大熊猫线粒体DNA的九种限制酶图谱

本文用9种限制性内切酶（BamHⅠ,BglⅠ,BglⅡ,EcoRⅠ,EcoRⅤ,PstⅠ,PvuⅡ,SalⅠ,XhoⅠ）分析大熊猫的线粒体DNA（mtDNA）。构建其中5种酶（BamHⅠ,EcoRⅠ,EcoRⅤ,PstⅠ,PvuⅡ）的mtDNA物理图谱。大熊猫mtDNA的分子大小约为16.4 Kb,酶切位点是随机分布。我们的结果为进一步研究大熊猫mtDNA进化提供了基础资料。     

Physical and gene mapping of chloroplast DNA from Atriplex triangularis and Cucumis sativa.

A rapid and simple method for constructing restriction maps of large DNAs (100-200 kb) is presented. The utility of this method is illustrated by mapping the Sal I, Sac I, and Hpa I sites of the 152 kb Atriplex triangularis chloroplast genome, and the Sal I and Pvu II sites of the 155 kb Cucumis sativa chloroplast genome. These two chloroplast DNAs are very similar in organization; both feature the near-universal chloroplast DNA inverted repeat sequence of 22-25 kb. The positions of four different genes have been localized on these chloroplast DNAs. In both genomes the 16S and 23S ribosomal RNAs are encoded by duplicate genes situated at one end of the inverted repeat, while genes for the large subunit of ribulose-1,5-bisphosphate carboxylase and a 32 kilodalton photosystem II polypeptide are separated by 55 kb of DNA within the large single copy region. The physical and genetic organization of these DNAs is compared to that of spinach chloroplast DNA.     

Chloroplast DNA differences between two species of Oenothera subsection Munzia: location in the chloroplast genome and relevance to possible interactions between nuclear and plastid genomes

Summary The chloroplast DNAs (cpDNAs) of Oenothera berteriana and Oe. odorata (subsection Munzia) were examined by restriction endonuclease analysis with Sal I, Pvu II, Kpn I, Pst I, Hind III, and Bam HI. The fragment patterns show that these cpDNAs have all 133 restriction sites in common as well as a lot of individual bands. Nevertheless the cpDNAs of the two species can be distinguished by distinct differences in size between a small number of fragments. The 42 cleavage sites produced by Sal I, Pvu II and Kpn I were mapped on the circular cpDNAs. This was achieved by an approach which combined experimental and mathematical procedures. The overall serial order of the fragments was found to be the same for both cpDNAs. The size differences of individual fragments in the Sal I, Pvu II and Kpn I patterns between Oe. berteriana and Oe. odorata cpDNA are located within five regions scattered along the plastid chromosome. Two of these regions have been localized in the larger and one in the smaller of the two single-copy parts of the cpDNA molecule. The remaining two overlap the borders between the large single-copy and each of the duplicated parts of the molecule. The positions of distinct restriction sites are altered among the two Oenothera plastome DNAs by 0.02–0.4 MDa (30–600 base pairs). These alterations probably result from insertions/deletions.Abbreviations cpDNA chloroplast, plastid DNA - Oe. Oenothera - MDa Megadalton - rRNA, rDNA ribosomal RNA, DNA Dedicated to Professor Berthold Schwemmle, Tübingen, on the occasion of his 60th birthday     

Substrate recognition by the Pvu II endonuclease: binding and cleavage of CAG5mCTG sites.

The Pvu II restriction endonuclease (R. Pvu II) cleaves CAG downward arrowCTG sequences as indicated, leaving blunt ends. Its cognate methyltransferase (M. Pvu II) generates N4-methylcytosine, yielding CAGN4mCTG, though the mechanism by which this prevents cleavage by R. Pvu II is unknown. The heterologous 5-methylcytosinemethylation CAG5mCTG has also been reported to prevent cleavage by R. Pvu II and this has been used in some cloning methods. Since this heterologousmethylation occurs at the native methylated base, it can provide insights into the detection of DNAmethylation by R. Pvu II. We found that the cloned gene for R. Pvu II could not stably transform cells protected only by M. Alu I (AG5mCT) and then determined that R. Pvu II cleaves CAG5mCTG in vitro, even when both strands are methylated. DNase I footprint analysis and competition experiments reveal that R. Pvu II binds to CAG5mCTG specifically, though with reduced affinity relative to the unmethylated sequence. These results provide biochemical support for the publishedstructures of R. Pvu II complexed with DNA containing CAGCTG and CAG5-iodoCTG and support a model for how methylation interferes with DNA cleavage by this enzyme.     

