Identification of a DNA restriction-modification system in Pectobacterium carotovorum strains isolated from Poland.

AIMS: Polish isolates of pectinolytic bacteria from the species Pectobacterium carotovorum were screened for the presence of a DNA restriction-modification (R-M) system. METHODS AND RESULTS: Eighty-nine strains of P. carotovorum were isolated from infected potato plants. Sixty-six strains belonged to P. carotovorum ssp. atrosepticum and 23 to P. carotovorum ssp. carotovorum. The presence of restriction enzyme Pca17AI, which is an isoschizomer of EcoRII endonuclease, was observed in all isolates of P. c. atrosepticum but not in P. c. carotovorum. The biochemical properties, PCR amplification, and sequences of the Pca17AI restriction endonuclease and methyltransferase genes were compared with the prototype EcoRII R-M system genes. Only when DNA isolated from cells of P. c. atrosepticum was used as a template, amplification of a 680 bp homologous to the gene coding EcoRII endonuclease. CONCLUSIONS: Endonuclease Pca17AI, having a relatively low temperature optimum, was identified. PCR amplification revealed that the nucleotide sequence of genes for EcoRII and Pca17AI R-M are different. Dcm methylation was observed in all strains of Pectobacterium and other Erwinia species tested. The sequence of a DNA fragment coding Dcm methylase in P. carotovorum was different from that of Escherichia coli. SIGNIFICANCE AND IMPACT OF THE STUDY: Pca17AI is the first psychrophilic isoschizomer of EcoRII endonuclease. The presence of specific Dcm methylation in chromosomal DNA isolated from P. carotovorum is described for the first time. A 680 bp PCR product, unique for P. c. atrosepticum strains, could serve as a molecular marker for detection of these bacteria in environmental samples.

Mesophilic cyanobacteria producing thermophilic restriction endonucleases.

When searching for the site-specific endonucleases in several strains of Phormidium we made the following observations. Among the 16 strains that originated from 15 species of Phormidium, 12 produced one or more restriction enzymes, of which two produced the highly thermophilic restriction endonucleases PtaI and PpaAII with their optimum activity at 65-80 degrees C, which is far above the lethal temperature for the host microorganism (40 degrees C). These two temperature-resistant enzymes are isoschizomers of known BspMII and TaqI endonucleases, respectively. The presence of the thermophilic TaqI isoschizomer does not seem to play any role in the mesophilic host microorganism, which does not even contain an active cognate methyltransferase. Among the remaining 10 strains, six produced isoschizomers of endonucleases which we first described in cyanobacteria, namely: PfaAII (NdeI), PinBII and PtaI (BspMII), PlaAII (RsalI), PpaAII, PpeI (ApaI). Two enzymes, PauAII (AhaIII) and PfaAII (NdeI), belong to a group of a very rarely occurring isoschizomers. Out of 21 cyanobacterial endonucleases investigated by us, four were active in a wide range of temperatures (from 15 to 60 degrees C) which also extended the optimal growth temperature of the hosts. We assume that our observation on the presence of temperature-resistant restriction enzymes in mesophilic hosts supports the idea of horizontal gene transfer. Restriction modification systems may be an excellent tool for investigation of that phenomenon.

Inhibition of hepadnaviral replication by polyethylenimine-based intravenous delivery of antisense phosphodiester oligodeoxynucleotides to the liver.

Antisense oligodeoxynucleotides (ODNs) appear as attractive anti-hepatitis B virus (HBV) agents. We investigated in vivo, in the duck HBV (DHBV) infection model, whether linear polyethylenimine (lPEI)-based intravenous delivery of the natural antisense phosphodiester ODNs (O-ODNs) can prevent their degradation and allow viral replication inhibition in the liver. DHBV-infected Pekin ducklings were injected with antisense O-ODNs covering the initiation codon of the DHBV large envelope protein, either in free form (O-ODN-AS2) or coupled to lPEI (lPEI/O-ODN-AS2). Following optimization of lPEI/O-ODN complex formulation, complete O-ODN condensation into a homogenous population of small (20-60 nm) spherical particles was achieved. Flow cytometry analysis showed that lPEI-mediated transfer allowed the intrahepatic delivery of lPEI/O-ODN-AS2 to increase three-fold as compared with the O-ODN-AS2. Following 9-day therapy the intrahepatic levels of both DHBV DNA and RNA were significantly decreased in the lPEI/O-ODN-AS2-treated group as compared with the O-ODN-AS2-treated, control lPEI/O-ODN-treated, and untreated controls. In addition, inhibition of intrahepatic viral replication by lPEI/O-ODN-AS2 was not associated with toxicity and was comparable with that induced by the phosphorothioate S-ODN-AS2 at a five-fold higher dose. Taken together, our results demonstrate that phosphodiester antisense lPEI/O-ODN complexes specifically inhibit hepadnaviral replication. Therefore we provide here the first in vivo evidence that intravenous treatment with antisense phosphodiester ODNs coupled to lPEI can selectively block a viral disease-causing gene in the liver.

