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© 1996 Oxford University Press 1045-1046

Footnote

Restriction enzyme Hin cII is sensitive to methylation of cytosine that occurs 5 ' to the recognition sequence

Restriction enzyme Hin cII is sensitive to methylation of cytosine that occurs 5 ' to the recognition sequence Kanduri Chandrasekhar* and Rajiva Raman

Cytogenetics Laboratory, Department of Zoology, Banaras Hindu University, Varanasi 221005, India

Received November 12, 1995; Revised and Accepted January 22, 1996

In this paper we demonstrate that the Hin cII restriction endonuclease, in addition to being sensitive to methylation of the 3' A and C residues, is also sensitive to methylation of a cytosine immediately 5' to the recognition sequence. Having encountered this property in one of the sites in the mouse c- fos gene, we confirmed the sensitivity of Hin cII to the 5' cytosine methylation in in vitro methylated pUC12, pBR322 and p fos -1 plasmids.

Hin cII is a six base-cutter which recognises the sequence GTPyPuAC , the cleavage site being between the Py/Pu. Its activity is known to be sensitive to the methylation of the A residue in the sequence ( 1 ). Recently, Bull et al. ( 2 ) used plasmid constructs having GTCGAC G sequence for Hin cII digestion and showed that the presence of methylated cytosine flanked by a G at the 3' end of recognition sequence inhibits Hin cII digestion. They also showed that methylation in the internal CpG did not affect the digestion. However, the Hin cII sensitivity to C methylation has not been universally accepted (see ref. 3 ). Here, we report that in the DNA motif C GTCGACC , Hin cII digestion is sensitive to methylation of the 5'C which is not a part of its recognition sequence. We also confirm that methylation of the internal C has no effect on the digestion by Hin cII.

While studying kinetics of methylation at individual CpGs in the c- fos gene during mouse development, one of the sites analysed was C GTCGAC C, present at the 3' end of the gene. Both Sal I and Hin cII cleave this sequence, except that Sal I is sensitive to the internal CpG methylation while Hin cII is not ( 2 - 4 ). However, not only Sal I but also Hin cII showed differential sensitivity patterns between the fetal and adult liver as well as brain (unpublished observations). As seen in Figure 1 , Hin cII digestion leads to two fragments (3.9 kb and expected 2.4 kb) in the fetal tissues (Fig. 1 , lanes 1 and 3) but in the adult, liver shows only 1 fragment (3.9 kb; Fig. 1 , lane 4), brain shows, in addition to the 3.9, a fainter 2.4 kb fragment (Fig. 1 , lane 2). In order to test whether this novel Hin cII pattern was due to methylation of the C residues in this sequence the following experiment was done.


Figure 1 . Southern hybridisation of Hin cII-digested genomic DNAs from fetal brain (lane 1), adult brain (lane 2), fetal liver (lane 3) and adult liver (lane 4) with 368 bp Msp I fragment from v- fos .


Figure 2 . Pvu II-linearized pBR322 plasmid DNA (lane 1), digested with Hin cII (lane 2) prior to methylation, and after methylation (lane 3).

