if you were to cut pbr322 with hindiii what would happen
In this newspaper we demonstrate that the HincII brake endonuclease, in addition to beingness 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 i of the sites in the mouse c-fos gene, we confirmed the sensitivity of HincII to the five′ cytosine methylation in in vitro methylated pUC12, pBR322 and pfos−1 plasmids.
HincII is a six base-cutter which recognises the sequence GTPyPuAC, the cleavage site beingness between the Py/Pu. Its activity is known to be sensitive to the methylation of the A residue in the sequence (1). Recently, Balderdash et al. (2) used plasmid constructs having GTCGAC Grand sequence for HincII digestion and showed that the presence of methylated cytosine flanked past a G at the 3′ end of recognition sequence inhibits HincII digestion. They also showed that methylation in the internal CpG did not affect the digestion. However, the HincII sensitivity to C methylation has non been universally accepted (meet ref. iii). Here, nosotros study that in the DNA motif C GTCGACC, HincII digestion is sensitive to methylation of the 5′C which is non a role of its recognition sequence. We too ostend that methylation of the internal C has no effect on the digestion past HincII.
While studying kinetics of methylation at individual CpGs in the c-fos gene during mouse development, i of the sites analysed was CGTCGACC, nowadays at the iii′ end of the gene. Both SalI and HincII cleave this sequence, except that SalI is sensitive to the internal CpG methylation while HincII is non (2–4). However, not merely SalI but also HincII showed differential sensitivity patterns between the fetal and adult liver as well as brain (unpublished observations). Equally seen in Figure 1, HincII digestion leads to two fragments (3.9 kb and expected 2.4 kb) in the fetal tissues (Fig. one, lanes 1 and 3) but in the adult, liver shows merely 1 fragment (three.9 kb; Fig. i, lane 4), brain shows, in add-on to the 3.ix, a fainter 2.iv kb fragment (Fig. one, lane 2). In order to test whether this novel HincII pattern was due to methylation of the C residues in this sequence the following experiment was done.
Plasmids pBR322 (4.36 kb), v-fos cloned in pBR322 (pfos−i clone, five.6 kb) and pUC12 (2.68 kb), which are known to have C GTCGAC, were in vitro methylated with SssI methyltransferase (10 µg DNA with 12 U Grand.SssI at 37°C for one h in the presence of 160 µM S-adenosyl methionine in fifty µl reaction book) and then digested overnight with excess amount of HincII (20 U/1 µg). The in vitro methylated pBR322, which has 2 HincII sites (5′-C GTCGACC-three′ at 651 bp, and 5′-C GTCAACC-3′ at 3905 bp positions), yielded six fragments (Fig. 2, lane 3) as confronting the expected three fragments in the unmethylated, linearised plasmid (Fig. 2, lane 2). This outcome clearly indicated that when methylated the 2 HincII sites were just partially cleaved fifty-fifty at high concentration of the HincII enzyme, and since both the sites were resistant to the enzyme, information technology was most likely due to methylation of the cytosine upstream to the HincII site. The possibility of interference in the HincII digestion by the internal CpG dinucleotide was ruled out by digesting the M.SssI treated pUC12, which harbours one HincII site (5′-AGTCGACC-3′), with HincII. pUC12 was completely digested (Fig. 3, lane 4). Since the present investigation was initiated in the proto-oncogene, c-fos, which contains ane HincII site 5′-C GTCGACC-3′ immediately downstream of the finish codon site (5), HincII digestion was tested in a v-fos gene cloned in pBR322 (pfos−1 clone). In this construct, there were three HincII sites, 1 of v-fos and ii of pBR322 and the sequence in 5-fos gene was similar to the 1 at 651 bp in pBR322. Following M.SssI methylation the v-fos site showed complete resistance to the enzyme, as seen in the genomic Deoxyribonucleic acid from adult tissues (Fig. 4, lane vii). The pBR322 sites showed partial cleavage as earlier observed with the pBR322 Dna. The efficiency of methylation by One thousand.SssI in the above reactions was checked by performing digestions with MspI and HpaII (Fig. 4, lanes 3 and four).
