Monomeric restriction endonuclease BcnI in the apo form and in an asymmetric complex with target DNA

Restriction endonuclease BcnI cleaves duplex DNA containing the sequence CC/SGG (S stands for C or G, / designates a cleavage position) to generate staggered products with single nucleotide 5'-overhangs. Here, we show that BcnI functions as a monomer that interacts with its target DNA in 1:1 molar ratio and report crystal structures of BcnI in the absence and in the presence of DNA. In the complex with DNA, BcnI makes specific contacts with all five bases of the target sequence and not just with a half-site, as the protomer of a typical dimeric restriction endonuclease. Our data are inconsistent with BcnI dimerization and suggest that the enzyme introduces double-strand breaks by sequentially nicking individual DNA strands, although this remains to be confirmed by kinetic experiments. BcnI is remotely similar to the DNA repair protein MutH and shares approximately 20% sequence identity with the restriction endonuclease MvaI, which is specific for the related sequence CC/WGG (W stands for A or T). As expected, BcnI is structurally similar to MvaI and recognizes conserved bases in the target sequence similarly but not identically. BcnI has a unique machinery for the recognition of the central base-pair.     

Structure of tomato bushy stunt virus: V. Coat protein sequence determination and its structural implications

We report the chemically determined sequence of most of the polypeptide chain of the coat protein of tomato bushy stunt virus. Peptide locations have been determined by comparison with the high-resolution electron density map from X-ray crystallographic analysis as well as by conventional chemical overlaps. Three small gaps remain in the 387-residue sequence. Positively charged side-chains are concentrated in the N-terminal part of the polypeptide (the R domain) as well as on inward-facing surfaces of the S domain. There is homology of S-domain sequences with structurally corresponding residues in southern bean mosaic virus.     

呼吸道合胞病毒B亚型分离株的G蛋白基因分析

对一株长春地区B亚型分离株(CC169)的G蛋白基因进行了 序列分析,结果表明：我国呼吸道合胞病毒(RSV)分离株CC169同RSV B亚型原型株CH18537的 核苷酸同源性为94%,核苷酸的有义突变率达65%。由核苷酸推导出氨基酸序列的同源性为894%,氨基酸的变异全部发生在胞外区,并主要集中在一个高度保守区的两端,胞内区和跨 膜区保守不变。氨基酸的变异导致了分离株既有糖基化位点的改变,又有蛋白长度的变异。 此外还初步探讨了我国RSV B 亚型分离株CC169的G蛋白基因同原型株之间的变异与疫苗研制 中的意义。     

In vivo DNA protection by relaxed-specificity SinI DNA methyltransferase variants

The SinI DNA methyltransferase, a component of the SinI restriction-modification system, recognizes the sequence GG(A/T)CC and methylates the inner cytosine to produce 5-methylcytosine. Previously isolated relaxed-specificity mutants of the enzyme also methylate, at a lower rate, GG(G/C)CC sites. In this work we tested the capacity of the mutant enzymes to function in vivo as the counterpart of a restriction endonuclease, which can cleave either site. The viability of Escherichia coli cells carrying recombinant plasmids with the mutant methyltransferase genes and expressing the GGNCC-specific Sau96I restriction endonuclease from a compatible plasmid was investigated. The sau96IR gene on the latter plasmid was transcribed from the araBAD promoter, allowing tightly controlled expression of the endonuclease. In the presence of low concentrations of the inducer arabinose, cells synthesizing the N172S or the V173L mutant enzyme displayed increased plating efficiency relative to cells producing the wild-type methyltransferase, indicating enhanced protection of the cell DNA against the Sau96I endonuclease. Nevertheless, this protection was not sufficient to support long-term survival in the presence of the inducer, which is consistent with incomplete methylation of GG(G/C)CC sites in plasmid DNA purified from the N172S and V173L mutants. Elevated DNA ligase activity was shown to further increase viability of cells producing the V173L variant and Sau96I endonuclease.     

BspLS2I--a new site-specific endonuclease from the thermophilic bacteria 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.     

