Demonstrating Xis is Unstable

Demonstrating Xis is Unstable

When lambda infects a cell and proceeds down the lysogenic pathway, ultimately it must integrate stably into the chromosome. If both Int and Xis proteins are synthesized, the phage DNA would repeatedly integrate and then excise from the chromosome until these proteins have been diluted away by growth or have become inactive. What ensures that the last event is integration rather than excision? The phage utilizes two mechanisms to maximize the probability that integration activity pre-dominates under these conditions. One is the preferential synthesis of Int protein via the p_I promoter which is activated by CII protein. The second is for Xis protein to be less stable than Int. Weisberg and Gottesman used the att² phage to simplify testing this hypothesis with an in vivo assay of the stability of Int and Xis proteins.

The test used a lysogen of λCI₈₅₇ briefly heat-pulsed to denature the thermolabile repressor and induce a burst of Int and Xis protein syn-thesis. The repressor then renatured, and the phage-encoded protein synthesis stopped. At times thereafter, portions of the culture were

Figure 18.11 The fraction ofλimm²¹att²int^-orxis^-as a functionof time that is converted to λimm²¹ by the Int and Xis proteins induced at zero time.

additionally infected with λimm²¹att²xis^- or λimm²¹att²int^-. Recall that an excision-type of reaction on λatt² removes the extra bacterial DNA from the phage. At early times after the heat pulsing, the Int and Xis protein already present from the first phage complemented the int or xis defect of the att²and catalyzed the excision reaction to produce thesmaller single attλimm²¹ from the λimm²¹att². At later times, however, the Int or Xis synthesized by the first phage had decayed, and therefore fewer wild-type λimm²¹ were produced (Fig. 18.11). Simply infecting at various times after the heat pulse and assaying for the number of non-att² lambda that were produced in the resulting phage burst al-lowed the kinetics of decay of the Int and Xis protein to be measured. The half-life of Int was greater than an hour, whereas the half-life of Xis was less than half an hour.