Hepatitis G virus: molecular organization, methods of detection, prevalence, and disease association.

This article reviews data on hepatitis G virus (HGV) prevalence and possible disease associations in various groups of patients. An important fraction of acute or chronic hepatitis cases probably have a viral etiology and are not attributable to known hepatitis viruses. Therefore, researchers continually are looking for new hepatitis viruses. Among the agents found are members of GB hepatitis viruses, including GB-C virus, or HGV. This review presents the history of the discovery of HGV, its molecular biology and some methods of detection; results of clinical and molecular studies of HGV infection also are discussed.

The FokI methyltransferase from Flavobacterium okeanokoites. Purification and characterization of the enzyme and its truncated derivatives.

The gene encoding the FokI methyltransferase from Flavobacterium okeanokoites was cloned into an Escherichia coli vector. The transcriptional start sites were mapped as well as putative -10 and -35 regions of the fokIM promoter. Enzyme overproduction was ensured by cloning the fokIM gene under the phi 10 promoter of phase T7. M.FokI was purified using a two-step chromatography procedure. M.FokI is a monomeric protein with a M(r) = 76,000 +/- 1,500 under denaturing conditions. It contains 21 Arg residues, and at least one of which is required for activity as shown by inhibition using 2,3-butanedione. Deletion mutants in the N- and C-terminus of M.FokI were isolated and characterized. The N-terminal derivative (M.FokIN) methylates the adenine residue within the sequence 5'-GGATG-3', whereas the C-terminal derivative (M.FokIC) modifies the adenine residue within the sequence 5'-CATCC-3'. Substrate-protection studies, utilizing chemical modification combined with data on the effect of divalent cations and pH on methylation activity, proved the existence of two catalytic centers within the FokI methyltransferase molecule. M.FokI and its truncated derivatives require S-adenosyl-L-methionine as the methyl-group donor, and they are strongly inhibited by divalent cations (Mg2+, Ca2+, Ba2+, Mn2+, and Zn2+) and S-adenosyl-L-homocysteine. The Km values for the methyl donor, S-adenosyl-L-methionine are 0.6 microM (M.FokI), 0.4 microM (M.FokIN), and 0.9 microM (M.FokIC) while the Km values for substrate lambda DNA are 1.2 nM (M.FokI), 1.4 nM (M.FokIN), and 1.3 nM (M.FokIC).

Identification, characterization and purification of the lantibiotic staphylococcin T, a natural gallidermin variant.

Staphylococcin T (StT), an antibacterial agent produced by a Staphylococcus cohnii T strain, was purified to homogeneity by ammonium sulphate precipitation, gel filtration, cation exchange and fast performance liquid chromatography (FPLC). The final yield was about 20%, and over a 1000-fold increase in the specific activity was obtained. Mass determination (2166 Da), amino acid sequencing (Ile-Ala-Xaa-Lys-Phe-Leu-Xaa-Xaa-Pro-Gly-Xaa-Ala-Lys-block) and DNA sequencing demonstrated that StT is identical to gallidermin, a lanthionine-containing antimicrobial peptide. StT has a broad spectrum of bactericidal activity against Gram-positive and some Gram-negative bacteria. StT appears to damage cell membrane, and as a result causes an efflux of ions and an immediate block in macromolecular synthesis. Moreover, electron microscopic observations reveal morphological changes, with a loss of ribosomes and condensation of the nucleoid DNA. These changes are followed by a dissolution of the cell contents resulting in a bacterial ghost composed of seemingly intact cell walls with remnants of the cytoplasmic membrane and internal structure. Since StT exhibits antimicrobial activity especially against the Staphylococcus species, this compound may be of use in the treatment of staphylococcal infections.

