## 2 Result and Discussions
### Influence of P-alteration, or O O OOP RNA Co-Expression on P-Iethality and Plasmid Retention
The co-expression of \(O\) would provide an interactive partner for P, possibly lowering the cellular level of unbound P. The 77 nucleotide antisense OOP RNA, which is transcribed from promoter \(pO\) in the opposite orientation to the \(pR\)-\(O\)-\(P\) mRNA (Figure 1A), can hybridize between \(pR\) and \(O\), forming an OOP-RNA: \(pR\)-\(O\)-\(P\)-mRNA hybrid that can provide a target for \(P\) mRNA degradation downstream from the OOP-binding site. The co-expression of \(O\)-\(P\) increased cell viability at 37-39 'C, but not plasmid retention. The constitutive or inducible expression of \(O\) without \(P\) did not influence cell viability or plasmid loss between 30-39 'C. Early studies [74] attempting to determine the coding sequence for \(O\), reported a longer protein (O') made by read-through of the normal stop codon, terminating downstream somewhere within the N-terminal sequence of \(P\). Plasmids were made that included the wild type \(\lambda\) sequence between the start of \(O\) through the UGA stop codon [75], plus 35 or 62 in-frame codons downstream into the \(P\) DNA sequence, so that if the stop codon for \(O\) was designated \"1\", the next in-frame stop codons would occur at positions 36 or 63 in frame with \(O\), even though the downstream reading frame for \(P\) differs from \(O\). While the plasmids expressing the intact \(O\) sequence were not toxic, the plasmids with O-36P or O-63P, when expressed at 39 'C, were toxic and caused plasmid loss. It is unclear if their toxicity depends on O-read-through downstream of its stop codon, or to polypeptides made from the N-terminal end of \(P\). Plasmids pclpR-_oop#1-O-P_-timm and pclpR-_oop#2-O-P_-timm (Figure 1A, Table 10), when compared to versions expressing only \(P\) or \(O\)-\(P\) significantly suppressed plasmid loss at 37-39 'C, and partially suppressed P-lethality at 37 'C, with the _oop#_1 plasmid showing stronger suppressing effects than the slightly larger #2 version. This data supports a hypothesis that \(P\) expression from the \(\lambda\) genome is reduced by OOP antisense micro RNA, possibly via a OOP:\(pR\)-mRNA complex serving as a target for mRNA degradation. We observed that the co-expression of \(O\) and \(P\), with the potential for formation of an O:P complex, did not suppress the P phenotypes. In contrast, placing the natural _oop-O-P_ sequence orientation downstream from \(pR\) in the pclpR-[ ]-timm plasmids markedly suppressed P-inhibition of ColE1 replication as monitored by assaying for plasmid curing. This is explained, possibly, by OOP micro-RNA serving in some manner as a competitor to RNA I made by the plasmid, resulting in the loss of plasmid copy control and higher plasmid copies per cell. We view this explanation as unlikely. Alternatively, OOP RNA may limit the adverse effects of \(P\) expression. It can bind to \(pR\)-promoted _cII_ mRNA and target its degradation in an RNaseIII-dependent reaction [76, 77], suggested to limit downstream \(O\)-\(P\) gene expression. What is the possibility that inefficient termination of \(pR\)-GOI transcription and consequent run-on transcription could cause problems with plasmid maintenance and explain the loss of pclpR-_P_-timm from cells, rather than a direct effect of P on plasmid maintenance? We have observed that the wild type _timm_ terminator prevents read through from the _cI-rex_ transcript into the _oL_ operator region or through into \(N\) when CII stimulated transcription from \(pE\) occurs at 30 to 100-fold the level of _cI-rex_ transcription from the _cI_ maintenance promoter \(pM\) (see [17] and contained references). This was the rationale for incorporating _timm_ into the design of pclpR-(GOI)-timm plasmids. Table 7 shows the influence of expression of lambda gene \(O\) from plasmids pclpR-\(O\)-timm and p434'pR-\(O\)-timm on plasmid loss at 30, 35, 37, 39 and 42 'C. The immunity properties of these two plasmids was shown in Table 4 of [36]. The expression of gene O is constitutive at all temperatures from plasmid p434'pR-_O_-timm.