ABSTRACT
The genes from Haemophilus parainfluenzae encoding the HpaI restriction-modification system were cloned and expressed in Escherichia coli. From the DNA sequence, we predicted the HpaI endonuclease (R.HpaI) to have 254 amino acid residues (Mr 29,630) and the HpaI methyltransferase (M.HpaI) to have 314 amino acid residues (37,390). The R.HpaI and M.HpaI genes overlapped by 16 base pairs on the chromosomal DNA. The genes had the same orientation. The clone, named E. coli HB101-HPA2, overproduced R.HpaI. R.HpaI activity from the clone was 100-fold that from H. parainfluenzae. The amino acid sequence of M.HpaI was compared with those of other type II methyltransferases.
Subject(s)
Deoxyribonucleases, Type II Site-Specific/genetics , Haemophilus/enzymology , Site-Specific DNA-Methyltransferase (Adenine-Specific)/genetics , Amino Acid Sequence , Base Sequence , Blotting, Southern , Cloning, Molecular , Deoxyribonucleases, Type II Site-Specific/chemistry , Gene Expression , Haemophilus/genetics , Molecular Sequence Data , Restriction Mapping , Site-Specific DNA-Methyltransferase (Adenine-Specific)/chemistrySubject(s)
DNA Restriction Enzymes/isolation & purification , Deoxyribonucleases, Type II Site-Specific/isolation & purification , Myxococcales/enzymology , Base Sequence , DNA Restriction Enzymes/analysis , DNA Restriction Enzymes/metabolism , Deoxyribonucleases, Type II Site-Specific/analysis , Deoxyribonucleases, Type II Site-Specific/metabolism , Electrophoresis, Polyacrylamide Gel , Molecular Sequence DataABSTRACT
Two genes, coding for the HincII from Haemophilus influenzae Rc restriction-modification system, were cloned and expressed in Escherichia coli RR1. Their DNA sequences were determined. The HincII methylase (M.HincII) gene was 1,506 base pairs (bp) long, corresponding to a protein of 502 amino acid residues (Mr = 55,330). The HincII endonuclease (R.HincII) gene was 774 bp long, corresponding to a protein of 258 amino acid residues (Mr = 28,490). The amino acid residues predicted from the R.HincII and the N-terminal amino acid sequence of the enzyme found by analysis were identical. These methylase and endonuclease genes overlapped by 1 bp on the H. influenzae Rc chromosomal DNA. The clone, named E. coli RR1-Hinc, overproduced R.HincII. The R.HincII activity of this clone was 1,000-fold that from H. influenzae Rc. The amino acid sequence of M.HincII was compared with the sequences of four other adenine-specific type II methylases. Important homology was found between tne M.HincII and these other methylases.