The ability of bacterial DNA methyltransferases to use methylcobalamine as a cofactor in DNA methylation reactions

The ability of bacterial DNA methyltransferases Alu I, Cfr I, Cfr 6, Cfr 10, Eco RI, Eco RII, Msp I, Mva I, Pvu I, Pvu II, and Sau 3A to use methylcobalamine and methylmethionine as cofactors of DNA methylation in vitro has been investigated. These bacterial DNA methyltransferases used methylcobalamine, but not methylmethionine for DNA methylation.     

Structure of pvu II DNA-(cytosine N4) methyltransferase, an example of domain permutation and protein fold assignment.

We have determined the structure of Pvu II methyltransferase (M. Pvu II) complexed with S -adenosyl-L-methionine (AdoMet) by multiwavelength anomalous diffraction, using a crystal of the selenomethionine-substituted protein. M. Pvu II catalyzes transfer of the methyl group from AdoMet to the exocyclic amino (N4) nitrogen of the central cytosine in its recognition sequence 5'-CAGCTG-3'. The protein is dominated by an open alpha/beta-sheet structure with a prominent V-shaped cleft: AdoMet and catalytic amino acids are located at the bottom of this cleft. The size and the basic nature of the cleft are consistent with duplex DNA binding. The target (methylatable) cytosine, if flipped out of the double helical DNA as seen for DNA methyltransferases that generate 5-methylcytosine, would fit into the concave active site next to the AdoMet. This M. Pvu IIalpha/beta-sheet structure is very similar to those of M. Hha I (a cytosine C5 methyltransferase) and M. Taq I (an adenine N6 methyltransferase), consistent with a model predicting that DNA methyltransferases share a common structural fold while having the major functional regions permuted into three distinct linear orders. The main feature of the common fold is a seven-stranded beta-sheet (6 7 5 4 1 2 3) formed by five parallel beta-strands and an antiparallel beta-hairpin. The beta-sheet is flanked by six parallel alpha-helices, three on each side. The AdoMet binding site is located at the C-terminal ends of strands beta1 and beta2 and the active site is at the C-terminal ends of strands beta4 and beta5 and the N-terminal end of strand beta7. The AdoMet-protein interactions are almost identical among M. Pvu II, M. Hha I and M. Taq I, as well as in an RNA methyltransferase and at least one small molecule methyltransferase. The structural similarity among the active sites of M. Pvu II, M. Taq I and M. Hha I reveals that catalytic amino acids essential for cytosine N4 and adenine N6 methylation coincide spatially with those for cytosine C5 methylation, suggesting a mechanism for amino methylation.     

结核分枝杆菌中插入序列的研究

用人型复合分枝杆菌的特异插入序列IS6110和IS1081制 备探针,对5种限制性内切酶消化的结核分枝杆菌DNA进行杂交。结果表明,经PvuⅡ酶消化 的结核分枝杆菌DNA,用IS6110制备的探针进行杂交呈现高度多态性,说明IS6110对于人型 复合分枝杆菌分型研究和结核病流行病学研究具有很大价值。用IS6110制备的317bp探针对4 6株人结核分枝杆菌分离株多态性分析研究证实,这些菌株呈现高度DNA多态性,而且所含拷 贝数也极为不同,一般含7～18个拷贝。     

Structural organization and evolution of the plastid genome of Vaucheria sessilis (Xanthophyceae)

The plastid DNAs of 18 Vaucheria sessilis strains from various habitats in western Europe were digested with the restriction endonucleases Eco RI, Sal I, Bam HI and Pvu II. Their restriction patterns showed variable fragment divergencies. Two main groups of plastid genomes were recognized, which were substantiated by morphological features. The differences among the restriction patterns could be attributed to the loss or appearance of restriction sites and to minor size variations caused by deletions/insertions. The Sal I and Bam HI restriction sites which together discriminate six different plastid genomes were mapped on the circular molecule of 124 kilobase paris (kbp). The plastid genomes of several Vaucheria sessilis strains were shown to exist in two inversion isomers caused by intramolecular recombination within the inverted repeat segments.     

Genomic variability within laboratory and wild isolates of the trichostrongyle mouse nematode Heligmosomoides polygyrus

PCR-RFLP techniques have been used to characterize wild and laboratory isolates of the trichostrongyle nematode Heligmosomoides polygyrus from the wood mouse Apodemus sylvaticus and the laboratory mouse Mus musculus respectively. Both isolates can be distinguished by eight endonuclease digestions of the ITS region of the rDNA repeat namely, Alu I, Dde I, Hpa II, Hae III, Hinf I, Hha I, Pvu II and Sal I. In two of the digests, Hinf I and Rsa I, a minor polymorphism was observed in the wild isolate of H. polygyrus which has been cultured in laboratory-bred A. sylvaticus for several generations when compared with H. p. polygyrus from wild A. sylvaticus. A minor polymorphism was also identified in further wild isolates of H. polygyrus collected from A. sylvaticus in a field site in Egham, Surrey. However no evidence of polymorphism was observed in the laboratory isolate of H. polygyrus from the CD1 strain of M. musculus and the laboratory-bred A. sylvaticus. Reasons for this are discussed and further studies on the population genetics of H. polygyrus are suggested.     

