## 2 Result and Discussions
### Transformation of Cells by Plasmids Expressing P, Complementation and Immunity Assays
Table 3 shows the transformation of nonlysogenic 594 _dnaB\({}^{+}\)_ cells (_dnaB_ was sequenced [34]) and of 594(\(\lambda\)nin5) lysogenic cells. The CI repressor made from the \(\lambda\)nin5 prophage is not Ts. The lysogenic cells expressing CI\({}^{+}\) were transformable by pclpR-\(P\)-timm at 37 \({}^{\circ}\)C. The nonlysogenic cells were transformed by pclpR-\(P\)-timm only between 25-34 \({}^{\circ}\)C, where CI[Ts] retains sufficient repressor activity to shut off \(P\) expression in transformed cells. CI[Ts] is reported [35] marginally active at \(\geq\)37 \({}^{\circ}\)C and would not fully repress \(P\) expression, explaining the absence of transformants arising in nonlysogenic cells when the transformation temperature is raised above a temperature where CI[Ts] repressor retains sufficient DNA binding activity. The 594[pclpR-\(P\)-timm] cells complement for P, at 30 \({}^{\circ}\)C, and very marginally (in comparison to the \(\Delta P\) plasmid) complement at 25 \({}^{\circ}\)C (Table 4), suggesting a low level of leaky \(P\) expression, even when CI[Ts] is active. The noninduced P-SPA fusion complements for P in cells grown at 30 and 37 \({}^{\circ}\)C (data not shown). 594[pclpR-\(P\)\({}^{\lambda 76}\)-timm] transformants were unable to complement for \(P\), suggesting that the region of \(P\) removed by the in-frame deletion of 76 codons, localized to the N-terminal end of the 233 amino acid P protein (fusing part of codon 9 with 86) is essential for the role of P in \(\lambda\) replication. The immune state, \begin{table}
\begin{tabular}{l l l} \hline
**Plating host** & **Incubation** & **EOP \({}^{\circ}\) of** \\  & **temperature** & \(\lambda\)_imm_434_P_am3** \\ \hline
594[pclpR-\(P\)-timm] & 25 \({}^{\circ}\)C & 0.0008 \\  & 30 & 0.82 \\  & 37 & 0.93 \\
594[pclpR-\(P\)\({}^{\lambda 76}\)-timm] & 30 & \(<\)0.00003 \\  & 37 & \(2\times 10^{-7}\) \\ \hline \end{tabular} \({}^{\mathrm{a}}\): EOP of 1.0 determined on host TC600 _supE_ at 30 \({}^{\circ}\)C.  
\end{table} 
Table 4: Complementation of \(\lambda\)_P_am mutation by plasmid cloned \(P\) gene. \begin{table}
\begin{tabular}{l c c c} \hline
**Cells** & **Plating** & **Transformation** & **Transformants** \\
**Transformed** & **Temp. \({}^{\circ}\)C** & **Frequency \({}^{\mathrm{a}}\)** & **per \(\mathrm{\SIUnitSymbolMicro g}\) DNA** \\ \hline
594 & 25 & \(5.2\times 10^{5}\) & \(1.5\times 10^{5}\) \\  & 37 & \(<\)\(1.7\times 10^{-8}\) & 0 \\
594(\(\lambda\) wt min5) & 25 & \(7.2\times 10^{-5}\) & \(4.7\times 10^{4}\) \\  & 37 & \(8.5\times 10^{-6}\) & \(5.5\times 10^{3}\) \\ \hline \end{tabular} \({}^{\mathrm{a}}\): Transformation frequency represents the average cell titer on TB+Amp\({}_{50}\) plates per average cell titer on TB plates.  
\end{table} 
Table 3: Cellular transformation by pclpR-\(P\)-timm plasmid. i.e., _imm_\(\lambda\)[Ts] phenotype, of the pcIpR-[ ]-timm plasmids was examined (Table 5, and [36]) by plating \(\lambda cI72\) on hosts with plasmids encoding intact \(P\) or \(O\) genes. The plasmid control p434'pR-\(O\)-timm with a hybrid \(\lambda\)/434 CI repressor does not block \(pR\) transcription, expresses \(O\) constitutively at all temperatures, and showed no immunity to an infecting _imm_\(\lambda\) phage. Plasmid pcIpR-\(O\)-timm confers high _imm_\(\lambda\) immunity in cells grown between 25 and 39 'C, and loses it at 42 'C where the CI[Ts]857 repressor is fully inactive. Additional immune characterization of these plasmids was reported (Table 4 in [36]). Although pcIpR-\(O\)-timm only carries \(oR\) operators for repressor binding, CI immune repression is maintained through 39 'C, but lost at 42 'C where the CI repressor is denatured. Thus, the pcIpR-(GOI)-timm plasmids, even though they carry only \(oR\) site, can maintain CI immune repression, and presumably block the expression of GOI positioned downstream of \(pR\), between 25-39 'C. However, the analogous plasmid pcIpR-\(P\)-timm exhibits _imm_\(\lambda\) immunity at 25-30 'C but the immune response is ineffective at and above 35 'C.