Cloning and expression of AatII restriction-modification system in Escherichia coli.

The genes encoding the AatII restriction endonuclease and methylase from Acetobacter aceti have been cloned and expressed in Escherichia coli. The nucleotide sequences of aatIIM and aatIIR genes were determined. The aatIIM and aatIIR genes are 996 bp and 1038 bp, respectively, encoding the 331-aa methylase with a predicted molecular mass of 36.9 kDa, and the 345-aa AatII restriction endonuclease with a predicted molecular mass of 38.9 kDa. The two genes overlap by 4 base pairs and are transcribed in the same orientation. The aatIIRM genes are located next to a putative gene for plasmid mobilization. A stable overproducing strain was constructed, in which the aatIIM gene was expressed from a pSC101-derived plasmid. The aatIIR gene was inserted into a modified T7 expression vector that carries transcription terminators upstream from the T7 promoter. The recombinant AatII restriction endonuclease was purified to near homogeneity by chromatography through DEAE Sepharose, Heparin Sepharose, and phosphocellulose columns.

The XmnI restriction-modification system: cloning, expression, sequence organization and similarity between the R and M genes.

The xmnIRM genes from Xanthomonas manihotis 7AS1 have been cloned and expressed in Escherichia coli. The nucleotide (nt) sequences of both genes were determined. The XmnI methyltransferase (MTase)-encoding gene is 1861 bp in length and codes for 620 amino acids (aa) (68660 Da). The restriction endonuclease (ENase)-encoding gene is 959 bp long and therefore codes for a 319-aa protein (35275 Da). The two genes are aligned tail to tail and they overlap at their respective stop codons About 4 x 10(4) units/g wet cell paste of R.XmnI was obtained following IPTG induction in a suitable E. coli host. The xmnIR gene is expressed from the T7 promoter. M.XmnI probably modifies the first A in the sequence, GAA(N)4TTC. The xmnIR and M genes contain regions of conserved similarity and probably evolved from a common ancestor. M.XmnI is loosely related to M.EcoRI. The XmnI R-M system and the type-I R-M systems probably derived from a common ancestor.

Cloning of a pair of genes encoding isoschizomeric restriction endonucleases from Bacillus species: the BspEI and BspMII restriction and modification systems.

The respective genes (R-M) encoding restriction and modification systems from two Bacillus species which recognise the same nucleotide sequence, 5'-TCCGGA, have been cloned and expressed in Escherichia coli. The BspEI R-M genes were cloned on a 3.6-kb HindIII fragment, whereas the BspMII R-M genes were cloned on three contiguous HindIII fragments totalling 9.8 kb. Upon thermal induction, E. coli carrying the bspEIR clones under the control of the phage lambda PL promoter, express high levels of R.BspEI (10(6) units/g wet cell paste). The bspMIIR gene, on the other hand, is only poorly expressed (about 4 x 10(3) units/g wet cell paste) following induction. Although the enzymes of both R-M systems recognize the same sequence and the restriction endonucleases (ENases) cleave DNA at the same position, the modification specified by the methyltransferases (MTases) differ. The internal cytosine is the site of M.BspMII modification (TCmeCGGA), whereas the external cytosine is modified by M.BspEI (TmeCCGGA). The two R-M systems probably evolved independently.

Cloning, analysis and expression of the HindIII R-M-encoding genes.

The genes encoding the HindIII restriction endonuclease (R.HindIII ENase) and methyltransferase (M.HindIII MTase) from Haemophilus influenzae Rd were cloned and expressed in Escherichia coli and their nucleotide (nt) sequences were determined. The genes are transcribed in the same orientation, with the ENase-encoding gene (hindIIIR) preceding the MTase-encoding gene (hindIIIM). The two genes overlap by several nt. The ENase is predicted to be 300 amino acids (aa) in length (34,950 Da); the MTase is predicted to be 309 aa (35,550 Da). The HindIII ENase and MTase activities increased approx. 20-fold when the genes were brought under the control of an inducible lambda pL promoter. Highly purified HindIII ENase and MTase proteins were prepared and their N-terminal aa sequences determined. In H. influenzae Rd, the HindIII R-M genes are located between the holC and valS genes; they are not closely linked to the HindII R-M genes.

Characterization of BcgI, a new kind of restriction-modification system.

The BcgI restriction enzyme from Bacillus coagulans is unusual in that it cleaves on both sides of its recognition site, CGAN6TGC, releasing a fragment that includes the site and several bases on each side. We report the organization and nucleotide sequences of the genes for the BcgI restriction-modification system and the properties of the proteins that they encode. The system comprises two adjacent, similarly oriented genes. The proximal gene, bcgIA, codes for a 637-amino acid protein (molecular mass = 71.6 kDa) that resembles certain m6A-specific DNA-methyltransferases, particularly those that constitute the modification subunits of type I restriction-modification systems. The distal gene, bcgIB, codes for a 341-amino acid protein (molecular mass = 39.2 kDa) that resembles none of the sequences in the sequence data bases. The two genes overlap by several nucleotides. Alone, neither protein restricts or modifies DNA, but, together, they form a complex in the proportion A2B that does both. DNA binding assays showed that the DNA-protein complex can be formed only in the presence of both subunits, suggesting that the association of inactive subunits generates the active BcgI enzyme that can bind DNA and then either cleaves or methylates at target site.

