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ZusammenfassungDie biostatistische Bewertung DNA-analytischer Befunde unterstützt Gerichte bei der Einschätzung des Beweiswertes einer Spur. In der Praxis werden dabei zunehmend Spuren mit minimaler DNA-Menge und möglichen „Drop-in“- und „Drop-out“-Ereignissen sowie komplexe Mischspuren analysiert. Solche Spuren sind mit einer klassischen „binären“ Berechnung biostatistisch häufig nicht oder nur eingeschränkt bewertbar.Die Entwicklung vollkontinuierlicher Modelle (VKM) macht eine Vielzahl dieser bisher nicht berechenbaren Spuren einer biostatistischen Bewertung zugänglich. Dabei werden nahezu sämtliche verfügbaren Informationen einer DNA-Spur in die Berechnung einbezogen. Während diese probabilistischen Verfahren international bereits vielfach zum Einsatz kommen, liegen hierzu im deutschsprachigen Raum nur wenige Erfahrungen vor.Um Funktionsweise, Möglichkeiten und Grenzen von VKM-Berechnungen zu erfassen, wurden Mischspuren bekannter Zusammensetzung mit 4 aktuell verfügbaren VKM-Programmen vergleichend analysiert. Bei der Auswertung wurden zentrale Aspekte betrachtet, wie beispielsweise die Konkordanz von Berechnungsergebnissen, der Einfluss von Drop-in- und Drop-out-Ereignissen auf die berechneten vollkontinuierlichen LR-Werte (LRfc) sowie die Ableitung recherchefähiger DNA-Profile mithilfe wahrscheinlichkeitsbasierter Prognosen (Deconvolution).Die im Rahmen dieser Arbeit gewonnenen Erfahrungen bilden, zusammen mit weiteren bereits international publizierten Studien, eine Basis für Empfehlungen zum Einsatz von VKM-basierter Software bei der biostatistischen Bewertung DNA-analytischer Befunde.

ZusammenfassungDie biostatistische Bewertung DNA-analytischer Befunde unterstützt Gerichte bei der Einschätzung des Beweiswertes hinsichtlich einer möglichen Spurenbeteiligung durch eine zu betrachtende Person (engl. „Person Of Interest“; POI). Um die Vergleichbarkeit derartiger Berechnungen auf Grundlage etablierter wissenschaftlicher Standards zu gewährleisten, wurden bereits in der Vergangenheit entsprechende Empfehlungen im nationalen Konsens formuliert.Mit Einführung sog. vollkontinuierlicher Modelle (VKM) für die probabilistische Genotypisierung, die u. a. die Signalintensitäten eines Elektropherogramms berücksichtigen, wurde eine Ergänzung zu den damaligen Empfehlungen erforderlich. VKM erlauben eine biostatistische Bewertung von Spuren mit möglichen Drop-in- und Drop-out-Ereignissen und wahrscheinlichkeitsbasierte Prognosen der zu einer Mischspur beitragenden Genotypen („Deconvolution“).Die vorliegende Veröffentlichung enthält Empfehlungen zum Einsatz VKM-basierter Software und zur Berichterstattung vollkontinuierlicher LR-Werte (engl. „Fully Continuous Likelihood Ratios“; LRfc). Sie empfiehlt bei schwierig zu interpretierenden Befunden eine VKM-Berechnung zur Bewertung einer Spurenlegerschaft. Die VKM-Berechnung ersetzt die bisher in Ausnahmefällen als hinnehmbar erachtete Vorgehensweise einer binären Berechnung unter Ausklammern einzelner Merkmalssysteme. Der Einsatz von VKM erfordert eine umfassende Anwenderschulung sowie eine Validierung und Verifizierung gemäß den Vorgaben der Programmanbieter. Mit der Empfehlung von LRfc-Schwellenwerten soll eine sichere, vergleichbare Anwendung von VKM gewährleistet werden.

The first complete primary structure of a mammalian sialate-pyruvate lyase, namely of the enzyme from porcine kidney, was elucidated by a combination of different PCR techniques followed by sequencing of the resulting fragments. The primers used were either deduced from four porcine lyase peptides or from an alignment of human and mouse expressed sequence tags (ESTs), which were found to be homologous to already known microbial lyase sequences, and cDNA alone or after ligation with a plasmid vector served as a template. The lyase primary structure consists of 319 amino acids with a calculated protein molecular mass of approximately 35 kDa, which fits well to the value determined for the native enzyme. The porcine lyase sequence made it possible to assemble several ESTs from mouse and man in order to obtain the complete putative lyase genes. The three mammalian sequences reveal a high degree of homology both on the nucleotide (83% of the nucleotides are identical between all three sequences) and on the amino-acid level (72% of the amino acids are identical between all three sequences), and thus form a tightly related group. In contrast, the identity between the lyase primary structures from pig kidney and the microbial enzyme from Clostridium perfringens is much less pronounced (25%). Thirty-one amino acids were found to be absolutely conserved in all lyase sequences. Among them are two amino acids (lysine 173 and tyrosine 143 in the porcine lyase) that are most important for the catalytic reaction. After expression cloning, recombinant enzyme activity was expressed in Escherichia coli BL21(DE3)pLysS, which confirms the identity of the cloned sequence and verifies one of the putative human and murine sequences. After SDS/PAGE of a cell extract of the expression clone, a band of 35kDa was stained on the gel.

