Search

Aim A collaborative exercise with several institutes was organized by the Forensic DNA Service (FDNAS) and the Institute of the Legal Medicine, 2nd Faculty of Medicine, Charles University in Prague, Czech Republic, with the aim to test performance of different laboratories carrying out DNA analysis of relatively old bone samples. Methods Eighteen laboratories participating in the collaborative exercise were asked to perform DNA typing of two samples of bone powder. Two bone samples provided by the National Museum and the Institute of Archaelogy in Prague, Czech Republic, came from archeological excavations and were estimated to be approximately 150 and 400 years old. The methods of genetic characterization including autosomal, gonosomal, and mitochondrial markers was selected solely at the discretion of the participating laboratory. Results Although the participating laboratories used different extraction and amplification strategies, concordant results were obtained from the relatively intact 150 years old bone sample. Typing was more problematic with the analysis of the 400 years old bone sample due to poorer quality. Conclusion The laboratories performing identification DNA analysis of bone and teeth samples should regularly test their ability to correctly perform DNA-based identification on bone samples containing degraded DNA and potential inhibitors and demonstrate that risk of contamination is minimized.

We show that in Dictyostelium discoideum an endogenous gene as well as a transgene can be silenced by introduction of a gene construct that is transcribed into a hairpin RNA. Gene silencing was accompanied by the appearance of sequence-specific RNA ∼23mers and seemed to have a limited capacity. The threeDictyostelium homologues of the RNA-directed RNA polymerase (RrpA, RrpB, and DosA) all contain an N-terminal helicase domain homologous to the one in the dicer nuclease, suggesting exon shuffling between RNA-directed RNA polymerase and the dicer homologue. Only the knock-out of rrpA resulted in a loss of the hairpin RNA effect and simultaneously in a loss of detectable ∼23mers. However, ∼23mers were still generated by theDictyostelium dsRNase in vitro with extracts from rrpA−, rrpB−, and DosA−cells. Both RrpA and a target gene were required for production of detectable amounts of ∼23mers, suggesting that target sequences are involved in ∼23mer amplification.

Publisher Summary As in most other organisms, sense-antisense hybrids cannot be detected by conventional hybridization experiments in Dictyostelium. A dsRNase may therefore cause the degradation of the postulated double-stranded RNA and might be a target for the regulation of antisense mechanisms. By using an assay to identify nucleases that specifically hydrolyze double-stranded RNA, a predominantly cytosolic dsRNase was identified in Dictyostelium extracts. This chapter describes a purification method for Dictyostelium dsRNase (DdsRNase) and presents a partial biochemical characterization of the enzyme. It is nonsequence specific and digests homopolymer duplexes as well as random sequence dsRNA targets generated from various Dictyostelium gene sequences; dsRNA digestion depends on divalent metal ions, with a vast preference for Mg 2+ . DdsRNase has been purified and elutes from a gel-filtration column as a large, approximately 450-kDa multicomponent complex. The enzyme is thus distinct from the RNase III family and other previously described dsRNase activities by structure and biochemical activity.