Two intertwined methylation activities of the MmeI restriction-modification class-IIS system from Methylophilus methylotrophus.

The class-IIS restriction endonuclease, R.MmeI, was isolated from Methylophilus methylotrophus. It was originally described as a monomeric enzyme, with the native Mr 105000+/-7000, which did not cleave DNA efficiently [Boyd et al. (1986) Nucleic Acids Res. 14, 5255-5274; Tucholski et al. (1995) Gene 157, 87-92]. However, it was discovered that R.MmeI endonucleolytic activity is enhanced by S-adenosyl-l-methionine (AdoMet) and sinefungin, an analogue of AdoMet. Surprisingly, the purified R.MmeI endonuclease was found to have a second enzymatic activity, namely methylation of the adenine residue to N6-methyladenine in the top strand of the MmeI-recognition sequence, 5'-TCCR*AC-3' (*A=meA. The R.MmeI methylating activity requires AdoMet and is increased in the presence of several divalent cations, 20-fold by Mg2+ or Ca2+, and less by Mn2+, Zn2+ and Co2+; however, methylation is inhibited entirely by sinefungin, at concentrations above 9microM. The latter observation shows that the enhancing effect of AdoMet or sinefungin on the DNA cleavage was not related to the process of DNA methylation. Furthermore, a second component of the MmeI restriction-modification system, a M.MmeI methyltransferase, was isolated and purified. The M.MmeI protein was found to have an Mr of 48000+/-2000 (under denaturing conditions) and to methylate both adenine residues (*A) in the MmeI-recognition sequence 5'-TCCR*AC-3'/3'-*AGGYTG-5'. Methylation of the top strand does not inhibit the DNA cleavage by R.MmeI, whereas methylation of both DNA strands blocks the cleavage process.

Amplification of erbB-4 oncogene occurs less frequently than that of erbB-2 in primary human breast cancer.

ErbB-4 protein is a recently discovered member of the ErbB family. The role of ErbB-4 protein in mammary-gland tissue has not been definitively established. To date, the expression of erbB-4 in breast tissue has been determined in only a few cases and, to the best of our knowledge, its amplification has not been examined. We therefore used the double differential polymerase chain reaction (ddPCR) for determination of the amplification profile of erbB-4 and erbB-2, another gene from the ErbB family, in human primary breast cancer specimens. We examined the amplification of the genes in 20 normal breasts and 176 invasive breast cancer samples. Amplification of erbB-2 was detected in 19% and erbB-4 in 13% of the samples studied. Co-amplification of the two oncogenes was found in only five out of 176 samples. Human breast cancer-derived cell lines in most cases overexpress both erbB-2 and erbB-4 (Beerli et al., 1995. Mol. Cell Biol. 15, 6496-6505; Han et al., 1995. Proc. Natl. Acad. Sci. USA 92, 9747-9751), but data on separate erbB-2 overexpression, without overexpression of erbB-4, were also reported (Wosikowski et al., 1997. Clin. Cancer Res. 3, 2405-2414). At the gene level, we found that co-amplification of the genes in the case of human breast cancer is rare. Moreover, an inverse association of the erbB-4 amplification with estrogen receptor activity and direct correlation with the tumor size were found. Due to these correlations, erbB-4 oncogene amplification can be assumed to be of prognostic or predictive value in the diagnosis of breast cancer.

Isolation and characterization of the restriction endonuclease PpeI from Phormidium persicinum.

PpeI is a type II restriction endonuclease isolated from cyanobacterial strain Phormidium persicinum. The endonuclease PpeI, an isoschizomer of ApaI, recognizes the hexanucleotide sequence (5'-GGGCC/C-3') and cleaves, after the second C, producing four nucleotide 3'-cohesive ends.

Interaction of the MboII restriction endonuclease with DNA.

The binding of the MboII restriction endonuclease (R.MboII; ENase) to DNA containing its recognition site was investigated using a mobility shift assay. R.MboII forms specific, stable and immunodetectable complexes with its canonical target sequence. The association constant (Ka) of R.MboII was calculated to be 2.8 x 10(9)/M, and is about 10(4)-fold higher than the Ka value for non-specific binding. Based on results obtained after sedimentation of the R.MboII-DNA complex in a glycerol gradient and measurement of the retardation of the complexes in polyacrylamide gels, we conclude that specific binding to the canonical sequence involves a monomer of R.MboII. DNase I footprinting has shown that the enzyme covers 16 nucleotides of DNA on the 5'-GAAGA-3' strand.

