Добірка наукової літератури з теми "Restriction Endonuclease KpnI"

A Pseudomonas aeruginosa bacteriophage, [Formula: see text], with extremely limited host range has been isolated. It belongs to the virus family Podoviridae, morphological type C1, and possesses a head diameter of 45 nm. The phage has a buoyant density in CsCl of 1.516 g/cm3, and its mass is 45 × 106 daltons. The phage particles are composed of double-stranded DNA (49.9 mol% G + C; 42.4 kilobase pairs) and 11 structural proteins (66% by weight). The major head protein, P5, has a Mr of 34 500. The DNA is not cut by SalI or XhoI restriction endonucleases, but is cut by PvuII (1 site), KpnI and BglII (2 sites), PvuI (4 sites), BamHI (7 sites), EcoRI (9 sites), and HindIII (12 sites). A restriction endonuclease map is presented.Key words: Pseudomonas, bacteriophage, DNA, restriction map, structural proteins, electron microscopy.

Ninety-two equine herpesvirus type 1 isolates were recovered from aborted, stillborn, or neonatal foals from Ontario, Canada, from 1986 to 1992. From this total, 32 strains were randomly chosen for further study. Four or 5 isolates from each winter were selected, each from a different premises, and characterized by restriction enzyme analysis using BamHI, KpnI, BglII, HindIII, and EcoRI. Additional isolates from 2 premises and from a zebra foal were also assessed. For the strains isolated in 1986 and 1989–1992, the DNA pattern of 18 strains was similar to that of type 1P (Kentucky D) for BamHI and KpnI. None of the 32 strains studied could be differentiated by HindIII or EcoRI. Using BglII, an inconsistent fragment pattern and distribution were observed. Of the 8 strains isolated in 1987 and 1988, 7 were assigned into the 1B prototype group. The geographic distribution of 17 type 1P and 12 1B isolates was random across southern Ontario. These findings suggest that both electropherotypes can be recovered from horses in Ontario. The patterns of the additional equine isolates from the same premises were identical. The zebra isolate was different from the prototype equine herpesvirus type 1 and type 4 patterns and from all other equine isolates.

Broiler carcasses (n = 325) were sampled at three sites along the processing line (prescalding, prechilling, and post-chilling) in a commercial poultry processing plant during five plant visits from August to October 2004. Pulsed-field gel electrophoresis (PFGE) was used to determine the genomic fingerprints of Campylobacter coli (n = 27), Campylobacter jejuni (n = 188), Arcobacter butzleri (n = 138), Arcobacter cryaerophilus 1A (n = 4), and A. cryaerophilus 1B (n = 31) with the restriction enzymes SmaI and KpnI for Campylobacter and Arcobacter, respectively. Campylobacter species were subtyped by the Centers for Disease Control and Prevention PulseNet 24-h standardized protocol for C. jejuni. A modification of this protocol with a different restriction endonuclease (KpnI) and different electrophoresis running conditions produced the best separation of restriction fragment patterns for Arcobacter species. Both unique and common PFGE types of Arcobacter and Campylobacter strains were identified. A total of 32.8% (57 of 174) of the Arcobacter isolates had unique PFGE profiles, whereas only 2.3% (5 of 215) of the Campylobacter isolates belonged to this category. The remaining Arcobacter strains were distributed among 25 common PFGE types; only eight common Campylobacter PFGE types were observed. Cluster analysis showed no associations among the common PFGE types for either genus. Each of the eight common Campylobacter types consisted entirely of isolates from one sampling day, whereas more than half of the common Arcobacter types contained isolates from different sampling days. Our results demonstrate far greater genetic diversity for Arcobacter than for Campylobacter and suggest that the Campylobacter types are specific to individual flocks of birds processed on each sampling day.

