6210 Nucleic Acids Research, 2005, Vol. 33, No. 19 recombination events between the cut site and the HEG inser- tion site has been suggested to increase the homing efﬁciency of the HEG by expanding the region to be replaced (13,28). If efﬁcient recombination were possible in the region between the cut and the HEG insertion site, repair would proceed without transfer of the HEG in the majority of repair events. If SegH recognition includes sequences downstream of the cleavage site the extensive sequence differences between HEG-containing and HEG-less alleles in this region could make segH-containing alleles resistant to cleavage. To test if SegH and Hef show any cleavage speciﬁcity for different phage DNA sequences, competition assays were performed. Partially puriﬁed SegH shows a higher cleavage activity towards T2 (without any HEG in the nrdDG region) and T4 (with mobC in the nrdDG region) compared to the LZ2 nrdDG region in a mix of equimolar concentrations of PCR ampliﬁed targets (Figure 4A). If this is due to differences in target binding or in cleavage activity remains to be determ- ined (work in progress). Competition assays with in vitro translated U5 Hef protein showed no preferential cleavage activity towards a speciﬁc target in a mixture of equimolar concentrations of PCR ampliﬁed nrdAB regions from T2 (without any HEG in the nrdAB region), T4 (with mobE in the nrdAB region) and U5 (Figure 4B) in accordance with the high degree of sequence similarity around the cleavage site between the phages. Homing of segH and mobE in mixed phage infections To assess if segH and hef can home to HEG-less phages and to further investigate the reported unidirectional inheritance of mobE (19), we performed mixed infections (two different phages infecting the same bacterium) using donor phages containing various combinations of mobE, segH and hef (Figure 5). If only neutral recombination occurs, the theoret- ical occurrence of an allele from either phage in the progeny of a mixed infection is 50%. However, homing endonucleases have been shown to cause a phenomenon termed localized marker exclusion where genetic markers from the HEG-less recipient phage are excluded in the progeny of the cross while the HEG is strongly over represented (13,14,30). In addition, some phages generally exclude other phage alleles to varying extent by still largely unknown mechanisms (30). We have shown previously that T2 generally excludes T6 and RB3 markers but that genes with homing properties (i.e. the td intron) can partly counteract this exclusion (19). The use of partially excluded phages as donors is a good set-up for hom- ing studies since the donor alleles found in the progeny are most likely from homing events. To mid-log phase bacteria we added equal amounts of donor and recipient phages in excess to minimise single infection events, and the relative frequency of occurrence of HEGs in the progeny was determined. In those phages where introns known to be homing proﬁcient were present the frequency of these in the progeny was also determined for comparison. To assess mobE and segH homing we used either T6, RB3 or LZ2 as donor phages while U5 was used as donor for homing studies of hef (Figure 5A). T2 was used as the recipient in all crosses. Figure 5B shows that all RB3 and LZ2 markers tested were strongly excluded by the T2 recipient. Interest- ingly, both RB3 and LZ2 segH, and in one cross also T6 segH, were more frequent than the respective td introns that are homing proﬁcient. This indicates that also segH is homing proﬁcient and can propagate itself in spite of exclusion. Not- ably, also mobE was found at a similar frequency as td in the T6/T2 crosses suggesting that mobE is homing proﬁcient. Progeny from the RB3/T2 crosses contained no mobE genes or nrdB introns in accordance with previous reports that this region of RB3 is very strongly excluded by T2 (12,19,30). In the U5/T2 crosses hef was strongly over-represented in the progeny (Figure 5B), and so were also the nearby td and nrdB introns, whereas, the nrdD intron with its non-functional HEG was found at a lower frequency. To analyse further if homing of the segH, mobE and hef genes has taken place we analysed the origin of the regions ﬂanking these genes in all progeny that contained the HEG, from the T2 crosses (Figures 5C and D). Recombination points between donor and recipient alleles were found close to the segH gene in all progeny screened from both RB3/T2 and LZ2/ T2 crosses (Figure 5C). This is strong evidence that homing has taken place for this gene and corroborates our earlier data with the td intron (19) showing that homing proﬁcient genes can overcome exclusion. Similarly, we found that 9 of the 12 progeny from the T6/T2 crosses had at least one recombination point close to the mobE gene strongly suggesting that T6 mobE is homing proﬁcient (Figure 5D). In a previous study we observed that the homing deﬁcient T4 nrdB intron was trans- mitted with the same frequency as the homing proﬁcient td and nrdD introns in T4, and speculated that T4 mobE mobilized the nrdB intron as well as its own gene (19). Similar observations were made by Liu et al. (14). Our current results from the T6/T2 crosses strongly support this hypothesis and suggest that T4 and T6 MobE may have a cleavage site early in the T2 nrdB gene. A similar screen of the progeny from the U5/T2 cross did not locate any T2 markers throughout the nrdA and nrdB genes (data not shown) making it impossible to deter- mine if hef has been transferred via homing. Generally, U5 seems to exclude most phages over large genetic regions (T2, T4, RB23, LZ1, our unpublished data) making the homing results in these regions uninformative as there appears to be little mixing of alleles between U5 and other phages. The same difﬁculty is apparent with T4 that strongly excludes most T-even-like phages over large regions (30,31). Further ana- lysis of homing of U5 hef as well as T4 mobC therefore has to await the construction of exact deletion variants of these strains for unbiased homing analyses (work in progress). DISCUSSION We have found that, like the T4 introns, the putative H-N-H endonuclease genes mobC and mobE of bacteriophage T4 are infrequently represented in closely related phages and that several phages have an unrelated gene at either of these posi- tions. Phages T6, RB3 and LZ2 have a gene homologous to the T4 seg-genes replacing T4 mobC between their nrdD and nrdG genes. This gene is the eighth seg-like gene found among the T-even-like phages and we therefore name it segH (10,13,14). We show here that segH, like several of its relat- ives in T4, encodes an endonuclease with a cleavage site close to its gene locus, a feature common to homing endonucleases. Analysis of the co-conversion region between the segH cut site and insertion site indicates that segH has been transferred to the T-even-like phages from a genetically quite distant source.