Cloning and expression of the Apa LI, Nsp I, Nsp HI, Sac I, Sca I, and Sap I restriction-modification systems in Escherichia coli

The genes encoding the ApaLI (5′-G^TGCAC-3′), NspI (5′-RCATG^Y-3′), NspHI (5′-RCATG^Y-3′), SacI (5′-GAGCT^C-3′), SapI (5′-GCTCTTCN1^-3′, 5′-^N4GAAGAGC-3′) and ScaI (5′-AGT^ACT-3′) restriction-modification systems have been cloned in E.?coli. Amino acid sequence comparison of M.ApaLI, M.NspI, M.NspHI, and M.SacI with known methylases indicated that they contain the ten conserved motifs characteristic of C5 cytosine methylases. NspI and NspHI restriction-modification systems are highly homologous in amino acid sequence. The C-termini of the NspI and NlaIII (5′-CATG-3′) restriction endonucleases share significant similarity. 5mC modification of the internal C in a SacI site renders it resistant to SacI digestion. External 5mC modification of a SacI site has no effect on SacI digestion. N4mC modification of the second base in the sequence 5′-GCTCTTC-3′ blocks SapI digestion. N4mC modification of the other cytosines in the SapI site does not affect SapI digestion. N4mC modification of ScaI site blocks ScaI digetion. A DNA invertase homolog was found adjacent to the ApaLI restriction-modification system. A DNA transposase subunit homolog was found upstream of the SapI restriction endonuclease gene.     

Substrate specificity and biochemical properties of M3.BstF5I DNA methyltransferase from the BstF5I restriction-modification system

Optimal conditions for DNA methylation by the M3.BstF5I enzyme from Bacillus stearothermophilus and kinetic parameters of λ phage DNA modification and that of a number of oligonucleotide substrates are established. Comparison of M1.BstF5I and M3.BstF5I kinetic parameters revealed that with similar temperature optima and affinity for DNA, M3.BstF5I has nearly fourfold lower turnover number (0.24 min⁻¹) and modifies the hemimethylated recognition site with lower efficiency under optimal conditions than the unmethylated one. In contrast to another three methylases of the BstF5I restriction-modification system, the M3.BstF5I enzyme is able to optionally modify the noncanonical 5′-GGATC-3′ DNA sequence with a rate more than one order of magnitude lower than the methylation rate of the canonical 5′-GGATG-3′ recognition site.     

A primer-based approach to genome walking

A genome walking strategy based on annealing and ligation of single-stranded DNA primers to 3′ overhangs following restriction endonuclease digestion was developed. A set of primers contains 4 nucleotides at the 3′ end that are complementary to overhangs formed by restriction endonucleasesApaI;PstI;SacI andSphI. Following ligation, 5′ end overhangs are formed on the DNA, which serves as sites for the adaptor primers and nested primers for PCR amplification in combination with the gene-specific primers. This strategy was verified by the amplification of up to 4 kb of a potato leafroll virus full-length infectious clone. The procedure could be adopted to target any upstream and downstream regions flanking known sequences within the plant genome.     

Isolation of a new restriction enzyme,ApaCI, an isoschizomer ofBamHI produced byAcetobacter pasteurianus

A new Type II restriction endonucleaseApaCI purified fromAcetobacter pasteurianus is an isoschizomer ofBamHI that cleaves at the nucleotide sequence 5′-G/GATCC-3′ of double-stranded DNA. The single restriction activity present in this strain permits rapidly purified 30 000 units of cleavage activity from 10 g of freshly harvested cells. The resultingApaCI preparation is free of contaminant nuclease activities that might interfere within vitro manipulation of DNA.     

