FIG. 3.

Effect of phage ΦW104 integration on ryeA/ryeB expression. Bulk RNA was extracted from strains LT2 and MA7833 (ΦW104 lysogen) as described in the legend to Fig. 2. (A) Primer extension analysis of RyeA RNA from the lysogenic strain (lane 1) and from wild-type LT2 (lane 2). Reverse transcriptase reactions were carried out using primer ppB12 (5′-CCCTGGACCGAATACAGGA-3′) as previously described (6). Sequencing reactions were performed with the fmol DNA cycle sequencing system from Promega according to the manufacturer's protocol. The template was a DNA fragment obtained by PCR amplification of chromosomal DNA from strain MA7833 with oligonucleotides pp490 (5′-TGGCGTCGTCATCTATTC-3′) and pp491 (5′-CAGGGACGCTATCACACA-3′) as primers. (B) Northern blot quantification of RyeA and RyeB levels in strains MA7833 (lane 1) and wild-type LT2 (lane 2). Bulk RNA was fractionated on an 8% polyacrylamide-8 M urea gel. Membranes were probed with ³²P-labeled oligonucleotides pp925 and ppB13 (see the legend to Fig. 2). 5S RNA and tmRNA probed with ppB10 (5′-ACACTACCATCGGCGCTACG-3′) and pp813 (5′-GCGGAGGCTAGGGAGAGAGG-3′), respectively, were used as internal controls. Due to the higher gel concentration, the two RyeA bands are less separated than in Fig. 2.