RNA trans-splicing: identification of components of a putative chloroplast spliceosome.

Group II introns with highly complex RNA structures have been discovered in both prokaryotes and eukaryotic organelles. Usually, excision of non-coding group II intron sequences occurs by cis-splicing, the intramolecular ligation of exons in the same precursor RNA, but some group II introns are excised by intermolecular ligation. This process is called trans-splicing, and genome sequencing predicted that this type of RNA processing occurs in more than 180 organelle genomes from eukaryotes. A well characterised trans-spliced intron RNA is represented by the chloroplast psaA gene of the model alga Chlamydomonas reinhardtii. The psaA gene is split into three exons, which are widely distributed over the plastome and transcribed independently. PsaA exons are flanked by sequences typical for group II introns and joined by trans-splicing via two transesterification reactions. Although it is known that some group II introns are able to splice autocatalytically, trans-splicing of the psaA RNA depends on several nucleus and chloroplast encoded factors. The phylogenetic relationship between group II introns and nuclear spliceosomal RNA led to the hypothesis that these factors are part of large multiprotein and ribonucleoprotein complexes akin to the nuclear spliceosome. Here, we give a concise overview of experimental strategies to identify novel factors involved in trans-splicing of psaA RNA and review recent results that have elucidated the composition and function of a putative chloroplast spliceosome involved in processing of chloroplast precursor RNAs.

Performance evaluation of the QIAGEN EZ1 DSP Virus Kit with Abbott RealTime HIV-1, HBV and HCV assays.

BACKGROUND: Automated sample preparation systems must meet the demands of routine diagnostics laboratories with regard to performance characteristics and compatibility with downstream assays. OBJECTIVES: In this study, the performance of QIAGEN EZ1 DSP Virus Kit on the BioRobot EZ1 DSP was evaluated in combination with the Abbott RealTime HIV-1, HCV, and HBV assays, followed by thermalcycling and detection on the Abbott m2000rt platform. STUDY DESIGN: The following performance characteristics were evaluated: linear range and precision, sensitivity, cross-contamination, effects of interfering substances and correlation. RESULTS: Linearity was observed within the tested ranges (for HIV-1: 2.0-6.0 log copies/ml, HCV: 1.3-6.9 log IU/ml, HBV: 1.6-7.6 log copies/ml). Excellent precision was obtained (inter-assay standard deviation for HIV-1: 0.06-0.17 log copies/ml (>2.17 log copies/ml), HCV: 0.05-0.11 log IU/ml (>2.09 log IU/ml), HBV: 0.03-0.07 log copies/ml (>2.55 log copies/ml)), with good sensitivity (95% hit rates for HIV-1: 50 copies/ml, HCV: 12.5 IU/ml, HBV: 10 IU/ml). No cross-contamination was observed, as well as no negative impact of elevated levels of various interfering substances. In addition, HCV and HBV viral load measurements after BioRobot EZ1 DSP extraction correlated well with those obtained after Abbott m2000sp extraction. CONCLUSIONS: This evaluation demonstrates that the QIAGEN EZ1 DSP Virus Kit provides an attractive solution for fully automated, low throughput sample preparation for use with the Abbott RealTime HIV-1, HCV, and HBV assays.