Multilocus sequence typing reveals high genetic diversity and epidemic population structure for the fish pathogen Yersinia ruckeri

Yersinia ruckeri is the causative agent of enteric redmouth in fish and one of the major bacterial pathogens causing losses in salmonid aquaculture. Previously typing methods, including restriction enzyme analysis, pulsed-field gel electrophoresis and multilocus enzyme electrophoresis (MLEE) have indicated a clonal population structure. In this work, we describe a multilocus sequence typing (MLST) scheme for Y.ruckeri based on the internal fragment sequence of six housekeeping genes. This MLST scheme was applied to 103 Y.ruckeri strains from diverse geographic areas and hosts as well as environmental sources. Sequences obtained from this work were deposited and are available in a public database (http://publmst.org/yruckeri/). Thirty different sequence types (ST) were identified, 21 of which were represented by a single isolate, evidencing high genetic diversity. ST2 comprised more than one-third of the isolates and was most frequently observed among isolates from trout. Two major clonal complexes (CC) were identified by eBURST analysis showing a common evolutionary origin for 94 isolates forming 21 STs into CC1 and for 6 isolates of 6 STs in the CC2. It was also possible to associate some unique ST with isolates from recent outbreaks in vaccinated salmonid fish.     

汉坦病毒糖蛋白G1酵母双杂交诱饵载体的构建及初步鉴定

拟利用Ras募集系统(RRS)构建并鉴定含汉坦病毒囊膜糖蛋白GI的诱饵载体。将汉坦病毒76—118株囊膜糖蛋白GI基因与Ras基因连接,构建嵌合基因Ras—G1及Ras—G1’(G1’无前导肽序列)。将两个嵌合基因克隆入酵母表达载体pMet25,并转染至酵母温度敏感株cdc25-2,检测其对RRS系统的激活作用。限制性内切酶酶切鉴定表明,Met—G1和Met—G1’载体构建正确。激活试验为阴性。说明Met—G1和Met—G1’载体可用于从cDNA库中筛选汉坦病毒受体。     

FseI, a new type II restriction endonuclease that recognizes the octanucleotide sequence 5'' GGCCGGCC 3''.

A Type II restriction endonuclease, designated FseI, has been partially purified from a Frankia species (NRRL 18528). This enzyme cleaves Adenovirus 2 DNA at three sites, but does not cleave the DNAs from bacteriophages lambda, T7, and phi X174, the animal virus SV40, pUC18 and pBR322. FseI recognizes the octanucleotide sequence 5' GGCCGG decreases CC 3' and cleaves as indicated by the arrow. The frequency of occurrence of FseI sites within sequenced regions of the human genome is similar to that for NotI sites.     

采用TEV酶法进行整合膜蛋白拓扑学分析

为了研究膜蛋白的跨膜结构,进行拓扑学分析是十分重要的.有许多分析膜蛋白拓扑结构的方法,本文采用烟草蚀斑病毒（TEV）酶特异性切割测试蛋白中跨膜片段的前段或后端所插入的tev识别序列EXXYXQ(S/G),如果TEV酶能够切割,表明该序列位于目标蛋白的细胞 质外.将Tev识别序列ENLYFQG 分别插入到拟南芥整合膜蛋白的的跨膜区域,然后转化进入酿酒酵母中. 消解酶(zymolyase)酶破除酵母的细胞壁后,TEV酶消化球状体,最后通过Western免疫印迹法来分析结果.有关该方法的注意事项在结果中进行了讨论.     

Size and physical map of the Campylobacter jejuni chromosome.

The chromosome of Campylobacter jejuni is circular and approximately 1700 kb in circumference. The size of the genome was determined by field inversion gel electrophoresis of restriction endonuclease fragments using lambda DNA concatamers and yeast chromosomes to calibrate the size of the fragments. In view of the low (32-35%) G + C content of the campylobacter genome, enzymes that recognizes GC-rich sequences were used. Of the enzymes tested BssHII (G/C(G)CGC), NciI (CC/CGCG) and SalI (G/TCGAC) appeared to be usable. Hybridization of labeled fragments with two or more fragments from digests with a different restriction enzyme gave the information to order the fragments on the C jejuni chromosome. The localization on the genome of the flagellin and ribosomal gene clusters was determined.     

Partial characterization of a DNA restriction endonuclease from Ruminococcus flavefaciens FD-1 and its inhibition by site-specific adenine methylation.