Organization of the interferon-inducible 2′,5′-oligoadenylate-dependent ribonuclease L (RNase L) gene of mouse

Interferons (IFNs) induce a 2′,5′-oligoadenylate (2-5A)-dependent ribonuclease L (RNase L) following virus-infection of mammalian cells. RNase L degrades both viral and cellular RNAs and restricts virus-proliferation. We have studied organization of RNase L gene in genomic DNA from the mouse liver by Southern blot analysis. Several BamHI, BglII, EcoRI, HincII, HindIII, NcoI, PstI, SacI, and XbaI restriction fragments hybridized to ³²P-labeled mouse RNase L cDNA and the 5′-proximal exon probes. Mouse RNase L gene exists as a single copy (>16 kb DNA) gene. A 5 kb HindIII and a 2.5 kb EcoRI DNA were detected as 5′-upstream DNA of the gene which may contain mouse RNase L promoter. Our results will help studying mouse RNase L gene promoter in further details.     

Localization of flagellin genes on the physical map of Methanococcus voltae

Methanococcus voltae DNA, digested individually with the restriction enzymes ApaI, SacII, BamHI, or EagI, was resolved by pulsed-field gel electrophoresis reproducing the previously published digestion patterns. Hybridization of a flagellin gene-specific probe to such gels dried down (unblots) resulted in the identification of one band per enzyme harboring the flagellin genes. These bands all overlapped, revealing that an approximately 15-kb BamHI/EagI DNA fragment should harbor the flagellin genes. Double digestion with BamHI and EagI resulted in the resolution of two bands in the 15-kb region of the gel. Separation of these two fragments prior to blotting and probing with a flagellar gene-specific probe revealed that one of these fragments possessed the flagellar sequences. The presence of an EagI restriction site in flaB3 localized the flagellin genes precisely at the junction of EagI fragments Ea2 and Ea5 at approximately the 1800-kb position of the physical map.     

Characterization of a restriction barrier and electrotransformation of the cyanobacterium Nostoc PCC 7121

We have investigated host restriction as a barrier to transformation and developed a method for gene transfer into the previously untransformable, heterotrophic cyanobacterium Nostoc PCC 7121. A restriction endonuclease, designated Nsp 7121I, has been partially purified by phosphocellulose chromatography of Nostoc cell extract. Comparisons of Nsp 7121I digests of bacteriophage lambda and plasmid DNAs with computer-generated restriction fragment profiles showed that Nsp 7121I is an isoschizomer of restriction endonucleases, such as Asu I, Nsp 7524IV, Sau 96I, and Eco 47II, that recognize the sequence GGNCC. Cleavage by Nsp 7121I within this sequence was confirmed by sequence analysis of DNA fragments cleaved at a unique Nsp 7121I site. These data further suggested that cleavage occurs after the first G (5-G/GNCC-3) in this site to generate a three base 5 overhang. Nsp 7121I degraded all plasmids used in previous transformation attempts but modification of these DNA molecules by Eco 47II methylase effectively prevented digestion by Nsp 7121I. Plasmids premethylated by passage through Escherichia coli carrying a plasmid encoded Eco 47II methylase have now been used in an electroporation procedure to transform Nostoc PCC 7121 to neomycin resistance at frequencies as high as one transformant per 10³ viable cells. Transformation, and stable replication within Nostoc of one of the transforming plasmids (pRL25), was confirmed by recovery of pRL25, in its original form, from transformants. Conjugal transfer of pRL25 from E. coli into Nostoc was also possible but at much lower efficiency than by electroporation. These findings establish the basis for genetic analysis of Nostoc PCC 7121, from which genes for photosynthetic electron transport have been cloned.     

Molecular cloning, sequence and expression of Aa-polB , a mitochondrial gene encoding a family B DNA polymerase from the edible basidiomycete Agrocybe aegerita