Biochemical transformation of thymidine kinase (TK)-deficient mouse cells by herpes simplex virus type 1 DNA fragments purified from hybrid plasmids.

The thymidine kinase (TK) gene of HSV-1 has been cloned in Escherichia coli K12 plasmids, pMH1, pMH1A, and pMH4. These plasmids contain a 1,92Obp HSV-1 TK DNA sequence, which replaces a 2,067 bp EcoR I to Pvu II sequence of plasmid pBR322 DNA. Superhelical DNAs of plasmids pMH1, pMH1A, and pMH4 as well as plasmid DNAs cleaved by EcoR I, Hinc II, Bg1 II, Sma I, and Pvu II transformed TK-deficient LM(TK-) cells to the TK+ phenotype. A 1,230bp EcoR I-Sma I fragment purified from pMH1 DNA (and from plasmid pAGO, DNA, the parent of pMH1) also transformed LM(TK-) cells. Serological and disc PAGE studies demonstrated that the TK activity expressed in biochemically transformed cells were HSV-1-specific. The experiments suggest that the HSV-1 TK coding region may be contained within a l.1kbp DNA sequence extending from about the Hinc II (or Bgl II) cleavage site to the Sma I site. 35S-methionine labeling experiments carried out on cell lines transformed by Hinc II-cleaved pMH1 DNA and by the EcoR I-Sma I fragment showed that the TKs purified from the transformed cells consisted of about 39-40,000 dalton polypeptides.     

The use of fractionation in a polyethylene glycol-dextran two-phase system for the testing and purification of restriction endonucleases

A simple procedure was developed for testing and purification of restriction endonucleases Msp I, Pst I, Bam HI, Pvu I, Pvu II that includes biomass destruction, fractionation of cell-free extracts in the aqueous two-phase (polyethylene glycol-dextran) system and chromatography oh phosphocellulose. Optimal conditions for the fractionation of Msp I, Pst I, Bam HI, Pvu II, EcoR I, EcoR II, BspR I, Alu I were chosen. For separation of Pvu I and Pvu II gel filtration through biogel A-0.5 m was additionally introduced.     

DNA of Epstein-Barr Virus. II. Comparison of the Molecular Weights of Restriction Endonuclease Fragments of the DNA of Epstein-Barr Virus Strains and Identification of End Fragments of the B95-8 Strain

Incubation of the DNA of the B95-8 strain of Epstein-Barr virus [EBV (B95-8) DNA] with EcoRI, Hsu I, Sal I, or Kpn I restriction endonuclease yielded 8 to 15 fragments separable on 0.4% agarose gels and ranging in molecular weight from less than 1 to more than 30 x 10(6). Bam I and Bgl II yielded fragments smaller than 11 x 10(6). Preincubation of EBV (B95-8) DNA with lambda exonuclease resulted in a decrease in the Hsu I A and Sal I A and D fragments, indicating that these fragments are positioned near termini. The electrophoretic profiles of the fragments produced by cleavage of the DNA of the B95-8, HR-1, and Jijoye strains of EBV were each distinctive. The molecular weights of some EcoRI, Hsu I, and Sal I fragments from the DNA of the HR-1 strain of EBV [EBV (HR-1) DNA] and of EcoRI fragments of the DNA of the Jijoye strain of EBV were identical to that of fragments produced by cleavage of EBV (B95-8) DNA with the same enzyme, whereas others were unique to each strain. Some Hsu I, EcoRI, and Sal I fragments of EBV (HR-1) DNA and Kpn I fragments of EBV (B95-8) DNA were present in half-molar abundance relative to the majority of the fragments. In these instances, the sum of the molecular weights of the fragments was in excess of 10(8), the known molecular weight of EBV (HR-1) and (B95-8) DNA. The simplest interpretation of this finding is that each EBV (HR-1), and possibly also (B95-8), DNA preparation contains two populations of DNA molecules that differ in the arrangement of DNA sequences about a single point, such as has been described for herpes simplex virus DNA. Minor fragments could also be observed if there were more than one difference in primary structure of the DNAs. The data do not exclude more extensive heterogeneity in primary structure of the DNA of the HR-1 strain. However, the observation that the relative molar abundance of major and minor fragments of EBV (HR-1) DNA did not vary between preparations from cultures that had been maintained separately for several years favors the former hypothesis over the latter.     