Intervening sequences in an Archaea DNA polymerase gene.

The DNA polymerase gene from the Archaea Thermococcus litoralis has been cloned and expressed in Escherichia coli. It is split by two intervening sequences (IVSs) that form one continuous open reading frame with the three polymerase exons. To our knowledge, neither IVS is similar to previously described introns. However, the deduced amino acid sequences of both IVSs are similar to open reading frames present in mobile group I introns. The second IVS (IVS2) encodes an endonuclease, I-Tli I, that cleaves at the exon 2-exon 3 junction after IVS2 has been deleted. IVS2 self-splices in E. coli to yield active polymerase, but processing is abolished if the IVS2 reading frame is disrupted. Silent changes in the DNA sequence at the exon 2-IVS2 junction that maintain the original protein sequence do not inhibit splicing. These data suggest that protein rather than mRNA splicing may be responsible for production of the mature polymerase.

Cloning the BamHI restriction modification system.

BamHI, a Type II restriction modification system from Bacillus amyloliquefaciensH recognizes the sequence GGATCC. The methylase and endonuclease genes have been cloned into E. coli in separate steps; the clone is able to restrict unmodified phage. Although within the clone the methylase and endonuclease genes are present on the same pACYC184 vector, the system can be maintained in E. coli only with an additional copy of the methylase gene present on a separate vector. The initial selection for BamHI methylase activity also yielded a second BamHI methylase gene which is not homologous in DNA sequence and hybridizes to different genomic restriction fragments than does the endonuclease-linked methylase gene. Finally, the interaction of the BamHI system with the E. coli Dam and the Mcr A and B functions, have been studied and are reported here.

Cloning type-II restriction and modification genes.

We have cloned into Escherichia coli the genes for 38 type-II bacterial modification methyltransferases. The clones were isolated by selecting in vitro for protectively modified recombinants. Most of the clones modify their DNA fully but a substantial number modify only partially. In approximately one-half of the clones, the genes for the corresponding endonucleases are also present. Some of these clones restrict infecting phages and others do not. Clones carrying endonuclease genes but lacking methyltransferase genes have been found, in several instances, to be viable.

Cloning and expression of the MspI restriction and modification genes.

The genes for the MspI restriction (R) and modification enzymes (recognition sequence CCGG) have been cloned into Escherichia coli using the vector pBR322. Clones carrying both genes have been isolated from libraries prepared with EcoRI, HindIII and BamHI. The smallest fragment that encodes both activities is a 3.6-kb HindIII fragment. Plasmids purified from the clones are fully resistant to digestion by MspI, indicating that the modification gene is functional in E. coli. The clones remain sensitive to phage infection, however, indicating that the endonuclease is dysfunctional. When the R gene is brought under the control of the inducible leftward promoter from phage lambda, the level of endonuclease increases and the level of methylase decreases, suggesting that the genes are transcribed in opposite directions.

Purification of RNA polymerase and transcription-termination factor Rho from Erwinia carotovora.

Erwinia carotovora RNA polymerase consists of the holoenzyme structure sigma 2 beta beta' sigma as found in Escherichia coli and other bacteria. E. carotovora RNA polymerase can synthesize RNA using lambda, T7 of T4 DNA as templates; however, it is two times less active on these templates and is more temperature-sensitive than the E. coli enzyme. The alpha subunit of the E.. carotovora enzyme is lower in molecular mass than its E. coli counterpart. The sigma factors from E. coli and E. carotovora are similar in size and in their ability to stimulate RNA synthesis by core enzyme on DNA templates such as T7 DNA. An additional protein of 115 000 Da molecular mass, termed gamma, is found associated with E. carotovora RNA polymerase. The gamma protein is tightly associated with the polymerase subunits as it is not dissociated by gel filtration in buffer containing 0.5 M NaCl. It can be purified by passing the Agarose 1.5 m enzyme through coupled Bio-Rex 70 and DEAE-cellulose columns. The gamma-protein, when present in excess over the sigma subunit, inhibits holoenzyme activity on T7 DNA but not on poly[d(A-T)]and may thus interfere with sigma activity. The gamma protein by itself cannot transcribe T7 DNA or poly[d(A-T)], nor does it stimulate core enzyme activity on T7 DNA. E. carotovora rho has a subunit molecular mass of 48 000 Da and exhibits RNA-dependent phosphohydrolysis of adenosine ribonucleoside triphosphate. E. coli and E. carotovora rho are indistinguishable immunologically, as total fusion of precipitin bands is observed. E. carotovora rho elutes from a phosphocellulose column at a salt concentration of about 0.21 M KCl, compared to that of 0.29 M KCl for E. coli rho. The poly(C)-dependent ATPase activity of E. carotovora rho is more-temperature sensitive and is six to ten times less active than that of E. coli rho. E. carotovora rho is capable of terminating RNA transcripts, as indicated by a decrease in RNA synthesis using lambda or T7 DNA as template and E. carotovora or E. coli polymerase as the transcribing-enzyme.