An ORF of 1716 nucleotides, putatively encoding a DNA polymerase, was characterized in the mitochondrial genome of the edible basidiomycete Agrocybe aegerita. The complete gene, named Aa-polB, and its flanking regions were cloned and sequenced from three overlapping restriction fragments. Aa-polB is located between the SSU rDNA (5′ region) and a gene for tRNA^Asn (3′ region), and is separated from these genes by two A+T-rich intergenic regions of 1048 (5′ region) and 3864 (3′ region) nucleotides, which lack repeated sequences of mitochondrial or plasmid origin. The deduced Aa-POLB protein shows extensive sequence similarity with the family B DNA polymerases encoded by genomes that rely on protein-primed replication (invertrons). The domains involved in the 3′→5′ exonuclease (Exo I to III) and polymerase (Pol I to Pol V) activities were localized on the basis of conserved sequence motifs. The alignment of the Aa-POLB protein (571 amino acids) with sequences of family B DNA polymerases from invertrons revealed that in Aa-POLB the N-terminal region preceding Exo I is short, suggesting a close relationship with the DNA polymerases of bacteriophages that have linear DNA. The Aa-polB gene was shown to be present in all wild strains examined, which were collected from a wide range of locations in Europe. As shown by RT-PCR, the Aa-polB gene is transcribed in the mitochondria, at a low but significant level. The likelihood of the coexistence of Aa-POLB and Pol γ in the A. aegerita mitochondrion is discussed in the light of recent reports showing the conservation of the nucleus-encoded Pol γ from yeast to human. Received: 13 October 1998 / Accepted: 21 December 1998     

The pilA gene of Xanthomonas campestris pv. citri is required for infection by the filamentous phage cf

 Host factors that are important for infection of Xanthomonas campestris pv. citri by the filamentous bacteriophage cf were investigated by transposon mutagenesis with Tn5tac1. A mutant, XT501, that was resistant to cf infection was recovered, showing that the gene inactivated by the transposon is required for infection by the phage but not for cf replication or assembly. A 1.7-kb SacI-ApaI DNA fragment from XT501 containing the bacterial DNA flanking one end of the transposon was cloned and shown to be required for cf infection. Nucleotide sequence analysis of the 1.7-kb fragment reveals the presence of an ORF that encodes a protein of 146 amino acids. This protein shows 42% identity to the type 4 prepilin encoded by the pilA genes of other bacteria. The pilA gene of X. campestris pv. citri is thus essential for infection by the bacteriophage cf. Received: 30 November 1998 / Accepted: 21 April 1999     

Multiplicity of site-specific DNA methyltransferases of theBstF5I restriction-modification system

A fragment located downstream of the genes for DNA methyltransferases ofBacillus stearothermophilus F5 (M.BstF5I-1 and M.BstF5I-2) was sequenced. The fragment contains a gene for another methylase, M.BstF5I-3, structurally and functionally similar to the N-terminal domain of M.FokI. Thus, in contrast to other restriction-modification systems, theBstF5I system includes three methylases, two being homologous to the individual M.FokI domains.     

Recombinant DNA-methyltransferase M1.Bst19I from <Emphasis Type="Italic">Bacillus stearothermophilus</Emphasis> 19: Purification,properties, and amino acid sequence analysis

The M1.Bst19I DNA-methyltransferase gene from restriction-modification system Bst19I (recognition sequence 5′-GCATC-3′) in Bacillus stearothermophilus 19 has been cloned in the expressing vector pJW that carries a tandem of thermo inducible promoters P _R /P _L from phage λ. Highly purified enzyme has been isolated by chromatography on various resins from Escherichia coli cells where it is accumulated in a soluble form. The study of M1.Bst19I properties has revealed that the enzyme has a temperature optimum at 50°C and demonstrates maximal activity at pH 8.0. M1.Bst19I modifies adenine in sequence 5′-GCATC-3′. Kinetic parameters of M1.Bst19I DNA methylation reaction have been determined as follows: Km for λ DNA is 0.68 ± 0.07 μM, Km for S-adenosyl-L-methionine is 2.02 ± 0.31 μM. Catalytical constant (k _cat) is 1.8 ± 0.05 min⁻¹. Comparative analysis of Target Recognition Domain amino acid sequences for M1.Bst19I and other α-N6-DNA-methyltransferases has allowed us to suggest the presence of two types of the enzymes containing ATG or ATC triplets in the recognition sequence.     

