4786 Nucleic Acids Research, 2005, Vol. 33, No. 15 DNA (55), we conducted a survey of strand bias in all CAN- CAG sequence in a number of different phage genomes (Table 1). In T7 and T7-related genomes, the CANCAG sequences show signiﬁcant strand bias. This bias is not wide- spread amongst other phage however, although some strand bias of the PstII sequence is observed in T3. The more detailed statistical analysis of Kru¨ ger et al. (55) identiﬁed seven hex- americ sequences which show signiﬁcant strand bias in T7. The two sequences with the greatest statistical bias were CAGCAG (EcoP15I) and CATCAG (PstII). Therefore, this earlier study had already predicted the existence of PstII. In a similar manner, all of the palindromic sequences identiﬁed as completely absent from T7 20 years ago (55) can now be assigned to a corresponding Type II activity (1). Sequence asymmetry in the T7 genome therefore acts as an historical imprint of restriction endonuclease activity (56). The other biased hexameric T7 sequences identiﬁed by Kru¨ ger et al. (55) as statistically signiﬁcant were CATAGC, CAGAGC, GAGCTT, GCACTT, GCAGAG and CATAGC. One limitation of the study was that the analysis was only concerned with sequences absent from the L strand but present on the H strand at high frequency (n > 16). Consequently, the CAACAG sequence (Table 2) was not recorded. Moreover, there are a large number of sequences which are not com- pletely absent from one or other strand but for which strand bias is still signiﬁcant, such as CACCAG (Table 2). With the discovery of a second Type III sequence which shows strand bias in T7 it is possible that selective pressure from additional, as yet unidentiﬁed, Type III enzymes has resulted in strand bias in many different sequences. Sequence asymmetry in genomes is a recognized feature of evolution in all organisms (65,66) but is particularly marked in T7 (67). Although the spontaneous mutational spectra of many phage should be similar, it seems that T7 has been particularly successful in harnessing evolutionary drift to prevent cleavage by both Type II and III restriction enzymes. To further safeguard against restriction by Type I enzymes, the antirestriction ocr gene 0.3 of T7 is the ﬁrst to be expressed during the early stages of internalization (68). ACKNOWLEDGEMENTS We thank Elaine Davies and Allan Brewer for technical assis- tance, Susan Milsom for supplying pSKfokI, pNAG4 and pAT153, Rich Roberts and his group for the REBASE suite of resources and Steve Halford, Louise Stanley and the Bristol DNA-Proteins Interactions Unit for discussions. This work was supported in Bristol by the Wellcome Trust and the BBSRC. G.G.W. thanks Donald Comb for continuous interest and sup- port. M.D.S. is a Wellcome Trust Senior Research Fellow in Basic Biomedical Sciences. 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