Ricky Ansell

A gene encoding sn‐glycerol 3‐phosphate dehydrogenase (NAD+) complements an osmosensitive mutant of Saccharomyces cerevisiae

Osmoregulatory mutants of Saccharomyces cerevisiae with a defect in their capacity to readjust the cell volume/buoyant density after osmotically induced dehydration were enriched by density gradient centrifugation. Colonies derived from cells that remained dense after dehydration were screened for sensitivity to high concentrations of NaCl and defects in their osmotically induced production and intracellular accumulation of glycerol. The isolated osg (osmosensitive gtycerol defective) mutants were recessive in heterozygous diploids and fell into four complementation groups (osg1‐osg4). The osg1‐1 mutant, described in this work, is unable to grow at low water potential and shows a decreased capacity for glycerol production and a strongly reduced activity of NAD+‐dependent sn‐glycerol 3‐phosphate dehydrogenase (GPD), an enzyme in the glycerol‐producing pathway. Complementation of the osg1‐1 salt sensitivity defect with a low copy yeast genomic library led to the cloning of GPD1, encoding an S. cerevisiae GPD consisting of 391 amino acids and sharing 47‐50% identity with GPD from other sources. Micro‐sequencing of the N‐terminus of purified S. cerevisiae GPD revealed a 20‐amino‐acid sequence that was identical to a nucleotide‐deduced amino acid sequence in GPD1, but indicated that the enzyme is produced with an N‐terminal extension that is removed from the functional enzyme. Subcellular fractionation does not indicate, however, that the putative pre‐sequence targets GPD to any organelle; the enzyme appears to be located in the cytoplasm. Chromoblot and tetrad analysis were used to position the GPD1 gene to chromosome IV, with a distance of about 18 cM from trp1.

Cloning and characterization of GPD2, a second gene encoding sn‐glycerol 3‐phosphate dehydrogenase (NAD+) in Saccharomyces cerevisiae, and its comparison with GPD1

We have cloned and characterized a homologue of the previously isolated GPD1 gene, encoding sn‐glycerol 3‐phosphate dehydrogenase (NAD+) in Saccharomyces cerevisiae. This second gene, called GPD2, encodes a protein of 384 amino acids that shares 69% sequence identity with GPD1. Like GPD1 it has an amino‐terminal extension of unknown function. GPD2 is located on chromosome VII and cross‐hybridizes with GPD1 at chromosome IV as well as with an unknown homologue at chromosome XV. Disruption of the GPD2 gene did not reveal any observable phenotypic effects, whereas overexpression resulted in a slight, but significant, increase of GPD enzyme activity in wild‐type cells. Analysis of gene transcription by a CAT‐reporter gene fused to the GPD promoters revealed decreased transcriptional activity of the GPD2 promoter in cells grown on non‐fermentable as opposed to fermentable carbon sources, and no induction in cells exposed to high osmolarity or heat shock. Similar analysis of GPD1 demonstrated an 8–17‐fold higher basal level of transcription compared to GPD2. Furthermore, such analysis revealed that the GPD1 promoter was it induced by increased osmolarity essentially independent of the type of stress solute used, the level of GPD1 transcription being increased about sevenfold in cells growing at 1.4M NaCl.

NADH-reductive stress in Saccharomyces cerevisiae induces the expression of the minor isoform of glyceraldehyde-3-phosphate dehydrogenase ( TDH1 )

Abstract A strain of Saccharomyces cerevisiae lacking the GPD2 gene, encoding one of the glycerol-3-phosphate dehydrogenases, grows slowly under anaerobic conditions, due to reductive stress caused by the accumulation of cytoplasmic NADH. We used 2D-PAGE to study the effect on global protein expression of reductive stress in the anaerobically grown gpd2Δ strain. The most striking response was a strongly elevated expression of Tdh1p, the minor isoform of glyceraldehyde-3-phosphate dehydrogenase. This increased expression could be reversed by the addition of acetoin, a NADH-specific redox sink, which furthermore largely restored anaerobic growth of the gpd2Δ strain. Additional deletion of the TDH1 gene (but not of TDH2 or TDH3) improved anaerobic growth of the gpd2Δ strain. We therefore propose that TDH1 has properties not displayed by the other TDH isogenes and that its expression is regulated by reductive stress caused by an excess of cytoplasmic NADH.

Improved forensic DNA analysis through the use of alternative DNA polymerases and statistical modeling of DNA profiles

DNA evidence, linking perpetrators to crime scenes, is central to many legal proceedings. However, DNA samples from crime scenes often contain PCR-inhibitory substances, which may generate blank or incomplete DNA profiles. Extensive DNA purification can be required to rid the sample of these inhibitors, although these procedures increase the risk of DNA loss. Most forensic laboratories use commercial DNA amplification kits (e.g., AmpFlSTR SGM Plus) with the DNA polymerase AmpliTaq Gold as the gold standard. Here, we show that alternative DNA polymerase-buffer systems can improve the quality of forensic DNA analysis and efficiently circumvent PCR inhibition in crime scene samples, without additional sample preparation. DNA profiles from 20 of 32 totally or partially inhibited crime scene saliva samples were significantly improved using Bio-X-Act Short, ExTaq Hot Start, or PicoMaxx High Fidelity instead of AmpliTaq Gold. A statistical model for unbiased quality control of forensic DNA profiles was developed to quantify the results. Our study demonstrates the importance of adjusting the chemistry of the PCR to enhance forensic DNA analysis and diagnostic PCR, providing an alternative to laborious sample preparation protocols.