M.BstF5I-2 and M.BstF5I-4 DNA Methyltransferases from BstF5I Restriction–Modification System of Bacillus stearothermophilus F5

The BstF5I restriction–modification system from Bacillus stearothermophilus F5 includes four site-specific DNA methyltransferases, thus differing from all known restriction–modification systems. Here we demonstrated for the first time that one bacterial cell can possess two pairs of methylases with identical substrate specificities (methylases BstF5I-1 and BstF5I-3 recognize GGATG, whereas methylases BstF5I-2 and BstF5I-4 recognize CATCC) that modify adenine residues on both DNA strands. Different chromatographic methods provide homogenous preparations of methylases BstF5I-2 and BstF5I-4. We estimated the principal kinetic parameters of the reaction of transfer of methyl group from the donor S-adenosyl-L-methionine to the recognition site 5"-CATCC-3" catalyzed by BstF5I-2 and BstF5I-4 DNA [N6-adenine]-methyl-transferases from the BstF5I restriction–modification system.     

Connection between foreign DNA replication and induced expression of the restriction-modification system inStreptomyces aureofaciens

Tetracycline-producing strains ofStreptomyces aureofaciens expressedSauLPI restriction-modification (R-M) system, which recognized specific DNA sequence 5′-GCCGGC-3′ (isoschizomerNaeI). The activation of the second R-M systemSauLPII (5′-GAGCTC-3′, isoschizomer ofXhoI), which was silent during the growth cycle, after a foreign DNA transfer into this strain was observed. This phenomenon was tentatively explained as a response of the cells against the exogenous DNA entering the cells. The involvement of a SOS-like response in induction of R-M system genes inS. aureofaciens strains has been considered.     

Characterization of a complex restriction-modification system detected in<Emphasis Type="Italic">Staphylococcus aureus</Emphasis> and<Emphasis Type="Italic">Streptococcus agalactiae</Emphasis> strains isolated from infections of domestic animals

Characterization of classic type II restriction-modification systems (RMS) (restriction endonucleases and modification methyltransferases) was carried out in isolates ofStaphylococcus aureus andStreptococcus agalactiae obtained from clinical material. Among the 100 isolates ofS. aureus two different RMS type II were detected. The first was expressed in isolates 32 and 33 (Sau32 I andSau33 I); the targeting sequence was determined as 5′-GGN CC-3′ (Sau96 I isoschizomer). The second was found in isolates no. 90, 93, 96*, and 98 (Sau90 I,Sau93 I,Sau96* I,Sau98 I) and enzymes recognized sequence 5′-CTY RAG-3′ (SmlI isoschizomer). Analysis of 40 isolates ofS. agalactiae revealed only one RMS; it was detected in two isolates (no. 16 and 23;Sag16 I andSag23 I). Restriction endonuclease expressed by these isolates cleaved DNA in sequence 5′-CTG CA/G-3′ (PstI isoschizomer). In RMS-positiveS. aureus andS. agalactiae isolates plasmid DNA capable of replication inEscherichia coli andBacillus subtilis was also detected and isolated. This research was supported by VEGA grant of theSlovak Academy of Sciences no. 2/2059/22 and grant no. 2003 SP27/0208E 02/028/0E02 of theMinistry of Agriculture of the Slovak Republic.     

TaqI polymorphism in the LDL receptor gene and a TaqI 1.5-kb band associated with familial hypercholesterolemia

