Nucleic Acids Research, Vol. 20, No. 14 3787
Acil, a new restriction endonuclease from Acinetobacter calcoaceticus recognizing 5'-A CGTT-3' Sergey Kh.Degtyarev, Murat A.Abdurashitov, Alexander A.Kolyhalov and Nadya I.Rechkunova Sibenzyme, 8 Lavrentiev str., Novosibirsk 630090, Russia Submitted May 26, 1992 AclI, a new restriction endonuclease, has been purified from Acinetobacter calcoaceticus M4. AclI recognizes the sequence 5'AAICGTT3' and cleaves DNA as indicated by the arrow. The enzyme was purified using the following chromatographic steps: 1) gel-filtration through biogel A-0.5 m, 2) DEAE-cellulose, 3) phosphocellulose, 4) heparin sepharose. The enzyme was free of contaminating nuclease activity. After 20-fold overdigestion on X DNA greater than 95 % of the DNA fragments can be ligated and then recut by AclI. Optimal conditions for AclI activity are 33 mM Tris-acetate (pH 7.9), 10 mM Mg-acetate, 50 mM K-acetate at 37°C. The fragments produced by AclI digestion of lambda, T7, Ad2 and pBR322 DNAs match those predicted by cleavage at the sequence AACGTT (Figure 1). AclI has 7, 19, 3 and 4 recognition sites on above mentioned DNAs, respectively. The cleavage site was determined according to earlier published procedure (1). DNA of pBR322 was digested by AvalI, reaction products were labelled from [a-32P]dATP or [a-32P]dTTP by Klenow fragment in the presence of dGTP. 222 bp DNA fragment was eluted from gel after electrophoresis in 6% PAAG, its structure and AclI hydrolysis site were determined (Figure 2). The results show that AclI cleaves DNA as indicated by arrows: 5'-AAICG TT-3' 3'-TT GCIAA-5'
1
Biotechnology (Russ.) 4, 618-620.
3
4
5
6
Figure 1. Digests of different substrate DNAs: lane 1, lambda + VspI; lane 2, lambda + AclI; lane 3, T7 + AclI; lane 4, Ad2 + AclI; lane 5, pBR322 + AclI; lane 6, lambda + AvaII.
b
a
AT G + + C GC
Aci
A I G + + C GC
Acli
4-
REFERENCE 1. Prihod'ko,G.G., Petrov,N.A., Chizhikov,V.E. and Degtyarev,S.Kh. (1988)
2
-G
C
-
_
A
^s
\
,
\
C G C
4. -
A
A
G TT T G C
-. t-W .__*
T
G
woo ._0 _,1do
.. .-^
Figure 2. Sequencing of the pBR322 DNA fragment: a, labelled from [cs-32P]dATP; b, labelled from [ax-32P]dTTP.
Acil, a new restriction endonuclease from Acinetobacter calcoaceticus recognizing 5'-A CGTT-3' Sergey Kh.Degtyarev, Murat A.Abdurashitov, Alexander A.Kolyhalov and Nadya I.Rechkunova Sibenzyme, 8 Lavrentiev str., Novosibirsk 630090, Russia Submitted May 26, 1992 AclI, a new restriction endonuclease, has been purified from Acinetobacter calcoaceticus M4. AclI recognizes the sequence 5'AAICGTT3' and cleaves DNA as indicated by the arrow. The enzyme was purified using the following chromatographic steps: 1) gel-filtration through biogel A-0.5 m, 2) DEAE-cellulose, 3) phosphocellulose, 4) heparin sepharose. The enzyme was free of contaminating nuclease activity. After 20-fold overdigestion on X DNA greater than 95 % of the DNA fragments can be ligated and then recut by AclI. Optimal conditions for AclI activity are 33 mM Tris-acetate (pH 7.9), 10 mM Mg-acetate, 50 mM K-acetate at 37°C. The fragments produced by AclI digestion of lambda, T7, Ad2 and pBR322 DNAs match those predicted by cleavage at the sequence AACGTT (Figure 1). AclI has 7, 19, 3 and 4 recognition sites on above mentioned DNAs, respectively. The cleavage site was determined according to earlier published procedure (1). DNA of pBR322 was digested by AvalI, reaction products were labelled from [a-32P]dATP or [a-32P]dTTP by Klenow fragment in the presence of dGTP. 222 bp DNA fragment was eluted from gel after electrophoresis in 6% PAAG, its structure and AclI hydrolysis site were determined (Figure 2). The results show that AclI cleaves DNA as indicated by arrows: 5'-AAICG TT-3' 3'-TT GCIAA-5'
1
Biotechnology (Russ.) 4, 618-620.
3
4
5
6
Figure 1. Digests of different substrate DNAs: lane 1, lambda + VspI; lane 2, lambda + AclI; lane 3, T7 + AclI; lane 4, Ad2 + AclI; lane 5, pBR322 + AclI; lane 6, lambda + AvaII.
b
a
AT G + + C GC
Aci
A I G + + C GC
Acli
4-
REFERENCE 1. Prihod'ko,G.G., Petrov,N.A., Chizhikov,V.E. and Degtyarev,S.Kh. (1988)
2
-G
C
-
_
A
^s
\
,
\
C G C
4. -
A
A
G TT T G C
-. t-W .__*
T
G
woo ._0 _,1do
.. .-^
Figure 2. Sequencing of the pBR322 DNA fragment: a, labelled from [cs-32P]dATP; b, labelled from [ax-32P]dTTP.
