PMC

Results of real-time quantitative PCR showing linear regressions of the cycle threshold (C_T values) and the log concentration of genomic DNA of G. intraradices isolates B3, C2, and C3. The analysis was performed with primers amplifying all previously found variants of the BiP gene and pseudogenes (A); ribosomal genes 18S (B), 5.8S (C), and 25S (D); and Rad15 (E). Real-time quantitative PCR was also performed on Rad32, but the data are not shown as they showed exactly the same pattern as that of Rad15. For all real-time quantitative PCR experiments, two replicate amplifications for each isolate were performed. Data points shown in the graphs represent the average C_T value of the two replicates.

Evidence for the segregation of BiP pseudogene variants among isolates of G. intraradices. A. Partial nucleotide sequence alignments of two BiP pseudogene variants isolated from five isolates of G. intraradices (DAOM181602, A4, B3, C2, and C3). The two different sequence types are shown in separate boxes. The number following each isolate code represents the pseudogene sequence type. B. Southern blot hybridization on genomic DNA from the isolate DAOM181602 of a probe that hybridizes to all BiP gene and pseudogene variants. Results with genomic DNA digested with EcoRV and XbaI are shown. The sizes of the bands are shown on the right. C. PCR amplification of the two BiP pseudogene variants using specific primers with DNA from the different G. intraradices isolates. The analysis shows results from an additional isolate (D1) from the same population that was not included in the cloning and sequencing experiments. The upper gel shows amplification of the BiP pseudogene type 1, using the reverse primer BiPT1.R. The lower gel shows amplification of the BiP pseudogene type 2, using the reverse primer BiPT2.R. The isolate C2 is shown to harbor both types 1 and 2 of the BiP pseudogene. The size standard is in the far left and far right lanes of both gels.