To analyze rickettsial transcriptional termination, we focused on the most likely location for termination, the intervening sequences between convergent genes. Of the 104 convergent gene pairs annotated in the R. prowazekii genome, we selected 12 genes, representing 6 well-separated gene pairs that, with two exceptions, met the following selection criteria. First, each gene is transcribed at detectable levels. This was evaluated using microarray data or by direct measurements using RPA. In addition, in the current study intragenic positive control probes were included to confirm gene transcription. Secondly, the gene products were detected by proteomic analysis, with the exception of RP826 and RP777. The latter gene was listed as a pseudogene in Madrid E and therefore not annotated or screened in proteomic analyses. Two of these gene pairs were also included due to the prediction by TransTermHP of a strong, bidirectional, intrinsic terminator within the intervening regions. Transcript detection was accomplished Tamibarotene using ribonuclease protection assays. RPA analysis uses single-strand, labeled RNA probes that are antisense to target mRNAs. If mRNA specific to the probe is present, it will hybridize to the probe and protect it from digestion with nucleases that specifically digest single strands. The protected probe can then be analyzed by gel electrophoresis. The extent of protection allows for the estimation of termination sites and reveals a complete picture of protected transcripts within the selected region. Conversely, if the transcript does not stop and reads through the intervening region, the probe will appear as fully protected. If there are non-site specific termination events through the intervening region, multiple bands of differing sizes will be visualized. In Table 2, the sizes of the intergenic regions, the probe size, and the amount the probe overlaps the coding regions of the genes are presented for Sulfamethazine the probes used in this study. We assayed rickettsial RNA extracted from rickettsiae grown in hen egg yolk sacs and in L929 mouse fibroblast cells. Previous studies had indicated that mRNA assayed at 34uC from rickettsiae grown in L929 cells had a half-life of approximately 15 minutes, a property that would preclude the isolation of mRNA from yolk sacs due to the 4–5 hours required to isolate rickettsiae from this source. However, we found that rickettsial mRNA is present in the egg yolk sac rickettsial RNA preparation and can be detected at levels comparable to mRNA isolated from rickettsiae grown in L929 cells. The recovery of mRNA from yolk sac grown rickettsiae is most likely due to performing all manipulations at 4uC during rickettsial purification. This permitted us to assay rickettsial RNA from different rickettsial host backgrounds. Rather, the presence of a diffuse banding pattern suggests non-site- specific termination throughout the intervening region. In contrast to the RP145 transcript, the probe targeted to the intervening region downstream of RP146 was fully protected demonstrating that the RP146 transcript extends into the RP145 coding region generating antisense RNA to RP145 transcripts. The RP495-RP496 gene pair exhibited a similar termination profile: RP495 transcripts exhibit no defined termination site and the probe specific for RP496 was fully protected. Once again this demonstrates that RP496 transcripts are extending into the RP495 coding region generating antisense RNA. The protection profile observed with the RP777-RP778 gene pair demonstrated that both gene transcripts exhibited a diffuse pattern indicative of non-site-specific termination throughout the intervening region.