We developed a 50 SNP-multiplex assay for detection on a MALDI-TOF MS platform based on the SNPs in the 52 SNP-multiplex assay recently developed by the SNP for ID Consortium. After PCR amplification, the products were purified on Qiagen columns and used as templates in one single base extension (SBE) multiplex reaction. Two different strategies were used to design the SBE multiplex: (1) Small 5′-tags (3–8nt) that increased the masses of the SBE primers without changing the annealing temperature; (2) Cleavable primers with one RNA nucleotide which was later cleaved by a mixture of RNases. The SBE primers were extended with biotin labelled ddNTPs and purified on avidin beads ensuring that only the extended SBE primers were isolated and spotted on the MALDI-TOF anchor target. Detection of the 50 extended primers from the SBE reaction was performed in a mass range between 3000 and 10,000 m / z .

Cellular ion homeostasis involves communication between the cytosol and the luminal compartment of organelles. This is particularly critical for metal ions because of their toxic potential. We have identified the yeast homologue of the prokaryotic ArsA protein, the homodimeric ATPase Arr4p, as a protein that binds to the yeast intracellular CLC chloride-transport protein, Gef1p. We show that binding of Arr4p to the C terminus of Gef1p requires the presence of yeast cytosol and is sensitive to a highly specific copper chelator in vitro and in vivo. Copper alone can substitute for cytosol to support the interaction of Arr4p with the C terminus of Gef1p. The migration behavior of Arr4p in nonreducing gel electrophoresis correlates with cellular copper deficiency, repletion, or stress. Our homology model of Arr4p shows that the antimony (arsenic) metal binding site of ArsA is not conserved in Arr4p. The model suggests that a pair of cysteines, Cys285 and Cys288, is located in the interface of the Arr4p dimer. These residues are required for Arr4p homodimerization and for binding to the C terminus of Gef1p. Whereas both proteins are required for normal growth under iron-limiting conditions, they play opposite roles when copper and heat stress are combined in an alkaline environment. Under these conditions, deltagef1 cells grow much better than wild type yeast, whereas deltaarr4 cells are unable to grow. Comparison of the deltaarr4 with the deltaarr4deltagef1 strain suggests that Arr4p antagonizes the function of Gef1p.

Classic studies of temperature-sensitive secretory (sec) mutants have demonstrated that secreted and plasma membrane proteins follow a common SEC pathway via the endoplasmic reticulum (ER), Golgi apparatus, and secretory vesicles to the cell periphery. The yeast protein Ist2p, which is synthesized from a localized mRNA, travels from the ER to the plasma membrane via a novel route that operates independently of the formation of coat protein complex II–coated vesicles. In this study, we show that the COOH-terminal domain of Ist2p is necessary and sufficient to mediate SEC18-independent sorting when it is positioned at the COOH terminus of different integral membrane proteins and exposed to the cytoplasm. This domain functions as a dominant plasma membrane localization determinant that overrides other protein sorting signals. Based on these observations, we suggest a local synthesis of Ist2p at cortical ER sites, from where the protein is sorted by a novel mechanism to the plasma membrane.

CLC chloride channels are a family of channel proteins mediating chloride transport across the plasma membrane and intracellular membranes. The single yeast CLC protein Gef1p is localized to the Golgi and endosomal system. Investigating epitope-tagged variants of Gef1p, we found that the channel is proteolytically processed in the secretory pathway. Proteolytic cleavage occurs in the first extracellular loop of the protein at residues KR136/137 and is carried out by the Kex2p protease. Fragments mimicking the N- and C-terminal products of the cleavage reaction are non-functional when expressed alone. However, functional channels can assemble when the two fragments are co-expressed.

The GS FLX pyrosequencing platform using parallel tagged sequencing was tested on 10 Somali individuals for sequencing of the complete mitochondrial genome. The amplicons were sequenced twice with increasing coverage to establish the minimum of coverage needed to produce reliable sequence reads. The genome sequences were compared to previously obtained control regions sequences with Sanger sequencing and 49 SNPs in coding regions of the mitochondrial genome. No discrepancy was found with the three methods except in a poly-C stretch that was estimated to be 16193.1C by sequencing with terminator chemistry and no insert was observed with pyrosequencing. The sequence lengths of both pyrosequencing runs were between 225 and 235 nucleotides. The coverage for the first pyrosequencing run was between 0 and 159 and 10–577 for the second one.