This occurred, for example, in the regions between ORFs 62755-63176 (overlapping ORFs), ORFs 66202-66625 (12 bp intergenic region) and ORFs 73676-74436 (139 bp intergenic region, MLN2238 order Figure 1, 2). Figure 2 Reverse transcriptase-PCR amplifications of the analyzed transcript GS-4997 connections indicated in Figure 1. Numbers above amplicons indicate the examined region in ICEclc numbering; numbers

below the calculated amplicon size. ‘Minuses’ are negative control reactions with PCR only without reverse-transcriptase step to verify DNA contamination. Different panels are reactions run on the same gel but not necessarily in consecutive lanes. Electronic images were auto-leveled and relevant lanes were placed side-by-side using Adobe Photoshop CS3. Std, DNA size standard (in kilobase-pairs, kb). At least one negative control was performed on every batch of purified RNA. On top of the RT-PCR analysis we mapped the length of detectable transcripts by Northern hybridizations of RNA isolated from P. knackmussii B13 cultures grown to stationary phase on 3-chlorobenzoate (Figure 3). Arguably, Northern hybridizations do not always produce clear-cut signals and often show multiple bands indicative for mRNA degradation

or processing, but for most of the transcript sizes and positions proposed by RT-PCR analysis supporting evidence was provided by Northerns (Figure 1, 3). Even the breakpoints detected between ORFs 62755-63176 coincided with two detectable transcripts of around 3.5 kb that could be positioned around the gap (Figure 1). The GSK2399872A molecular weight longest detected transcript seems to be formed by an estimated 8.5 kb polycistronic mRNA that would start upstream of ORF81655 and ending at ORF74436. It is possible, as we will argue below, that this transcript is actually

synthesized as a much longer one, but cleaved somewhere in the area of the gap identified by RT-PCR between ORF73676 and 74436. The downstream part would be formed by a 6 kb mRNA that was detectable by probes for the ORFs 68987 and 73029 (Figure 3). Although a -10 promoter region was predicted upstream of ORF73676 by bioinformatic analysis, several others were predicted in this 8.5 kb region as well (see below and Table S1). Therefore, promoter prediction was CHIR-99021 supplier not sufficiently accurate to support or refute the hypothesis for the 8.5 and 6 kb regions being transcribed as a single polycistronic mRNA. Figure 3 Compiled Northern analysis of transcript sizes in the ICE clc core region on RNA isolated from cells grown to stationary phase on 3-chlorobenzoate. Probe used in hybridization for a respective panel is indicated as the ORF number above and the probe number below, corresponding to the indications in Figure 1. Black triangles point to the largest size determined for the hybridizing transcript.