Several phytoplasmas have been reported to be associated with phyllody of strawberry fruit, including clover yellow edge, clover proliferation, clover phyllody, eastern and western aster yellows, STRAWB2, strawberry multicipita, and Mexican periwinkle virescence phytoplasmas. Plant symptoms in addition to phyllody may include chlorosis, virescence, stunting, or crown proliferation. In this report we describe a new phytoplasma in association with strawberry leafy fruit (SLF) disease in Maryland. Diseased plants exhibited fruit phyllody, floral virescence, leaf chlorosis, and plant stunting. Phytoplasmal 16S rDNA was amplified from SLF diseased plants by using the polymerase chain reaction (PCR) primed by primer pair P1/P7 and was reamplified in nested PCR primed by primer pair R16F2n/R2 (F2n/R2) as previously described (1). These results indicated the presence of a phytoplasma, designated SLF phytoplasma. Identification of SLF phytoplasma was accomplished by restriction fragment length polymorphism (RFLP) analysis of DNA amplified in PCR primed by F2n/R2, using endonuclease enzyme digestion with AluI, HhaI, KpnI, HaeIII, MseI, HpaII, RsaI, and Sau3AI. Phytoplasma classification was done according to the system of Lee et al. (2). RFLP analyses of rDNA amplified in three separate PCRs gave identical patterns. On the basis of collective RFLP patterns of the amplified 16S rDNA, the SLF phytoplasma was classified as a member of group 16SrIII (group III, X-disease phytoplasma group). The HhaI RFLP pattern of SLF 16S rDNA differed from that of the apparently close relative, clover yellow edge (CYE) phytoplasma, and all other phytoplasmas previously described in group III. Based on these results, SLF phytoplasma was classified in a new subgroup, designated subgroup K (III-K), within group III. The 1.2 kbp DNA product of PCR primed by primer pair F2n/R2 was sequenced, and the sequence deposited in GenBank under Accession No. AF 274876. Results from putative restriction site analysis of the sequence were in agreement with the results from actual enzymatic RFLP analysis of rDNA amplified from phylloid strawberry fruit. Although the sequence similarity between the 1.2-kbp fragment from the 16S rDNA of SLF phytoplasma and that of CYE phytoplasma was 99.9%, the Hha1 RFLP pattern of SLF rDNA supports the conclusion that the SLF phytoplasma may be closely related to, but is distinct from, CYE and other strains that are classified in group III. These findings contribute knowledge about the diversity of phytoplasmas affiliated with group III and the diversity of phytoplasmas associated with diseases in strawberry. References: (1) R. Jomantiene et al. Int. J. Syst. Bacteriol. 48:269, 1998. (2) I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998.

Phytoplasma strains that belong to group 16SrI (aster yellows phytoplasma group), subgroup A (I-A, North American tomato big bud phytoplasma subgroup) were discovered in diverse plant species in Lithuania. Plants in which the strains were found exhibited symptoms characteristic of infections by phytoplasma. Carrot (Daucus sativus) with carrot proliferation disease exhibited symptoms of proliferation of the crown, chlorosis of young leaves, and reddening of mature leaves. Diseased phlox (Phlox paniculata) exhibited symptoms of virescence and leaf chlorosis. Diseased sea-lavender (Limonium sinuatum) exhibited abnormal proliferation of shoots, chlorosis of young leaves, reddening of mature leaves, and degeneration of flowers. Diseased hyacinth (Hyacinthus orientalis) exhibited chlorosis of leaves and undeveloped flowers. Diseased Aconitum sp. exhibited proliferation of shoots. Phytoplasma-characteristic ribosomal (r) DNA was detected in the plants by use of the polymerase chain reaction (PCR). The rDNA was amplified in PCR primed by primer pair P1/P7 and reamplified in nested PCR primed by primer pair R16F2n/R16R2 (F2n/R2), as previously described (1). The phytoplasmas were classified through restriction fragment length polymorphism (RFLP) analysis of 16S rDNA, amplified in the nested PCR primed by F2n/R2, using single endonuclease enzyme digestion with AluI, MseI, KpnI, HhaI, HaeIII, HpaI, HpaII, RsaI, HinfI, TaqI, and Sau3AI. Collective RFLP patterns indicated that all detected phytoplasma strains were affiliated with subgroup I-A. The 16S rDNA amplified from the phytoplasma (CarrP phytoplasma) in diseased carrot was cloned in Escherichia coli, sequenced, and the sequence deposited in the GenBank data library (GenBank accession no. AF291682). The 16S rDNAs of CarrP and tomato big bud (GenBank acc. no. AF222064) phytoplasmas shared 99.8% nucleotide sequence similarity. Phytoplasmas belonging to group 16SrIII (3), group 16SrV (D. Valiunas, unpublished data), and subgroup I-C in group 16SrI (2,3) occur in Lithuania. This report records the first finding of a subgroup I-A phytoplasma in the Baltic region and expands the known plant host range of this phytoplasma subgroup. References: (1) R. Jomantiene et al. Int. J. Syst. Bacteriol. 48:269, 1998. (2) Jomantiene et al. Phytopathology 90:S39, 2000. (3) Staniulis et al. Plant Dis. 84:1061, 2000.

