ABSTRACT
We report the organization of the HpaII restriction and modification (R-M) system from Haemophilus parainfluenzae (recognition sequence: 5'...CCGG...3'), the sequence of the gene coding for the HpaII restriction endonuclease, and the sequence of the upstream flanking DNA. The HpaII system comprises two genes, hpaIIM, coding for the methyltransferase (MTase; 358 amino acids (aa), 40.4 kDa: product, Cm5CGG), and hpaIIR, coding for the restriction endonuclease (ENase; 358 aa, 40.9 kDa: product, C'CGG). The genes are adjacent, they have the same orientation, and they occur in the order hpaIIM then hpaIIR. The ENase bears little as sequence similarity to the isoschizomeric R.BsuFI and R.MspI ENases. Upstream of, and partly overlapping hpaIIM is the coding sequence for a 141-aa protein that resembles the very-short-patch-repair endonuclease (Vsr) of Escherichia coli. Upstream of that is the coding sequence for a protein that resembles valyl-tRNA synthetase (ValS).
Subject(s)
DNA-Cytosine Methylases/genetics , Deoxyribonucleases, Type II Site-Specific/genetics , Haemophilus/enzymology , Amino Acid Sequence , Base Sequence , Cloning, Molecular , DNA, Bacterial , DNA-Cytosine Methylases/metabolism , Deoxyribonuclease HpaII , Deoxyribonucleases, Type II Site-Specific/metabolism , Haemophilus/genetics , Molecular Sequence Data , Mutation , Restriction Mapping , Sequence Homology, Amino Acid , TemperatureSubject(s)
Bacteria/enzymology , DNA Modification Methylases/metabolism , DNA/metabolism , Oligodeoxyribonucleotides/isolation & purification , S-Adenosylmethionine/metabolism , Base Sequence , Chromatography/methods , Chromatography, Thin Layer/methods , Cloning, Molecular , DNA/chemistry , DNA/genetics , DNA Modification Methylases/genetics , DNA Modification Methylases/isolation & purification , Electrophoresis, Polyacrylamide Gel/methods , Escherichia coli/enzymology , Escherichia coli/genetics , Kinetics , Phosphorus Radioisotopes , Radioisotope Dilution Technique , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Substrate Specificity , Sulfur Radioisotopes , TritiumABSTRACT
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.
Subject(s)
Bacterial Proteins/genetics , Cloning, Molecular/methods , DNA Modification Methylases/genetics , Deoxyribonucleases, Type II Site-Specific/genetics , Genes, Bacterial , Recombinant Proteins/genetics , DNA, Bacterial/metabolism , DNA, Viral/metabolism , Escherichia coli/geneticsABSTRACT
The genes for the HhaI (Roberts et al., 1976) and HinPI (Roberts, 1987) restriction-modification (R-M) systems have been cloned in pBR322. The HhaI system was isolated on a 9-kb PstI fragment, and the HinPI system was isolated on two PstI fragments of 1.5 and 4.6 kb in length. The clones were isolated by selecting for recombinant molecules that had protectively modified themselves. The HhaI and HinPI R-M systems recognize the same sequence, GCGC, but hybridization between the DNA fragments encoding them does not take place.