Summary The low density lipoprotein (LDL) receptor gene was analyzed in 67 unrelated healthy Japanese and 38 members of six consecutive families with familial hypercholesterolemia (FH) by Southern blot hybridization with TaqI, an LDL receptor cDNA fragment containing exons 1 to 8 being used as a probe. A new TaqI RFLP at the LDL receptor locus was detected with allele frequencies of 0.67 and 0.33. The data obtained with smaller cDNA subfragment probes revealed that the TaqI RFLP site is located within 1.1 kb of the 5 side of the EcoRI site of exon 5. The TaqI RFLP was in linkage disequilibrium with the PstI RFLP but showed no significant linkage disequilibrium with the RFLPs for AvaII, ApaLI/I15, PvuII, NcoI, and ApaLI/3. Among the seven RFLPs at the LDL receptor locus, the TaqI RFLP was the only useful genetic marker in one of the six families with FH. Furthermore, the association of an additional TaqI 1.5-kb band with a mutant LDL receptor gene was observed in another family with FH in which the proband was homozygous for all of the seven RFLPs. The data obtained with various restriction enzymes and smaller cDNA subfragments probes suggested that a minor change in nucleotide sequences in the region including exons 5 to 8 is present in the mutant gene. These data suggest that the TaqI RFLP is a useful genetic marker at the LDL receptor locus and that TaqI serves for the analysis of some mutant LDL receptor genes, when used with small LDL receptor cDNA probes.     

Effects of different irradiances on the photosynthetic process during ex-vitro acclimation of Anoectochilus plantlets

Six months old in vitro-grown Anoectochilus formosanus plantlets were transferred to ex-vitro acclimation under low irradiance, LI [60 μmol(photon) m⁻² s⁻¹], intermediate irradiance, II [180 μmol(photon) m⁻² s⁻¹], and high irradiance, HI [300 μmol(photon) m⁻² s⁻¹] for 30 d. Imposition of II led to a significant increase of chlorophyll (Chl) b content, rates of net photosynthesis (P _N) and transpiration (E), stomatal conductance (g _s), electron transfer rate (ETR), quantum yield of electron transport from water through photosystem 2 (Φ_PS2), and activity of ribulose-1,5-bisphosphate carboxylase/ oxygenase (RuBPCO, EC 4.1.1.39). This indicates that Anoectochilus was better acclimated at II compared to LI treatment. On the other hand, HI acclimation led to a significant reduction of Chl a and b, P _N, E, g _s, photochemical quenching, dark-adapted quantum efficiency of open PS2 centres (F_v/F_m), probability of an absorbed photon reaching an open PS2 reaction centre (F_v′/F_m′), ETR, Φ_PS2, and energy efficiency of CO₂ fixation (Φ_CO2/Φ_PS2). This indicates that HI treatment considerably exceeded the photo-protective capacity and Anoectochilus suffered HI induced damage to the photosynthetic apparatus. Imposition of HI significantly increased the contents of antheraxanthin and zeaxanthin (ZEA), non-photochemical quenching, and conversion of violaxanthin to ZEA. Thus Anoectochilus modifies its system to dissipate excess excitation energy and to protect the photosynthetic machinery.     

Characterization of thePac25I Restriction-Modification Genes Isolated from the Endogenous pRA2 Plasmid ofPseudomonas alcaligenesNCIB 9867

Genes for the class IIPseudomonas alcaligenesNCIB 9867 restriction-modification (R-M) system,Pac25I, have been cloned from its 33-kb endogenous plasmid, pRA2. ThePac25I endonuclease and methylase genes were found to be aligned in a head-to-tail orientation with the methylase gene preceding and overlapping the endonuclease gene by 1 bp. The deduced amino acid sequence of thePac25I methylase revealed significant similarity with theXcyI,XmaI,Cfr9I, andSmaI methylases. High sequence similarity was displayed between thePac25I endonuclease and theXcyI,XmaI, andCfr9I endonucleases which cleave between the external cytosines of the recognition sequence (i.e., 5′-C↓CCGGG-3′) and are thus perfect isoschizomers. However, no sequence similarity was detected between thePac25I endonuclease and theSmaI endonuclease which cleaves between the internal CpG of the recognition sequence (i.e., 5′-CCC↓GGG-3′). Both thePac25I methylase and endonuclease were expressed inEscherichia coli.An open reading frame encoding a protein which shows significant similarity to invertases and resolvases was located immediately upstream of thePac25I R-M operon. In addition, a transposon designated Tn5563was located 1531 bp downstream of the R-M genes. The location on a self-transmissible plasmid as well as the close association with genes involved in DNA mobility suggests horizontal transfer as a possible mode of distribution of this family of R-M genes in various bacteria.