The whole human cytomegalovirus strain AD169 genome was cloned into plasmid pAT153 in the form of 25 HindIII fragments. Double and triple digestions of the recombinant plasmids with restriction endonucleases BamHI, BglII, ClaI, DraI, EcoRI, EcoRV, HindIII, HpaI, KpnI, PaeR7, PstI, SphI and XbaI yielded a detailed restriction map of human cytomegalovirus DNA. Knowing the exact position of numerous restriction sites in the viral DNA molecule, we have been able to examine very closely the heterologous region between the long and the short segments of the human cytomegalovirus genome. Key words: DNA, physical map, cytomegalovirus, restriction endonucleases, HCMV.

Restriction modification (RM) systems are important components of bacterial immune system. Their primary function is to protect the bacteria from invading bacteriophages. Based on the subunit composition, enzyme properties and cofactor requirements, they are classified into four different types (Type I to Type IV). The Type II group comprises of two enzymes with contrasting activities- a restriction endonuclease (REase) and a methyltransferase (MTase). A Type II REase recognizes and cleaves specific DNA sequences on incoming foreign DNA while the MTase recognizes and methylates the base within the DNA sequence and protects the bacterial genome. These REases bind DNA in a sequence specific manner and cleave in the presence of metal ions. The work presented in this thesis deals with R.KpnI, which is isolated from Klebsiella pneumoniae OK8. The enzyme recognizes a palindromic double stranded DNA sequence, GGTAC↓C, and cleaves as indicated. It belongs to the HNH superfamily of nucleases and is characterized by the presence of a ββα- Me finger motif. R.KpnI has inherent promiscuous activity in the presence of Mg2+. It also shows activity in the presence of both the alkaline earth and transition metal ions. However, the enzyme catalyses very high fidelity DNA cleavage in the presence of Ca2+ unlike other REases. The basis for this property is yet to be investigated. The in vivo promiscuous activity of the enzyme could be advantageous for the bacteria to target the incoming foreign DNA. However, the importance of the promiscuous activity of REases in bacterial physiology is not completely understood. The work presented in this thesis describes the mechanism of Ca2+ mediated cleavage specificity and understanding the biological significance of the promiscuous activity of REases. Chapter 1 provides a general introduction and overview of the literature on Type II REases. It deals with general features of Type II REases, DNA binding, mechanism of phosphodiester bond hydrolysis and also the role of the metal ions in modulating specificity of these enzymes. New developments in engineering of new specificities in the REases are dealt with in this chapter. The wide prevalence and diversity of RM systems indicate that they might have additional biological roles and such functions have been discussed at the end of this chapter. One of the unusual features of R.KpnI is Ca2+ mediated specific DNA cleavage. Chapter 2 describes the mechanism of unusual Ca2+-mediated activity of R.KpnI. Substitution of residues in a putative Ca2+ binding motif, E132xD134xD136, showed decreased levels of DNA cleavage and Ca2+ coordination. However, the invariant His of the catalytic HNH motif acts as a general base for nucleophile activation, and the other two active site residues, D148 and Q175, also participate in Ca2+-mediated cleavage. Insertion of a 10- amino acid linker to disrupt the spatial organization of the ExDxD and HNH motifs impairs Ca2+ binding and affects DNA cleavage by the enzyme. This study showed the role of distinct Ca2+ binding motif in conferring site specific cleavage upon Ca2+ binding; the motif is needed for the promiscuous activity of the Mg2+- bound enzyme. From this study, it is evident that metal ion-mediated modulation of DNA cleavage is one of the key features of R.KpnI. The effect of altering the metal ion binding residues on the cleavage specificity of the enzyme is presented in Chapter 3. A conservative mutation of the metal-coordinating residue D148 to E results in the elimination of the Ca2+-mediated cleavage but imparts high cleavage fidelity with Mg2+. High cleavage fidelity of the mutant D148E is achieved through better discrimination of the target site at the binding and cleavage steps. Biochemical experiments and molecular dynamics simulations suggest that the mutation inhibits Ca2+-mediated cleavage activity by altering the geometry of the Ca2+-bound HNH active site. The present study showed that plasticity in active site to accommodate different metal ions is related to promiscuous activity, and altering of the metal ion coordination is a plausible way to reduce the promiscuous activity of metalloenzymes. The in vivo promiscuous activity of R.KpnI may have additional cellular functions. In Chapter 4 a new intracellular function of the promiscuous REase and elucidation of its biological significance is presented. Induction of programmed cell death in bacteria by the REase is described. Bacterial survival studies and microscopic analysis were carried out to examine the role of enzyme promiscuity in bacterial cell death. It is observed that REase triggered DNA damage leads to cell death, releasing nutrients during stationary phase which supports the growth of rest of the population. These observations may open up new avenues to understand the additional roles of REases in bacterial physiology.