Triple-base changes were made in each of the five Integrase (Int) arm-type binding sites of bacteriophage lambda. These triple changes, called ten mutants, were compared with single-base changes (hen mutants) for their effects on integrative and excisive recombination. The presence of ten or hen mutations in the P1, P'2, or P'3 sites inhibited integration, but the ten P'3 mutant was 10-fold more defective than the analogous hen mutant. The results with these mutants suggest that the P1, P'2, P'3, and possibly the P'1 sites are required for integration. In wild-type E. coli, the ten P'1 mutant reduced the frequency of excision 5-fold, whereas the hen P'1 mutant had no effect. The presence of ten mutations in the P2, P'1, or P'2 sites inhibited lambda excision in an E. coli strain deficient in the production of FIS, while hen mutations in the P2 and P'2 sites had little or no effect. The results with the ten mutants suggest that the P2, P'1, and P'2 sites are required for excision. The differences in the severity of the effects between the ten and hen mutations may be due to the inability of cooperative interactions among Int, IHF, Xis, and FIS to overcome the disruption of Int binding to sites with triple-base changes compared to sites with single-base changes.

We have performed a mutational analysis of the xis gene of bacteriophage lambda. The Xis protein is 72 amino acids in length and required for excisive recombination. Twenty‐six mutants of Xis were isolated that were impaired or deficient in lambda excision. Mutant proteins that contained amino acid substitutions in the N‐terminal 49 amino acids of Xis were defective in excisive recombination and were unable to bind DNA. In contrast, one mutant protein containing a leucine to proline substitution at position 60 and two truncated proteins containing either the N‐terminal 53 or 64 amino acids continued to bind lambda DNA, interact cooperatively with FIS and promote excision. However, these three mutants were unable to bind DNA cooperatively with Int. Cooperativity between wild‐type Xis and Int required the presence of FIS, but not the Int core‐type binding sites. This study shows that Xis has at least two functional domains and also demonstrates the importance of the cooperativity in DNA binding of FIS, Xis and Int in lambda excision.

The bacteriophage P22-based challenge-phage system was used to study the binding of Xis and FIS to their sites in aitP of bacteriophage lambda. Challenge phages were constructed that contained the Xl, X2, and F sites within the P22 Pa.,, promoter, which is required for expression of antirepressor. During lysogenic development, bacteriophage X integrates into the Escherichia coli chromosome via site-specific recombination (for a review, see reference 21). Integration occurs by recombination between specific sites in the X and E. coli chromosomes called attP and attB, respectively, and generates the two hybrid prophage sites, attL and attR. The lysogenization reaction requires the X-encoded integrase (Int) protein and the E. coli-encoded integration host factor (IHF) protein. Upon induction of a X lysogen, the prophage excises its DNA from the E. coli chromosome and commences lytic growth. Excisive recombination occurs between attL and attR and requires Int, IHF, and a second bacteriophage-encoded protein excisionase (Xis). In addition, the E. coli protein factor for inversion stimulation (FIS) enhances excision (32,40).Each of these proteins binds to specific targets within the att sites. In footprinting experiments using nuclease protection with attR, Xis protected a 40-bp region of DNA that contained two imperfect 13-bp repeats, designated the Xl and X2 sites (Fig.

We report the case of a 23-month-old girl who presented with poor growth and delayed attainment of gross-motor milestones. Elevated creatine phosphokinase (CPK) indicated rhabdomyolysis, ultimately attributed to severe, acquired autoimmune hypothyroidism. Growth data and bone-age suggest the onset of hypothyroidism occurred at or before 12 months of age. Acquired hypothyroidism is rare before age 3 years, and rhabdomyolysis due to hypothyroidism has not previously been reported as a cause of delayed gross-motor development in toddlerhood. Despite the early onset of hypothyroidism, cognitive function appeared to be unaffected. Adequate thyroid hormone replacement quickly normalized the CPK in our patient, and gross motor development rapidly improved. Although rare, rhabdomyolysis secondary to hypothyroidism should be in the differential diagnosis of delayed gross-motor development in infancy and toddlerhood.

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