Purification and characterization of the restriction endonuclease AvcI from Actinomyces cristalomycini.

The restriction endonuclease AvcI, an isoschizomer of Sau96I [Sussenbach et al., Nucleic Acids Res. 5 (1978) 1153-1163], was purified from Actinomyces cristalomycini. AvcI recognizes a 5-bp palindromic sequence, 5'-G decreases GNCC and cleaves it after the first G residue producing a 3-nucleotide 5'-overhang.

Purification and characterization of two restriction endonucleases isolated from Phormidium inundatum.

We have isolated two restriction endonucleases, PinBI and PinBII, from the cyanobacterial strain Phormidium inundatum, and identified them as isoschizomers of AvaIII and BspMII, respectively.

SacNI, an isoschizomer of BanII isolated from Streptomyces achromogenes recognizes the 5'-GRGCY/C sequence.

SacNI, an isoschizomer of the restriction endonuclease, BanII [Sugisaki et al., Nucleic Acids Res. 10 (1982) 5747-5752], has been isolated from Streptomyces achromogenes N-J-H. SacNI recognizes the palindromic sequence, 5'-GRGCY/C, and cleaves within the recognition sequence, generating a 3' protruding RGCY end (where R = A or G, and Y = C or G).

MmeI, a class-IIS restriction endonuclease: purification and characterization.

Two restriction endonucleases, MmeI and MmeII, from Methylophilus methylotrophus were purified to homogeneity. Both enzymes belong to the class-II restriction endonucleases (ENases) but exhibit very different enzymatic and physical properties. MmeII is a typical member of class-II ENases. It is a polymeric protein composed of 50-kDa subunits. In contrast to MmeII, MmeI is a monomeric protein of 101 kDa, cleaving a DNA molecule 20/18 nucleotides away from the asymmetric recognition sequence (5'-TCCRAC-3'); therefore, it is classified as a member of subclass-IIS. MmeI has an pI of 7.85 and is active in the pH range 6.5 to 10 with the optimum at 7 to 8. Increasing salt concentration creates an inhibitory effect on MmeI: 40 mM KCl decreases activity by 50%, 100 mM completely inhibits DNA cleavage. Tris.HCl (pH 7.5) at a concentration exceeding 20 mM inhibits MmeI activity. Mg2+ stimulates MmeI in the range of 0.2 to 35 mM, with the optimum between 0.5 and 10 mM.

Atypical DNA-binding properties of class-IIS restriction endonucleases: evidence for recognition of the cognate sequence by a FokI monomer.

The DNA-binding properties of the FokI restriction endonuclease were studied using the gel-mobility-shift assay. Specific recognition of the cognate sequence and cleavage of DNA are distinguishable functions and can be separated. FokI binds to its recognition site predominantly as a monomer. At high concentrations, FokI exhibits a cooperative recognition sequence-dependent aggregation. In 20 mM KCl/10 mM Tris.HCl buffer, the binding constant of FokI to its cognate site is equal 6.0-7.9 x 10(8)/mol and is lower than the values for most gene-regulatory proteins. FokI binding is 600-1500 times weaker to non-cognate double-stranded DNA than to the GGATG site, and 30,000 times weaker to single-stranded DNA or tRNA. The method of Bading [Nucleic Acids Res. 16 (1988) 5241-5248], used for determining the stoichiometry of protein bound to DNA by gel-mobility-shift assay, is extended.

Purification and properties of the MboII, a class-IIS restriction endonuclease.

After five purification steps a homogeneous preparation of endonuclease MboII was obtained, and several properties of the enzyme were determined. MboII is a monomer, with Mr under native and denaturing conditions being 47-49 x 10(3) Da. Endonuclease MboII is a basic protein (pI 8.3) which remains active when Mg2+ is replaced by Mn2+, Co2+, Ca2+, or Fe2+. MboII exhibits a star activity in the presence of some of the following reagents or ions: DMSO, glycerol, ethanol (and Co2+ or Mn2+ at pH 6). MboII does not bend DNA and is heat sensitive, losing activity after 15 min at 50 degrees C.