The principal DNA restriction-modification system of the cellulolytic ruminal bacterium Ruminococcus flavefaciens FD-1 is described. The restriction endonuclease RflFI could be separated from cell extracts by phosphocellulose and heparin-sepharose chromatography. Restriction enzyme digests utilizing RflFI alone or in combination with SalI, a restriction enzyme isolated from Streptomyces albus G, showed that the DNA sequence recognized by RflFI either overlapped or was the same as that recognized by SalI. DNA sequence analysis confirmed that RflFI was identical in activity to SalI, with the recognition sequence being 5'-GTCGAC-3' and cleavage occurring between G and T. Adenine methylation within this sequence can be catalyzed in vitro by TaqI methylase, and this inhibited the cleavage of plasmid DNA molecules by RflFI and SalI. Chromosomal DNA from R. flavefaciens FD-1 is also methylated within this DNA sequence because neither restriction endonuclease could degrade this DNA substrate. These findings provide a means to protect plasmid molecules from degradation prior to gene transfer experiments with R. flavefaciens FD-1.     

Partial characterization of a DNA restriction endonuclease from Ruminococcus flavefaciens FD-1 and its inhibition by site-specific adenine methylation.

The principal DNA restriction-modification system of the cellulolytic ruminal bacterium Ruminococcus flavefaciens FD-1 is described. The restriction endonuclease RflFI could be separated from cell extracts by phosphocellulose and heparin-sepharose chromatography. Restriction enzyme digests utilizing RflFI alone or in combination with SalI, a restriction enzyme isolated from Streptomyces albus G, showed that the DNA sequence recognized by RflFI either overlapped or was the same as that recognized by SalI. DNA sequence analysis confirmed that RflFI was identical in activity to SalI, with the recognition sequence being 5'-GTCGAC-3' and cleavage occurring between G and T. Adenine methylation within this sequence can be catalyzed in vitro by TaqI methylase, and this inhibited the cleavage of plasmid DNA molecules by RflFI and SalI. Chromosomal DNA from R. flavefaciens FD-1 is also methylated within this DNA sequence because neither restriction endonuclease could degrade this DNA substrate. These findings provide a means to protect plasmid molecules from degradation prior to gene transfer experiments with R. flavefaciens FD-1.     

DNA binding and cleavage selectivity of the Escherichia coli DNA G:T-mismatch endonuclease (vsr protein).

The Escherichia coli vsr endonuclease recognises T:G base-pair mismatches in double-stranded DNA and initiates a repair pathway by hydrolysing the phosphate group 5' to the incorrectly paired T. The gene encoding the vsr endonuclease is next to the gene specifying the E. coli dcm DNA-methyltransferase; an enzyme that adds CH3 groups to the first dC within its target sequence CC[A/T]GG, giving C5MeC[A/T]GG. Deamination of the d5MeC results in CT[A/T]GG in which the first T is mis-paired with dG and it is believed that the endonuclease preferentially recognises T:G mismatches within the dcm recognition site. Here, the preference of the vsr endonuclease for bases surrounding the T:G mismatch has been evaluated. Determination of specificity constant (kst/KD; kst = rate constant for single turnover, KD = equilibrium dissociation constant) confirms vsr's preference for a T:G mismatch within a dcm sequence i.e. CT[A/T]GG (the underlined T being mis-paired with dG) is the best substrate. However, the enzyme is capable of binding and hydrolysing sequences that differ from the dcm target site by a single base-pair (dcm star sites). Individual alteration of any of the four bases surrounding the mismatched T gives a substrate, albeit with reduced binding affinity and slowed turnover rates. The vsr endonuclease has a much lower selectivity for the dcm sequence than type II restriction endonucleases have for their target sites. The results are discussed in the light of the known crystal structure of the vsr protein and its possible physiological role.     

Sse8387I, a new type-II restriction endonuclease that recognizes the octanucleotide sequence 5''-CCTGCAGG-3''.

