6206 Nucleic Acids Research, 2005, Vol. 33, No. 19 was performed in 100 mM NaCl, 50 mM Tris–HCl (pH 7.5), 10 mM MgCl2, 0.1 mg/ml BSA and 3 mg RNase A at 37 C, and samples were withdrawn for analyses as indicated. Samples were immediately puriﬁed using QIAquick PCR puriﬁcation kit (Qiagen). Cleavage results were analysed on agarose gels by excitation at 473 nm and ﬁltered at 520 nm (FujiFilm FLA-3000); ImageGauge v3.45 (FujiFilm) was used for the quantiﬁcation of cleavage. Mixed phage infections and PCR screening of homing Equal amounts of two phage strains (2.5 · 109 p.f.u.) were mixed with 5 · 108 cells from a mid log-phase culture giving a multiplicity of infection of ﬁve of each phage. Incubation was continued for 90 min at 37 C with vigorous shaking. Cells were lysed with chloroform and progeny phage plated on new bacteria. For each cross 24 plaques were picked and resuspen- ded in 20 ml of water. A 5 ml aliquot of the suspension was used as template in a 25 ml PCR with 1 U of Taq polymerase (Fermentas) according to the manufacturer’s recommenda- tions. Primers for screening of HEG presence were for td intron: tdIVSA and tdIVSB, for nrdB intron: nrdBIVSA and nrdBIVSB, for U5 nrdD intron: nrdDIVSA and nrdDIV- SU5B, for hef: U5.1 and U5.2, for segH: segH1 and segH rev, for mobE: A2 and BS20, and for mobC: D2 and G2 (Supplementary Table 1). Screening for markers flanking HEGs Ampliﬁcation of the nrdD-nrdG region from progeny phage was performed as described above using primers D8 and G4 and for the nrdA-nrdB region BS40 and 43B (Supplementary Table 1). Phage speciﬁc markers were screened by restriction cleavage at sites indicated in Figure 5C, for the nrdG gene of RB3/T2 crosses by direct sequencing using the G4 primer, and in Figure 5D by direct sequencing using primers BS40 and 43B. RESULTS Screen for mobC and mobE among T-even-like bacteriophages The T4 mobC gene is inserted between genes nrdD and nrdG coding for the anaerobic ribonucleotide reductase and its activ- ator protein, respectively. In a PCR screen of the region bet- ween nrdD and nrdG among 20 T-even-like phages we found that only T4 produced a fragment of mobC size. Phages T6, RB3 and LZ2 produced slightly larger PCR products and the remaining phages produced fragments indicating no additional gene inbetween nrdD and nrdG (data not shown). Surpris- ingly, sequencing of the PCR fragments from T6, RB3 and LZ2 showed that they all contain a completely different gene in this position with similarity to the T4 seg-genes (Figure 1A). We have named the new gene segH. The T6, RB3 and LZ2 segH sequences are very similar with the protein products only differing at ﬁve out of 276 amino acid residues (data not shown). The putative H-N-H endonuclease gene mobE in T4 is loc- ated between the genes coding for the large (nrdA) and the small (nrdB) subunits of the aerobic ribonucleotide reductase. In a PCR screen of 18 T-even-like phages we found that phages T6, RB2, RB3, RB15 and LZ7 had insertions matching the length of T4 mobE in this region while phage U5 had a much longer insertion. Sequencing of the U5 region shows that it encodes a protein of 544 amino acids with no similarity to any known homing endonuclease, but containing a domain of unknown function (20) that appears to be related to a diverse group of endonucleases (Figure 1C). We have named the U5 gene hef for homing endonuclease-like function (see below). The remaining phages in the screen did not contain any gene inbetween nrdA and nrdB. Sequencing of the nrdA-mobE-nrdB region in T4 showed that mobE differed from previously published sequences (21,22) by two frame shifts that brought two previously annot- ated open reading frames (ORF) into a continuous open read- ing frame from the stop codon of nrdA to the start codon of nrdB (19). Sequencing of T6, RB2, RB3, RB15 and LZ7 conﬁrms that they also contain continuous mobE ORFs between nrdA and nrdB with somewhat varying sequences (Figure 1B). The genome sequence of the schizo-T-even phage Aeh1 also encodes a mobE gene of similar size in this region (Figure 1B). All in all this indicates that the original sequence of T4 mobE obtained from a cloned genome frag- ment (21,22) was of a non-functional gene (perhaps generated during the cloning procedure). Indeed, our attempts to clone T4 mobE have produced several frame shifted and mutated versions. However, we have now managed to clone T4 mobE and the gene product is highly toxic when expressed in E.coli (data not shown), a feature shared with numerous proven Figure 2. (A) PAGE gel 8% of the in vitro translation products. Proteins were labelled with [35S]Methionine during in vitro translation and bands were visualized by phosphoimager analysis. (B) Agarose gel from cleavage assay with in vitro translated T4 MobC (lane 2), LZ2 SegH (lane 3), T4 MobE (lane 5) and U5 Hef (lane 6); control incubations with in vitro translation mix without template DNA (lanes 1, 4). DNA targets were labelled at one end with Flourescein-labelled primer thereby generating only one labelled band upon cleavage.