鲫不同种系线粒体DNA物理图谱的构建

用１３种限制性内切酶对鲫鱼三个亚种共七个品系的线粒体ＤＮＡ进行分析,其中有９种酶在种系间或种系内产生限制性片断长度多态性,发现了１６个线粒体ＤＮＡ组合单倍型,通过双酶切分析,构建了１６个线粒体ＤＮＡ组合单倍型的１３种限制性内切酶的发点的物理图谱。     

A new method for the rapid identification of genes encoding restriction and modification enzymes.

We have constructed derivatives of Escherichia coli that can be used for the rapid identification of recombinant plasmids encoding DNA restriction enzymes and methyltransferases. The induction of the DNA-damage inducible SOS response by the Mcr and Mrr systems, in the presence of methylated DNA, is used to select plasmids encoding DNA methyltransferases. The strains of E. coli that we have constructed are temperature-sensitive for the Mcr and Mrr systems and have been further modified to include a lacZ gene fused to the damage-inducible dinD locus of E. coli. The detection of recombinant plasmids encoding DNA methyltransferases and restriction enzymes is a simple, one step procedure that is based on the induction at the restrictive temperature of the lacZ gene. Transformants encoding DNA methyltransferase genes are detected on LB agar plates supplemented with X-gal as blue colonies. Using this method, we have cloned a variety of DNA methyltransferase genes from diverse species such as Neisseria, Haemophilus, Treponema, Pseudomonas, Xanthomonas and Saccharopolyspora.     

Analysis of the genome of the five Bifidobacterium breve strains: plasmid content, pulsed-field gel electrophoresis genome size estimation and rrn loci number

Abstract The genomes of the five Bifidobacterium breve strains available from culture collections were compared by restriction endonuclease analysis. Electrophoretic migration of undigested DNA allowed us to detect a 5.6-kb circular plasmid in two of these strains. A restriction map of this plasmid was constructed using 10 enzymes. With Dra I endonuclease, pulsed-field gel electrophoresis has allowed the determination of the five B. breve genome sizes to 2.1 Mb. This estimation was further confirmed for CIP 6469 (type strain) and ATCC 15698 using Xba I and Spe I enzymes. In addition, rRNA gene regions were used as probes for strain characterization and suggest that there are at least three rrn loci in B. breve .     

Periodic classification of DNA sequences recognized by restrictases: properties of symmetry and regularity

A circular periodic map of short palindromic DNA sequences is constructed using the sequence homology and symmetry. It is applied to compare sequences recognised by class II restriction and modification enzymes with other similar DNA sequences. All known restriction sites have two strong properties: they are enriched by GC pairs, and clustered (purine-purine, pyrimidine-pyrimidine) bonds predominant upon alternating ones. The preference of AT/GC alternation is only slight. These properties were compared quantitatively with the help of suggested numerical methods among different groups of restriction enzymes. The map is applied for prediction of new specificities of restriction modification systems. Possible mechanism of DNA sequence recognition by these enzymes and their evolution are discussed.     

Recognition of Leuconostoc oenos strains by the use of DNA restriction profiles

The chromosome of 41 Leuconostoc oenos strains obtained from collections in different countries was analysed with the aim of differentiating the strains. Pulsed field electrophoresis (TAFE) was used to separate large DNA fragments created by the restriction enzymes NotI, SfiI and ApaI, which specifically recognize guanines or cytosines. The genomic DNA of 11 strains was analysed initially with NotI and only four different restriction profiles were observed. The genome size ranged from 1.8 to 2.1 megabase pairs (Mbp). Constant field electrophoresis applied to DNA treatment with 19 different restriction enzymes showed that the size of the fragments obtained increased proportionally to the percentage G+C present at the site of restriction. EcoRI and HindIII profiles revealed that the zone between 9 and 23 kbp allowed differentiation of the strains tested. Thus, the 41 strains fell into 30 restriction groups using only two enzymes. Hybridization with a non-radioactive DNA probe coding for 16S rRNA revealed that there were two 16S genes on the chromosome. Correspondence to: C. Diviès     

Restriction map of the single-stranded DNA genome of Kilham rat virus strains 171, a nondefective parvovirus.

We constructed a physical map of Kilham rat virus strains 171 DNA by analyzing the sizes and locations of restriction endonuclease-generated fragments of the replicative-form viral DNA synthesized in vitro. BglI, KpnI, BamHI, SmaI, XhoI, and XorII did not appear to have any cleavage sites, whereas 11 other enzymes cleaved the genome at one to eight sites, and AluI generated more than 12 distinct fragments. The 30 restriction sites that were mapped were distributed randomly in the viral genome. A comparison of the restriction fragments of in vivo- and in vitro-replicated replicative-form DNAs showed that these DNAs were identical except in the size or configuration of the terminal fragments.     

