Nucleic Acids Research, Vol. 18, No. 20 6161
Isolation and identification of restriction endonuclease BssAI M.Rina, I.Stratidakis and V.Bouriotis* Institute of Molecular Biology and Biotechnology, Enzyme Technology Division, P.O.Box 1515, Heraklion 711 10, Crete, Greece Submitted August 13, 1990
BssAI, an isoschizomer of CfrlOI (1) has been purified from Bacillus species. BssAI recognises the sequence 5'...RCCGGY... 3' (R= A or G, Y= C or T), cleaves between R and C and is sensitive to 5'C and 3'C methylation. The enzyme was purified using the following Chromatographic steps: 1. Phosphocellulose, 2. Heparin Agarose, 3. ds DNA cellulose. The enzyme was free of contaminating nuclease activity. After 100 fold overdigestion on lambda DNA greater than 95% of the DNA fragments can be ligated and greater than 95 % can be recut by BssAI. Optimal conditions for enzyme activity are 100 mM KCI, 20 mM Tris-HCl (pH8.5), 3 mM MgCl2, 0.04% Triton X-100 at 650C The fragments produced by BssAI digestion of lambda DNA, Adeno 2, 4X174, SV40, pBR322 and pUC19 match those predicted by cleavage at the sequence RCCGGY (figure 1, lanes 2-8). A pUC 19 recombinant clone which contained a recognition site for the enzyme was digested by the enzyme BssAI then annealed with forward or reverse sequencing primers and extended with Klenow enzyme in the presence of c32P-dATP. -
1
2 3 4 5 6 7
To whom correspondence should be addressed
5 ... R4CCGG Y 3 . Y GGCCtR a
... ---
3 5
REFERENCES 1. Janulaitis,A., Stakenas,B. and Berlin,Yu.A. (1983) FEBS Lett., 161, 210-212. 2. Sanger,F., Nicklen,S. and Coulson,A.R. (1977) Proc. Natl. Acad. Sci. USA 74. 43-5467.
8
Figure 1. BssAI digests; lanes 2 and 3: lambda DNA, 4: Adeno 2, 5: FX174, 6: SV40, 7: pBR322, 8: pUC19, 1: lambda-Hind III size standard.
*
Dideoxy sequencing reactions were performed at this region with the same primers and run in parallel with the extended products (2). Results in figure 2 show that the extended products of the forward (lane F) and the reverse (lane R) sequencing primers comigrate with the band corresponding to the 3'G in the 5'...ACCGGT... 3' sequence. Therefore BssAI recognises and cleaves the following sequence:
T F
Figure 2.
Isolation and identification of restriction endonuclease BssAI M.Rina, I.Stratidakis and V.Bouriotis* Institute of Molecular Biology and Biotechnology, Enzyme Technology Division, P.O.Box 1515, Heraklion 711 10, Crete, Greece Submitted August 13, 1990
BssAI, an isoschizomer of CfrlOI (1) has been purified from Bacillus species. BssAI recognises the sequence 5'...RCCGGY... 3' (R= A or G, Y= C or T), cleaves between R and C and is sensitive to 5'C and 3'C methylation. The enzyme was purified using the following Chromatographic steps: 1. Phosphocellulose, 2. Heparin Agarose, 3. ds DNA cellulose. The enzyme was free of contaminating nuclease activity. After 100 fold overdigestion on lambda DNA greater than 95% of the DNA fragments can be ligated and greater than 95 % can be recut by BssAI. Optimal conditions for enzyme activity are 100 mM KCI, 20 mM Tris-HCl (pH8.5), 3 mM MgCl2, 0.04% Triton X-100 at 650C The fragments produced by BssAI digestion of lambda DNA, Adeno 2, 4X174, SV40, pBR322 and pUC19 match those predicted by cleavage at the sequence RCCGGY (figure 1, lanes 2-8). A pUC 19 recombinant clone which contained a recognition site for the enzyme was digested by the enzyme BssAI then annealed with forward or reverse sequencing primers and extended with Klenow enzyme in the presence of c32P-dATP. -
1
2 3 4 5 6 7
To whom correspondence should be addressed
5 ... R4CCGG Y 3 . Y GGCCtR a
... ---
3 5
REFERENCES 1. Janulaitis,A., Stakenas,B. and Berlin,Yu.A. (1983) FEBS Lett., 161, 210-212. 2. Sanger,F., Nicklen,S. and Coulson,A.R. (1977) Proc. Natl. Acad. Sci. USA 74. 43-5467.
8
Figure 1. BssAI digests; lanes 2 and 3: lambda DNA, 4: Adeno 2, 5: FX174, 6: SV40, 7: pBR322, 8: pUC19, 1: lambda-Hind III size standard.
*
Dideoxy sequencing reactions were performed at this region with the same primers and run in parallel with the extended products (2). Results in figure 2 show that the extended products of the forward (lane F) and the reverse (lane R) sequencing primers comigrate with the band corresponding to the 3'G in the 5'...ACCGGT... 3' sequence. Therefore BssAI recognises and cleaves the following sequence:
T F
Figure 2.
