6204 Nucleic Acids Research, 2005, Vol. 33, No. 19 endonuclease activity or homing capability (17). The different seg-, and mob-genes are only scarcely represented in phages closely related to T4 (10,15,16,18). It is intriguing that T4 contains so many seemingly non-essential putative homing endonuclease genes while they are absent in many of its close relatives. In a previous study of the distribution of group I introns among T4-related phages we PCR screened and sequenced the td-nrdAB and nrdDG regions from more than 20 phage strains (19). In T4 these regions contain the three well- characterized intron-encoded HEGs (I-TevI in td, I-TevII in nrdD and I-TevIII in nrdB) and also two freestanding, putative HEGs containing the H-N-H motif, mobE between nrdA and nrdB, and mobC between nrdD and nrdG (cf. Figure 5A). These two freestanding putative HEGs have pre- viously only been found in T4 (10,15,16,18). Here we show that mobE genes are present in ﬁve additional T-even-like phages besides T4. No additional phages with mobC were found. Interestingly, some of the phages that lacked either mobE or mobC had completely unrelated genes inserted at the corresponding sites. One novel gene was found replacing mobC in three phages and shows similarity to the GIY- YIG seg-genes of T4 and a second gene was found replacing mobE in phage U5 and has no similarity to any known homing endonuclease. Both novel gene products displayed endonuclease activity with cleavage sites close to their gene loci in the phage genomes, a feature characteristic for homing endonucleases. We have therefore named the novel seg- gene segH and the U5 gene hef (for homing endonuclease- like function). Homing studies in mixed phage infections show that segH displays site-speciﬁc homing. In addition we have obtained evidence for homing of the mobE gene in phage T6. MATERIALS AND METHODS Bacteria and phages Strains of the original T-evens, T2H, T4D and T6 were kindly provided by Elisabeth Hagga˚rd, Dept. of Genetics, Stockholm University. Strains RB2, RB3, RB27, RB32, RB49, RB69, LZ1, LZ7, TuIa and U5 were kindly provided by Karin Carlson, Dept. of Cell and Molecular Biology, Uppsala Uni- versity. RB14, RB15, RB23 and RB51 are from our stocks, originally a gift from Sean Eddy and phage LZ2 was kindly provided for this study by Sean Eddy, Dept. of Genetics, Washington University of Medicine. Escherichia coli B0 was used as host strain for all phage work and is from our stocks. Screen for mobC and mobE genes Ampliﬁcations were performed using 2.5 U of proofreading Pfu DNA polymerase (Stratagene) for 30 cycles according to the manufacturer’s recommendations. Primers for PCR amp- liﬁcation were for mobE: A2 and BS20, and for mobC: D2 and G4 (Supplementary Table 1). PCR products were analysed on agarose gels and puriﬁed using the QIAquick PCR Puriﬁcation kit (Qiagen) to remove buffers, nucleotides and primers before sequencing. PCR fragments were sequenced on both strands using DYEnamic ET Terminator Cycle Sequencing for MEGA Bace from Amersham Pharmacia Biotech. All sequences obtained in this study have been reported to GenBank , either as updates to previously reported ﬁles AY262125–AY262139, AY262142 and AY262144–AY262145, or as novel accessions nos DQ178119–DQ178121. In vitro expression of proteins Targets for in vitro translations were ampliﬁed directly from phage using the following primer pairs with T7 promoter sequence and reticulocyte ribosome binding sequence: Retic T4 mobE T7 primer together with T4, T6, RB2, RB15 mobE end primer, Retic RB3/T6 mobE T7 primer together with T4, T6, RB2, RB15 mobE end primer or RB3 mobE end primer, Retic T4 mobC T7 primer together with G2, Retic segH T7 primer together with segH 30 primer and Retic U5 hef T7 primer together with U5.1 (Supplementary Table 1). PCR products were puriﬁed using QIAquick PCR puriﬁcation kit (Qiagen) and used as templates in in vitro translation reactions using the reticulocyte in vitro translation kit TNT T7 Quick for PCR DNA (Promega) according to the manufacturers’ recommendations. Radiolabelled [35S]-Met was included in the reactions and products were separated on 12% poly- acrylamide gels and analysed by PhosphoImager (FujiFilm FLA-3000). In vivo expression and purification of SegH A PCR fragment of segH from LZ2 was cloned into pET21(+) (Stratagene) and veriﬁed by DNA sequencing (MWG- biotech). Expression of C-terminally His-tagged segH was performed in E.coli strain BL21(DE3) codon plus, and cells were harvested after 2 h induction with 1 mM isopropyl- b-D-thiogalactopyranoside (IPTG). Cells were disrupted in a X-press chamber (AB Biox) and extracted in sodium phos- phate buffer (pH 7.4), containing 20 mM imidazol. The sus- pension was centrifuged and the supernatant was loaded onto a HisTrap HP column (Amersham Biosciences) and washed according to the manufacturer’s recommendations. SegH was eluted at 116 mM imidazol using a stepwise increase of imidazol concentration. In vitro endonuclease assays All targets for cleavage assays were ampliﬁed from T2 by PCR and puriﬁed using QIAquick PCR puriﬁcation kit (Qiagen). Primers for target ampliﬁcations shown in Figure 2B were: Pat11, G2(F), BS10 and A2(F) (Supplementary Table 1); (F) denotes primers that were Fluorescein-labelled in the 50 end. In vitro translation products were used directly in cleavage reactions. Reaction conditions were as follows: up to 800 fmol target DNA, 5 ml in vitro translation reaction and 3 mg RNase A in a total volume of 50 ml were incubated at 37 C for 30 min in 66 mM K-acetate, 33 mM Tris-acetate (pH 7.9), 10 mM Mg- acetate, 0.1 mg/ml BSA (for MobC and SegH) and in 100 mM NaCl, 50 mM Tris–HCl (pH 7.5), 10 mM MgCl2, 0.1 mg/ml BSA (for MobE and Hef). Incubations with target DNA and in vitro translation mix without primer DNA served as neg- ative controls. 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. Primers for target ampliﬁcations shown in Figures 3A and B were: D3, D4, G4, H2, A2, U5.1 and 43B (Table 1,