Genome organization of RNA tumor viruses II. Physical maps of in vitro-synthesized Moloney murine leukemia virus double-stranded DNA by restriction endonucleases.

Physical maps of the genome of Moloney murine leukemia virus (M-MLV) DNA were constructed by using bacterial restriction endonucleases. The in vitro-synthesized M-MLV double-stranded DNA was used as the source of the viral DNA. Restriction endonucleases Sal I and Hind III cleave viral DNA at only one site and, thus, generate two DNA fragments. The two DNA fragments generated by Sal I are Sal IA (molecular weight, 3.5 x 10(6)) and Sal IB (molecular weight, 2.4 x 10(6)) and by Hind III are Hind IIIA (molecular weight, 3.6 x 10(6) and Hind IIIB (molecular weight, 2.3 x 10(6)). Restriction endonuclease Bam I generates four fragments of molecular weights of 2.1 x 10(6) (Bam IA), 2 X 10(6) (Bam IB), 1.25 X 10(6) (Bam IC), and 0.24 x 10(6) (Bam ID), whereas restriction endonuclease Hpa I cleaves the M-MLV double-stranded DNA twice to give three fragments of molecular weights of 4.4 x 10(6) (Hpa IA), 0.84 X 10(6) (Hpa IB), and 0.74 x 10(6) (Hpa IC). Digestion of M-MLV double-stranded DNA with restriction endonuclease Sma I produces four fragments of molecular weights of 3.9 x 10(6) (Sma IA), 1.3 X 10(6) (Sma IB), 0.28 X 10(6) (Sma IC), and 0.21 x 10(6) (Sma ID). A mixture of restriction endonucleases Bgl I and Bgl II (Bgl I + II) cleaves the viral DNA at four sites generating five fragments of approximate molecular weights of 2 x 10(6) (Bgl + IIA), 1.75 X 10(6) (Bgl I + IIB), 1.25 X 10(6) (Bgl I + IIC), 0.40 X 10(6) (Bgl I + IID), and 0.31 x 10(6) (Bgl I + IIE). The order of the fragments in relation to the 5' end and 3' end of the genome was determined either by using fractional-length M-MLV double-stranded DNA for digestion by restriction endonucleases or by redigestion of Sal IA, Sal IB, Hind IIIA, and Hind IIIB fragments with other restriction endonucleases. In addition, a number of other restriction endonucleases that cleave in vitro-synthesized M-MLV double-stranded DNA have also been listed.     

Induction of sister-chromatid exchanges by restriction endonucleases

Restriction endonucleases Cfo 1, Pvu II, Sma I, Hpa II, Taq I and Hae III were tested for their ability to induce SCEs in CHO cells. The results indicate that the DNA double-strand breaks induced during S-phase by these enzymes lead to an increase in the frequencies of SCEs.     

Isolation of DNA fragment containing phoS gene of Escherichia coli K-12

The DNA fragment containing the phoS gene, a regulatory gene for alkaline phosphatase, has been isolated from Escherichia coli K-12 chromosomal DNA by cutting off the DNA with Hind III restriction enzyme and by cloning the gene with plasmid vector pTP 4 which was constructed in this study. The isolated fragment was of about 12.3 kbp and seemed to contain the phoT, glmS, and bgl genes. The 12.3 kbp Hind III fragment was subjected to restriction enzymes EcoR I, BamH I, Sal I, and Pst I, and was found to possess two EcoR I, no BamH I, a Sal I, and four Pst I sites. Partial deletion using these restriction enzymes suggested that the about 6 kbp Hind III-Pst I fragment contained the phoS and phoT genes. Further analysis with other restriction enzymes revealed that the 6 kbp Hind III-Pst I fragment contained a BstE II, two Mlu I and four Hpa I sites. The deletion of these restriction sites using single-strand-specific nuclease S1 suggested that the BstE II and one of Mlu I sites were in the phoT gene, and the BstE II and two Mlu I sites were not in the phoS gene.     

Restriction analysis of the plasmid and chromosomal DNA of the strain Streptomyces fradiae--a producer of neomycin

Plasmid DNA with a molecular weight of 53-55 Md was isolated from Streptomyces fradiae strain 676 producing neomycin on centrifugation of the DNA preparation at the density gradient of cesium chloride-ethidium bromide. The extrachromosomal DNA had 6 recognition sites for Bgl II, more than 13 recognition sites for Bam HI, more than 14 recognition sites for Kpn I, more than 18 recognition sites for Pst I, more than 20 recognition sites for Sac I, more than 21 recognition sites for Pvu II and no recognition sites for Eco RI, Eco RV, Hind III and Sla I.