolecular hydrogen bonds in methyl beta-cellobioside.

The existence of an HO-3...O-5' intramolecular hydrogen bond in methyl beta-cellobioside in solution in Me2SO-d6 and H2O-CD3OD (4:1 w/w) was studied by 500-MHz 1H-NMR spectroscopy and MD simulations. Temperature coefficients for the chemical shift of the hydroxyl resonances in these solvents were determined and the rates of proton exchange in the latter solvent were obtained from NOE data. With H2O-CD3OD as the solvent, the HO-3...O-5' hydrogen bond was insignificant, but its presence in Me2SO-d6 was confirmed.     

A study of substrate specificity of DNA-methylases from Flavobacterium okeanokoites

The specificity of DNA methylase M. FokI towards oligonucleotides containing sequence 5'...GGATG.../3'...CCTAC... was investigated, and N6-methyladenine in the GGATG chain was shown to be the only product of the modification.     

转基因香石竹中F3’5’H基因的克隆、表达和免疫学鉴定

在研究转基因香石竹品系月之霓裳（Moonshade）、月之伊人（Moonlite）中外源基因F3’5’H的表达中,本文克隆了F3’5’H全长基因1.5kb,构建获得工程菌株Escherichia coli BL21（DE3）（＋F3＇5＇H）。SDS-PAGE分析的结果显示,该菌株高效表达出F3’5’H重组蛋白,约占菌体总蛋白的30%。用经纯化的F3’5’H重组蛋白作为抗原,制备F3’5’H重组蛋白的抗血清,经ELISA免疫学分析表明,该抗血清的效价为1：25600。Western blot结果表明F3’5’H重组蛋白具有良好的IgG结合活性,且抗血清与转基因香石竹品系月之霓裳和月之伊人中的外源基因F3’5’H所表达的蛋白发生明显的抗原抗体反应。这样,月之霓裳和月之伊人用于评价转基因香石竹品系的环境安全性在我国也得到了验证。     

Methylase-limited partial NotI cleavage for physical mapping of genomic DNA.

Partial cleavage of DNA with the restriction endonuclease NotI (5'...GC/GGCCGC...3') is an important technique for genomic mapping. However, partial genomic cleavage with this enzyme is impaired by the agarose matrix in which the DNA must be suspended. To solve this problem we have purified the blocking methylase M. BspRI (5'...GGmCC...3') for competition digests with NotI. The resulting methylase-limited partial DNA cleavage is shown to be superior to standard techniques on bacterial genomic DNA. Abbreviations: bp, base-pair; kb, one thousand base-pairs; Mb, one million base-pairs; Tris, Tris(hydroxy-methyl)aminomethane; EDTA, (ethylenedinitrilo)tetraacetic; beta-ME, beta-mercaptoethanol; PMSF, phenyl methyl-sulfonyl fluoride; PEG, polyethyleneglycol (MW = 8000); 3H, tritium; SAM, S-adenosylmethionine; KGB, potassium glutamate buffer; DTT, dithiothreitol; IPTG, isopropyl-beta-D-thiogalactopyranoside; BSA, bovine serum albumin.     

Two restriction endonucleases from Bacillus globiggi.

The sites of action of the restriction enzyme Bgl II on lambda DNA are mapped. This enzyme recognises the sequence 5' ...AGATCT...3' and makes staggered cuts producing sticky ends. In lambda DNA, the second A in this sequence is methylated about 50% of the time by a bacterial methylase absent in E. coli dam. In contrast to Bgl II, Bgl I makes many cuts in lambda DNA and produces 5' terminals which are not substrates for polynucleotide kinase.     

Use of pulsed-field agarose gel electrophoresis to size genomes of Campylobacter species and to construct a SalI map of Campylobacter jejuni UA580.

To determine the physical length of the chromosome of Campylobacter jejuni, the genome was subjected to digestion by a series of restriction endonucleases to produce a small number of large restriction fragments. These fragments were then separated by pulsed-field gel electrophoresis with the contour-clamped homogeneous electric field system. The DNA of C. jejuni, with its low G+C content, was found to have no restriction sites for enzymes NotI and SfiI, which cut a high-G+C regions. Most of the restriction enzymes that were used resulted in DNA fragments that were either too numerous or too small for genome size determination, with the exception of the enzymes SalI (5' ... G decreases TCGAG ... 3'), SmaI (5' .... CCC decreases GGG .... 3'), and KpnI (5' ... GGTAC decreases C .... 3'). With SalI, six restriction fragments with average values of 48.5, 80, 110, 220, 280, and 980 kilobases (kb) were obtained when calibrated with both a lambda DNA ladder and yeast Saccharomyces cerevisiae chromosome markers. The sum of these fragments yielded an average genome size of 1.718 megabases (Mb). With SmaI, nine restriction fragments with average values ranging from 39 to 371 kb, which yielded an average genome size of 1.726 Mb were obtained. With KpnI, 11 restriction fragments with sizes ranging from 35 to 387.5 kb, which yielded an average genome size of 1.717 Mb were obtained. A SalI restriction map was derived by partial digestion of the C. jejuni DNA. The genome sizes of C. laridis, C. coli, and C. fetus were also determined with the contour-clamped homogeneous electric field system by SalI, SmaI, and KpnI digestion. Average genome sizes were found to be 1.714 Mb for C. coli, 1.267 Mb for C. fetus subsp. fetus, and 1.451 Mb for C. laridis.