COMAP: a comigrating analysis program for estimating the relationship of adenoviruses on the genome level.

The computer program COMAP is described that enables one to evaluate the relationship among adenovirus 6 strains by comigration analysis with regard to the genome. The required data were obtained by restriction analysis with several enzymes. The program is also important for identifying further adenovirus strains by DNA restriction analysis and to compare new restriction fragment patterns with stored data of known adenovirus strains. We believe that in this manner the DNA restriction analysis will become a valuable diagnostic procedure.     

New restriction enzymes discovered from Escherichia coli clinical strains using a plasmid transformation method

The presence of restriction enzymes in bacterial cells has been predicted by either classical phage restriction-modification (R-M) tests, direct in vitro enzyme assays or more recently from bacterial genome sequence analysis. We have applied phage R-M test principles to the transformation of plasmid DNA and established a plasmid R-M test. To validate this test, six plasmids that contain BamHI fragments of phage lambda DNA were constructed and transformed into Escherichia coli strains containing known R-M systems including: type I (EcoBI, EcoAI, Eco124I), type II (HindIII) and type III (EcoP1I). Plasmid DNA with a single recognition site showed a reduction of relative efficiency of transformation (EOT = 10(-1)-10(-2)). When multiple recognition sites were present, greater reductions in EOT values were observed. Once established in the cell, the plasmids were subjected to modification (EOT = 1.0). We applied this test to screen E.coli clinical strains and detected the presence of restriction enzymes in 93% (14/15) of cells. Using additional subclones and the computer program, RM Search, we identified four new restriction enzymes, Eco377I, Eco585I, Eco646I and Eco777I, along with their recognition sequences, GGA(8N)ATGC, GCC(6N)TGCG, CCA(7N)CTTC, and GGA(6N)TATC, respectively. Eco1158I, an isoschizomer of EcoBI, was also found in this study.     

Quick identification of Type I restriction enzyme isoschizomers using newly developed pTypeI and reference plasmids

Although DNA-recognition sequences are among the most important characteristics of restriction enzymes and their corresponding methylases, determination of the recognition sequence of a Type-I restriction enzyme is a complicated procedure. To facilitate this process we have previously developed plasmid R-M tests and the computer program RM search. To specifically identify Type-I isoschizomers, we engineered a pUC19 derivative plasmid, pTypeI, which contains all of the 27 Type-I recognition sequences in a 248-bp DNA fragment. Furthermore, a series of 27 plasmids (designated 'reference plasmids'), each containing a unique Type-I recognition sequence, were also constructed using pMECA, a derivative of pUC vectors. In this study, we tried those vectors on 108 clinical E. coli strains and found that 48 strains produced isoschizomers of Type I enzymes. A detailed study of 26 strains using these 'reference plasmids' revealed that they produce seven different isoschizomers of the prototypes: EcoAI, EcoBI, EcoKI, Eco377I, Eco646I, Eco777I and Eco826I. One strain EC1344 produces two Type I enzymes (EcoKI and Eco377I).     

人乙型肝炎病毒(HBV)adr亚型基因组的多态性

由乙型肝炎adr亚型病毒(HBVadr)携带者26人的混合血清,得到了HBVadr基因组克隆株(PADR)158株,对这些克隆株进行四种限制性内切酶(BglⅡ,HindⅢ,PstⅠ,XhoⅠ)切点测定,并对其中S株的13种限制性内切酶图谱进行比较研究,发现同为adr亚型病毒,其基因组的限制性酶切图谱存在差异。另外,通过HindⅢ)切点得到的12个克隆株(PADR-H),也进行了酶切图谱分析。在这170个克隆株中,已经发现了5种类型的HBVadr基因组限制性酶切图谱,其中有6种酶(AvaⅠ,EglⅠ,BglⅡ,HincⅡ,HindⅢ,HpaⅠ)的7个变异点。本文报道了HBVadr基因组的多态性现象。     

Restriction endonuclease DNA analysis of Leptospira interrogans serovars Icterohaemorrhagiae and Copenhageni

