# Implementation Studies of Bacteriophage \(\lambda\) O
## 2 Results
### Replicative Killing Selection and Mutants  
The selection of RK\({}^{-}\) mutants with defects in \(\lambda\) replication initiation were categorized (Figure 1) as follows: RK\({}^{-}\) clones designated Hd\({}^{-}\) (host defects), representing about 4% to 6% of selected spontaneous RK\({}^{-}\) clones [22], are not lysed by \(\lambda\)_vir_. These mutants are arbitrarily considered to have an altered host gene whose product participates in vegetative \(\lambda\) growth, or a \(\lambda\)-fragment mutation whose effect is to complement negatively for the growth of \(\lambda\)_vir_. The RK\({}^{-}\) clones that are lysed by \(\lambda\)_vir_, retain the \(imm\lambda\) phenotype at 30 \({}^{\circ}\)C, and were FI\({}^{+}\), were designated RK\({}^{-}\) ilr (initiation of \(\lambda\) replication defective). The FI, or functional immunity assay [11; 41], represents a stab of an RK\({}^{-}\) CFU to a lawn of cells lysogenized with \(\lambda\)_imm_434T to which is added free \(\lambda\)_imm_434_c_I. If \(imm\lambda\) double recombinant phage can be generated (indicated by a lysis area forming around the RK\({}^{-}\) clone stabbed to the overlay plate), this is taken to indicate that the \(imm\lambda\) region encoding o\(\lambda\)/_p_L -_cl_- \(\alpha_{\text{R}}\)/_p_R is functional both in the RK\({}^{-}\) mutant and in the \(imm\lambda\) recombinant. The RK\({}^{-}\) FI\({}^{-}\) Imm\({}^{-}\) isolates mainly have had large deletions (\(>\)10 Kb) [26; 27; 28]. In an examination of the spontaneous RK\({}^{-}\) mutants/mutations arising from four RK\({}^{+}\)\(N^{+}\) selector strains, 256/650 RK\({}^{-}\) isolates were the RK\({}^{-}\) ilr type [22].  
Figure 2 shows eight sequences for RK\({}^{-}\) ilr mutants falling within \(O\)-\(P\) that were derived from induced \(N^{+}\) prophage, along with mutants derived from induced \(N^{-}\) prophage, including ori-95, -96, -98 obtained by [PERSON] [20] and ti12 from the Dove laboratory [18]). The sequences for ori95 and ori98 were not previously reported [42]. The ori96 mutation was a 15 bp deletion of \(\lambda\) bases 39139-39153 (not 39138-39152 as reported [42]). Mutation ori98 removed the entire ieron-ITN4 region, and ori95 and ori96 each deleted part of the High-AT rich region within \(O\). Except for ilr541c, which included a stop codon that eliminated translation of the last 35 codons of \(O\), the remaining RK\({}^{-}\) ilr \(O\) mutations (Figure 2) represented small deletions within \(O\) or insertions that could exert a polar effect on downstream \(P\) expression. Each of the ilr mutants were initially scored as being \(O^{+}\), which clearly was not proved correct by sequence analysis. None of the ilr mutations had in-frame deletions within \(O\) as with those obtained in [PERSON]'s \(\lambda\)\(N^{-}\) selection.  
Figure 2: RK\({}^{-}\) ilr mutations characterized within \(\lambda\) genes \(O\)-\(P\). The minimal \(ori\lambda\) size was suggested to include a HIGH-AT-rich region to the right of four ieron sequences, ITN’s1-4 [43; 44], which each contain an 18 bp inverted repeat of hyphenated symmetry, joined by adenine residues that can cause \(ori\lambda\) to assume a bent structure [45]. The mutants shown designated ori95, ori96 and ori98 were obtained from WD from prophage with \(r\)-mutants \(r\)-95, \(r\)-96, and \(r\)-98. Note that [PERSON], et al., [46] sequenced the \(r\)-mutants \(r\)-99, \(r\)-96 and \(r\)93 which represent \(\Delta\)12 bp (39120–39131), \(\Delta\)15 bp (39138–39152) and \(\Delta\)24 bp (39092–39115) [42]. Our sequence localization for ori96 (\(r\)96) differs by one bp from that assigned in [42].