FIG. 6.
Effect of an antisense construct of ORF2 on activation of P15A10. ORF2 was amplified from P15A10 by using one primer consisting of nucleotides 200 to 221 and one primer complementary to nucleotides 640 to 655 and was cloned behind the strong, constitutive P6 promoter (10) in pNZ18. The T7 terminator was cloned behind the P6::anti-ORF2 cassette. The T7 terminator was amplified from the E. coli expression vector pET28a (Novagen) by using a 5′ primer consisting of 5′-GAGAAGCCCGAAAGGAAGC-3′ and a 3′ primer consisting of 5′-ATCCGGATATAGTTCCTC-3′. (A) β-Gal activity when the antisense construct was combined with pTRK391 (P15A10::lacZ.st) (27) in L. lactis subsp. lactis NCK203 both before and after phage infection. β-Gal levels reported are the average of assays performed at least three separate times. For each assay, time point determinations were performed in duplicate. (B) Slot blot Northern analysis of RNA isolated at various points in the φ31 lytic cycle and probed with 32P-labeled lacZ.st.