Leptospira interrogans serovar icterohaemorrhagiae strains Ictero No. I and RGA and serovar copenhageni strains M20, Shiromizu and Shibaura were examined by restriction endonuclease DNA analysis. Fifteen endonucleases (AluI, BamHI, BglII, EcoRI, HaeIII, HhaI, HindIII, KpnI, PstI, SacI, SalI, SmaI, StyI, XbaI and XhoI) were used as the digesting enzymes. Strain Ictero No. I showed endonuclease cleavage patterns which differed from those of the other four strains only when it was digested with enzymes KpnI and HindIII. When digested with KpnI, an extra band of about 5.4 kb was clearly produced, and when digested with HindIII, an extra band of about 25 kb was produced. When the other 13 enzymes were used, no differences were found between the endonuclease cleavage patterns among the five strains. Moreover, strains RGA, M20, Shiromizu and Shibaura could not be distinguished by the restriction endonuclease DNA analysis using all 15 endonucleases. In addition, six newly isolated leptospires from patients with leptospirosis and from Rattus norvegicus were compared with the Ictero No. I and M20 strains, by restriction endonuclease DNA analysis using enzymes KpnI and HindIII. Three leptospires belonging to serovar icterohaemorrhagiae showed the same endonuclease cleavage patterns as the M20 strain. The other three strains, which belong to serovar copenhageni, showed almost the same endonuclease cleavage patterns as the M20 strain; only the Kai ima 702 strain produced an extra band which was not identical to the Ictero No. I-specific extra band when digested with HindIII. The leptospiral restriction endonuclease DNA analysis has revealed taxonomic structures that are unrecognized by serology alone.     

Genotypic differentiation of twelve Clostridium species by polymorphism analysis of the triosephosphate isomerase (tpi) gene

Housekeeping genes encoding metabolic enzymes may provide alternative markers to 16S ribosomal DNA (rDNA) for genotypic and phylogenetic characterization of bacterial species. We have developed a PCR-restriction fragment length polymorphism (PCR-RFLP) assay, targeting the triosephosphate isomerase (tpi) gene, which allows the differentiation of twelve pathogenic Clostridium species. Degenerate primers constructed from alignments of tpi sequences of various gram-positive bacteria allowed the amplification of a 501 bp target region in the twelve Clostridium type strains. A phylogenetic tree constructed from the nucleotidic sequences of these tpi amplicons was well correlated with that inferred from analysis of 16S rDNA gene sequences. The analysis of tpi sequences revealed restriction sites of enzyme AluI that could be species-specific. Indeed, AluI digestion of amplicons from the twelve type strains provided distinct restriction patterns. A total of 127 strains (three to sixteen strains for each species) was further analyzed by PCR-RFLP of the tpi gene, and confirmed that each species could be characterized by one to three restriction types (RTs). The differences between RTs within species could be explained by point mutations in AluI restriction sites of the tpi sequences. PCR-restriction analysis of the tpi gene offers an accurate tool for species identification within the genus Clostridium, and provides an alternative marker to 16S rDNA for phylogenetic analyses.     

Natural transformation of an engineered Helicobacter pylori strain deficient in type II restriction endonucleases

Restriction-modification (RM) systems are important for bacteria to limit foreign DNA invasion. The naturally competent bacterium Helicobacter pylori has highly diverse strain-specific type II systems. To evaluate the roles of strain-specific restriction in H. pylori natural transformation, a markerless type II restriction endonuclease-deficient (REd) mutant was constructed. We deleted the genes encoding all four active type II restriction endonucleases in H. pylori strain 26695 using sacB-mediated counterselection. Transformation by donor DNA with exogenous cassettes methylated by Escherichia coli was substantially (1.7 and 2.0 log(10) for cat and aphA, respectively) increased in the REd strain. There also was significantly increased transformation of the REd strain by donor DNA from other H. pylori strains, to an extent corresponding to their shared type II R-M system strain specificity with 26695. Comparison of the REd and wild-type strains indicates that restriction did not affect the length of DNA fragment integration during natural transformation. There also were no differentials in cell growth or susceptibility to DNA damage. In total, the data indicate that the type II REd mutant has enhanced competence with no loss of growth or repair facility compared to the wild type, facilitating H. pylori mutant construction and other genetic engineering.     

Integrated Maps of the Chromosomes in Dictyostelium Discoideum

Detailed maps of the six chromosomes that carry the genes of Dictyostelium discoideum were constructed by correlating physically mapped regions with parasexually determined linkage groups. Chromosomally assigned regions were ordered and positioned by the pattern of altered fragment sizes seen in a set of restriction enzyme mediated integration-restriction fragment length polymorphism (REMI-RFLP) strains each harboring an inserted plasmid that carries sites recognized by NotI, SstII, SmaI, BglI and ApaI. These restriction enzymes were used to digest high molecular weight DNA prepared from more than 100 REMI-RFLP strains and the resulting fragments were separated and sized by pulsed-field gels. More than 150 gene probes were hybridized to blots of these gels and used to map the insertion sites relative to flanking restriction sites. In this way, we have been able to restriction map the 35 mb genome as well as determine the map position of more than 150 genes to with ~40 kb resolution. These maps provide a framework for subsequent refinement.     