Plasmids pBR322 (4.36 kb), v- fos cloned in pBR322 (p fos -1 clone, 5.6 kb) and pUC12 (2.68 kb), which are known to have C GTCGAC , were in vitro methylated with Sss I methyltransferase (10 [mu]g DNA with 12 U M. Sss I at 37oC for 1 h in the presence of 160 [mu]M S -adenosyl methionine in 50 [mu]l reaction volume) and then digested overnight with excess amount of Hin cII (20 U/1 [mu]g). The in vitro methylated pBR322, which has two Hin cII sites (5'- C GTCGAC C-3' at 651 bp, and 5'- C GTCAAC C-3' at 3905 bp positions), yielded six fragments (Fig. 2 , lane 3) as against the expected three fragments in the unmethylated, linearised plasmid (Fig. 2 , lane 2). This result clearly indicated that when methylated the two Hin cII sites were only partially cleaved even at high concentration of the Hin cII enzyme, and since both the sites were resistant to the enzyme, it was most likely due to methylation of the cytosine upstream to the Hin cII site. The possibility of interference in the Hin cII digestion by the internal CpG dinucleotide was ruled out by digesting the M. Sss I treated pUC12, which harbours one Hin cII site (5'-A GTCGAC C-3'), with Hin cII. pUC12 was completely digested (Fig. 3 , lane 4). Since the present investigation was initiated in the proto-oncogene, c- fos , which contains one Hin cII site 5'- C GTCGAC C-3' immediately downstream of the stop codon site ( 5 ), Hin cII digestion was tested in a v- fos gene cloned in pBR322 (p fos -1 clone). In this construct, there were three Hin cII sites, one of v- fos and two of pBR322 and the sequence in v- fos gene was similar to the one at 651 bp in pBR322. Following M. Sss I methylation the v- fos site showed complete resistance to the enzyme, as seen in the genomic DNA from adult tissues (Fig. 4 , lane 7). The pBR322 sites showed partial cleavage as earlier observed with the pBR322 DNA. The efficiency of methylation by M. Sss I in the above reactions was checked by performing digestions with Msp I and Hpa II (Fig. 4 , lanes 3 and 4).


Figure 3 . Digestion of Xmn I-linearized pUC12 plasmid DNA (lane 1), with Hin cII (lane 2), in the presence of S -adenosyl methionine alone (lane 3), and after methylation with M. Sss I (lane 4). Lane 5 contains M. Sss I treated Sal I digested pUC12 DNA.


Figure 4 . Hin cII digestion pattern of the p fos -1 clone ( Bgl II- Pvu II fragment of v- fos gene cloned in pBR322). Lanes 1 and 8 contain reference molecular weights consisting of Hinf I-digested pUC13 plasmid DNA (1419, 517, 396, 214, 75 and 65 bp) and Hin dIII-digested lambda DNA, respectively. Lane 2, Hin dIII-linearized p fos -1 clone; lane 3, Msp I-digested M. Sss I-treated p fos -1 clone; lane 4, Hpa II-digested M. Sss I-treated p fos -1 clone; lane 5, Hin cII digested unmethylated p fos -1 clone; lane 6, Hin cII digestion of p fos -1 clone in the presence of S -adenosyl methionine alone and lane 7, Hin cII-digested p fos -1 clone after treatment with M. Sss I.


The above results provide strong evidence in favour of Hin cII sensitivity to the methylation of cytosine occurring 5' to its recognition sequence. Viewed together with the observation of Bull et al. ( 2 ), which shows Hin cII sensitivity to the methylation of terminal cytosine in the 5'- GTCGAC G-3' sequence, it is clear that Hin cII is sensitive not only to the 3' flank CpG methylation, but also to the 5' flank CpG methylation. Therefore, care is warranted while using Hin cII restriction enzyme in methylation studies.

ACKNOWLEDGEMENTS

Authors are pleased to record their appreciation of Dr R.J. Roberts, NEB, USA for his interest and advice, and New England BioLabs, USA for the gift of Sss I methyltransferase. Funding for the work was provided by the Department of Science & Technology, New Delhi (to R.R.). K.C. is grateful to the Council of Scientific & Industrial Research for the Senior Research Fellowship.

REFERENCES

1 Kessler,C. and Manta,V. (1990) Gene, 92, 1-248.

2 Bull,L.N., Hewitt,J.E., Cox,D.R. and Myers,R.M. (1993) Nucleic Acids Res., 21, 2021.

3 McClelland,M., Nelson,M. and Raschke,E. (1994) Nucleic Acids Res., 22, 3640-3659.

4 Brooks,J.E. and Roberts,R.J. (1982) Nucleic Acids Res., 10, 913-914. MEDLINE Abstract

5 Curran,T., Peters,G., Van Beveren,C., Teich,N.M. and Verma,I.M. (1982) J. Virol., 44, 674-682.

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