A type II restriction endonuclease designated Sse8387I was partially purified from Streptomyces sp. 8387. This enzyme cleaved adenovirus 2 DNA at three sites, lambda phage DNA at five sites, and pUC18 and M13mp18 RF DNA at one site each, but did not cleave the DNAs from pBR322, SV40, or phi X174. Sse8387I recognized the octanucleotide sequence 5'-CCTGCA decreases GG-3', cleaving where shown by the arrow. Sse8387I is the first restriction endonuclease to be reported that recognizes an octanucleotide sequence consisting of all four nucleotides, G, A, T, and C. The frequency of occurrence of Sse8387I sites within sequenced regions of primate genomes was 2.4 times that of NotI sites.     

Strong structural effect of the position of a single acetylaminofluorene adduct within a mutation hot spot.

The NarI restriction enzyme recognition site, G1G2CG3CC, has been identified as a hotspot for -2 frameshift mutations induced by N-2-acetylaminofluorene (AAF) on the basis of a forward mutation assay in plasmid pBR322 in the bacterium Escherichia coli. AAF binds primarily to the C-8 position of guanine residues, and the three guanines of the NarI site are similarly reactive. Despite this similar chemical reactivity, only binding of AAF to the G3 residue causes the -2 frameshift mutations. To study the mechanisms underlying the specificity of the mutagenic processing further, we monitored the structural changes induced by a single AAF adduct within the NarI site by means of CD spectroscopy and thermal denaturation. The NarI sequence was studied as part of the 12-mer ACCGGCGCCACA. The purification and characterization of the three isomers having a single AAF adduct covalently bound to one of the three guanines of this 12 mer are described. The analysis of the melting profiles of the duplexes formed when these three isomers are annealed with the oligonucleotide of complementary sequence shows the same destabilizing effect of the AAF adduct on the three DNA helices. It is also shown, from the CD spectra, that modification of guanine G1 or G2 by AAF does not induce major changes in the helical structure of DNA. On the other hand, modification of guanine G3 induces a change in the CD signal that suggests the formation of a local left handed structure within the 12-mer duplex. These results show the polymorphic nature of the DNA structure in the vicinity of an AAF adduct.     

Construction and use of chimeric SPR/phi 3T DNA methyltransferases in the definition of sequence recognizing enzyme regions.

Multispecific DNA methyltransferases (Mtases) of temperate Bacillus subtilis phages SPR and phi 3T methylate the internal cytosine of the sequence GGCC. They differ in their capacity to methylate additional sequences. These are CCGG and CC(A/T)GG in SPR and GCNGC in phi 3T. Introducing unique restriction sites at equivalent locations within the two genes facilitated the construction of chimeric genes. These expressed Mtase activity at a level comparable to that of the parental genes. The methylation specificity of chimeric enzymes was correlated with the location of chimeric fusions. This analysis, which also included the use of mutant genes, showed that domains involved in the recognition of target sequences unique to each enzyme [CCGG, CC(A/T)GG or GCNGC] are represented by the central non-conserved parts of the proteins, whilst recognition of the sequence (GGCC), which is a target for both enzymes, is determined by an adjacent conserved region.     

Studies of the recognition sequence of phi X174 gene A protein. Cleavage site of phi X gene A protein in St-1 RFI DNA.

It is already known that phi X gene A protein converts besides phi X RFI DNA also the RFI DNAs of the single-stranded bacteriophages G4, St-1, alpha 3 and phi K into RFII DNA. We have extended this observations for bacteriophages G14 and U3. Restriction enzyme analysis placed the phi X gene A protein cleavage site in St-1 RF DNA in the HinfI restriction DNA fragment F10 and in the overlapping HaeIII restriction DNA fragment Z7. The exact position and the nucleotide sequence at the 3'-OH end of the nick were determined by DNA sequence analysis of the single-stranded DNA subfragment of the nicked DNA fragment F10 obtained by gelelectrophoresis in denaturing conditions. A stretch of 85 nucleotides of St-1 DNA around the position of the phi X gene A protein cleavage site was established by DNA sequence analysis of the restriction DNA fragment Z7F1. Comparison of this nucleotide sequence with the previously determined nucleotide sequence around the cleavage site of phi X gene A protein in phi X174 RF DNA and G4 RF DNA revealed an identical sequence of only 10 nucleotides. The results suggest that the recognition sequence of the phi X174 gene A protein lies within these 10 nucleotides.