Construction of a chromosome map for the phage group II Staphylococcus aureus Ps55.

The genome size and a partial physical and genetic map have been defined for the phage group II Staphylococcus aureus Ps55. The genome size was estimated to be 2,771 kb by pulsed-field gel electrophoresis (PFGE) using the restriction enzymes SmaI, CspI, and SgrAI. The Ps55 chromosome map was constructed by transduction of auxotrophic and cryptic transposon insertions, with known genetic and physical locations in S. aureus NCTC 8325, into the Ps55 background. PFGE and DNA hybridization analysis were used to detect the location of the transposon in Ps55. Ps55 restriction fragments were then ordered on the basis of genetic conservation between the two strains. Cloned DNA probes containing the lactose operon (lac) and genes encoding staphylococcal protein A (spa), gamma hemolysin (hlg), and coagulase (coa) were also located on the map by PFGE and hybridization analysis. This methodology enabled a direct comparison of chromosomal organization between NCTC 8325 and Ps55 strains. The chromosome size, gene order, and some of the restriction sites are conserved between the two phage group strains.     

A general method to optimize the amount of enzyme in restriction and DNA modification reactions using the beta galactosidase blue-white plaques assay

We propose a simple and economical method for assaying the activity of restriction and other modifying enzymes. The method involves assaying the use of the blue and white colored phenotypes of bacterial colonies obtained by digesting the polylinker sequence of M13 bacteriophage vectors followed by transformation in appropriate strains on X-gal/IPTG plates. In conjunction with restriction enzymes and DNA ligases, the method can evaluate polymerase activity and can be applied to test 3'...5' exonuclease activities such as that of T4 DNA polymerase, without having to use expensive radioisotopes. We describe its application in the assessment of restriction enzymes, DNA ligase and DNA polymerase activities.     

Restriction fragment length polymorphism of Azospirillum strains

Abstract: Total DNA of various Azospirillum strains representing different species was digested with restriction enzymes, Southern blotted and hybridized with four A. brasilense probes. Pairwise comparison of the conserved hybridization fragments was applied to calculate sequence divergence and to group the strains using the unweighted pair group method. The resulting dendrogram grouped the strains according to the known species indicating that the analysis of the restriction fragment length polymorphism is an useful tool for characterizing Azospirillum isolates.     

Identification and characterization of Streptococcus thermophilus strains by pulsed-field gel electrophoresis

The chromosomal DNA of a number of strains of the lactic acid bacterium Streptococcus thermophilus was analysed with the aim of rapidly differentiating and assessing the characteristics of each strain. Pulsed-field gel electrophoresis was used to separate large DNA fragments formed by the restriction enzymes Sma I, Sfi I or Apa I. Hybridization with a non-radioactive DNA probe confirmed the identification of strains as Strep. thermophilus and analysis of the electrophoretic patterns differentiated some strains from others. A more extensive study of the pulsed-field electrophoresis restriction patterns of new isolates of Strep. thermophilus may facilitate assessment of their technological properties by comparison of their restriction patterns with those of reference strains.     

Genotypic analysis of strains of mutans streptococci by pulsed-field gel electrophoresis

The species and serotypes of various strains of S. mutans and S. sobrinus were characterized by pulsed-field gel electrophoresis after the genomic DNA from the various strains had been digested with five restriction enzymes (EcoR I, Xba I, Hind III, Sfi I and BssH II) separately. Among these restriction enzymes, BssH II was very useful for the characterization of species and serotypes and, in particular, digestion discriminated between serotypes d and g. The restriction patterns obtained from the genomic DNA of isolates isolated from children's saliva were essentially identical to those from the genomic DNA of the standard laboratory strains. Patterns of BssH II digests of the genomic DNA of 10 isolates identified as S. sobrinus were characteristic of serotype g of the standard laboratory strains. Our results indicate that digestion with BssH II and subsequence analysis by pulsed-field gel electrophoresis should be useful for the characterization of species and serotypes and for epidemiological studies of mutans streptococci.     

A method for detecting new strains producing DNA modification and restriction enzymes--isoschizomers and isomethylomers of known restrictases and methylases

The paper describes a technique for the detection of new strains producing enzymes which mediate DNA modification and restriction, and isoschizomers and isomethylomers of the known restriction endonucleases and methylases. Three Bacillus subtilis strains whose DNA carries a BamH1 modification have been found. Two of these strains exert the restrictase activity with an R BamH1 specificity.