Effigy 1
Southern hybridisation of HincII-digested genomic DNAs from fetal brain (lane ane), adult brain (lane two), fetal liver (lane 3) and adult liver (lane 4) with 368 bp MspI fragment from five-fos.
Figure i
Southern hybridisation of HincII-digested genomic DNAs from fetal encephalon (lane 1), adult brain (lane ii), fetal liver (lane 3) and developed liver (lane 4) with 368 bp MspI fragment from 5-fos.
Effigy two
PvuTwo-linearized pBR322 plasmid DNA (lane 1), digested with HincII (lane ii) prior to methylation, and subsequently methylation (lane 3).
Figure 2
PvuTwo-linearized pBR322 plasmid DNA (lane 1), digested with HincII (lane ii) prior to methylation, and later on methylation (lane 3).
Figure 3
Digestion of XmnI-linearized pUC12 plasmid Dna (lane 1), with HincII (lane 2), in the presence of S-adenosyl methionine alone (lane iii), and afterward methylation with M.SssI (lane four). Lane 5 contains Grand.SssI treated SalI digested pUC12 DNA.
Figure three
Digestion of XmnI-linearized pUC12 plasmid DNA (lane 1), with HincII (lane 2), in the presence of S-adenosyl methionine solitary (lane 3), and afterward methylation with M.SssI (lane 4). Lane 5 contains M.SssI treated SalI digested pUC12 Dna.
The to a higher place results provide strong evidence in favour of HincII sensitivity to the methylation of cytosine occurring five′ to its recognition sequence. Viewed together with the ascertainment of Bull et al. (2), which shows HincII sensitivity to the methylation of terminal cytosine in the 5′-GTCGACM-3′ sequence, it is clear that HincII is sensitive not merely to the 3′ flank CpG methylation, but also to the 5′ flank CpG methylation. Therefore, care is warranted while using HincII restriction enzyme in methylation studies.
Figure 4
HincII digestion pattern of the pfos−i clone (BglIi-PvuTwo fragment of v-fos gene cloned in pBR322). Lanes 1 and 8 incorporate reference molecular weights consisting of HinfI-digested pUC13 plasmid DNA (1419, 517, 396, 214, 75 and 65 bp) and HindIII-digested lambda Deoxyribonucleic acid, respectively. Lane 2, HindIII-linearized pfos−1 clone; lane 3, MspI-digested Grand.SssI-treated pfos−1 clone; lane 4, HpaIi-digested G.SssI-treated pfos−one clone; lane five, HincII digested unmethylated pfos−i clone; lane half-dozen, HincII digestion of pfos−1 clone in the presence of S-adenosyl methionine alone and lane 7, HincII-digested pfos−1 clone later on treatment with Yard.SssI.
Effigy iv
HincII digestion blueprint of the pfos−1 clone (BglII-Pvu2 fragment of 5-fos factor cloned in pBR322). Lanes one and 8 comprise reference molecular weights consisting of HinfI-digested pUC13 plasmid Deoxyribonucleic acid (1419, 517, 396, 214, 75 and 65 bp) and HindIII-digested lambda Deoxyribonucleic acid, respectively. Lane 2, HindIII-linearized pfos−1 clone; lane 3, MspI-digested M.SssI-treated pfos−ane clone; lane four, HpaII-digested Yard.SssI-treated pfos−1 clone; lane 5, HincII digested unmethylated pfos−one clone; lane 6, HincII digestion of pfos−1 clone in the presence of S-adenosyl methionine alone and lane 7, HincII-digested pfos−1 clone after treatment with M.SssI.
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 souvenir of SssI methyltransferase. Funding for the piece of work was provided by the Department of Science & Engineering, New Delhi (to R.R.). One thousand.C. is grateful to the Council of Scientific & Industrial Research for the Senior Enquiry Fellowship.
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© 1996 Oxford University Printing
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