European Network of Forensic Science Institutes (ENFSI): Evaluation of new commercial STR multiplexes that include the European Standard Set (ESS) of markers

To support and to underpin the European initiative to increase the European set of standard markers (ESS), by the addition of five new loci, a collaborative project was organised by the European Network of Forensic Science Institutes (ENFSI) DNA working group in order to assess the new multiplex kits available. We have prepared allele frequency databases from 26 EU populations. Concordance studies were carried out to verify that genotyping results were consistent between kits. Population genetics studies were conducted and it was estimated that F(ST)<0.001. The results showed that the kits were comparable to each other in terms of performance and major discrepancy issues were highlighted. We provide details of allele frequencies for each of the populations analysed per laboratory.

A Swedish Perspective

The Nuffield Report is well-written, clear, extensive and up to date, and it covers most of the major ethical issues in the field of forensic DNA analysis and database searching. The ethical analysis is thorough and based on solid theoretical ground.

Evaluation of amylase testing as a tool for saliva screening of crime scene trace swabs.

Amylase testing has been used as a presumptive test for crime scene saliva for over three decades, mainly to locate saliva stains on surfaces. We have developed a saliva screening application for crime scene trace swabs, utilising an amylase sensitive paper (Phadebas(®) Forensic Press test). Positive results were obtained for all tested dried saliva stains (0.5-32 μL) with high or intermediate amylase activity (840 and 290 kU/L). Results were typically obtained within 5 min, and all samples that produced DNA profiles were positive. However, salivary amylase activities, as well as DNA concentrations, vary significantly between individuals. We show that there is no correlation between amylase activity and amount of DNA in fresh saliva. Even so, a positive amylase result indicates presence of saliva, and thereby presence of DNA. Amylase testing may be useful for screening in investigations where the number of DNA analyses is limited due to cost, e.g., in volume crime.

A fast analysis system for forensic DNA reference samples

On January 1st, 2006, the Swedish legislation on obtaining DNA reference samples from suspects and the recording of DNA profiles in databases was changed. As a result the number of samples analysed at the Swedish National Laboratory of Forensic Science (SKL) increased from about 4500 in 2005 to more than 25,000 in 2006. To meet this challenge, SKL launched a new analysis system to create an unbroken chain, from sampling to incorporation of a profile in the national DNA database and subsequent automatic generation of digitally signed hit reports. The system integrates logistics, digital data transfer, new functions in LIMS (ForumDNA Version 4, Ida Infront AB) and laboratory automation. Buccal swab samples are secured on a FTA card attached to an identity form, which is barcoded with a unique sample ID. After sampling, the police officer sends a digital request to SKL. The sample is automatically registered in LIMS and processed on delivery. The resulting DNA profiles are automatically classified according to quality using a custom-made expert system. Building the evaluation around mathematical rules makes it reproducible, standardised and minimises manual work and clerk errors. All samples are run in duplicate and the two profiles are compared within LIMS before incorporation in the database. In the first year of operation, the median time for completion of an analysis was 3 days, measured from delivery of the sample to incorporation of the profile in the national DNA database. In spite of the dramatic increase in the number of reference samples there was no backlog.

Swedish population data and concordance for the kits PowerPlex® ESX 16 System, PowerPlex® ESI 16 System, AmpFlSTR® NGM™, AmpFlSTR® SGM Plus™ and Investigator ESSplex.

Abstract The European Standard Set of loci (ESS) has been extended with five additional short tandem repeat (STR) loci following the recommendations of the European Network of Forensic Science Institutes (ENFSI) and the European DNA Profiling Group (EDNAP) to increase the number of loci routinely used by the European forensic community. Subsequently, a new extended Swedish population database, based on 425 individuals, has been assembled using the new STR multiplex kits commercially available. Allele frequencies and statistical parameters of forensic interest for 15 autosomal STR loci (D3S1358, TH01, D21S11, D18S51, D10S1248, D1S1656, D2S1338, D16S539, D22S1045, vWA, D8S1179, FGA, D2S441, D12S391 and D19S433) were obtained from the analysis of the PowerPlex ® ESX 16 System kit (Promega Corporation, USA). According to the data no evidence of deviations from Hardy–Weinberg equilibrium was found. The observed heterozygosity varies between 0.755 (TH01) and 0.892 (D1S1656). The power of discrimination was smallest for D22S1045 (0.869) and largest for D1S1656 (0.982) while the power of exclusion was smallest for TH01 (0.518) and largest for D1S1656 (0.778). A concordance study was performed on the five amplification systems: PowerPlex ® ESX 16 System, PowerPlex ® ESI 16 System (Promega Corporation, USA), AmpF l STR ® NGM™, AmpF l STR ® SGM Plus™ (Applied Biosystems, USA) and Investigator ESSplex (Qiagen, Germany) to reveal null alleles and other divergences between the kits. For the 425 DNA profiles included, AmpF l STR ® NGM™ revealed two null alleles, AmpF l STR ® SGM Plus™ revealed one, and Investigator ESSplex revealed a micro-variant, while the rest of the alleles showed full concordance between the kits tested.

Collaborative EDNAP exercise on the IrisPlex system for DNA-based prediction of human eye colour

The IrisPlex system is a DNA-based test system for the prediction of human eye colour from biological samples and consists of a single forensically validated multiplex genotyping assay together with a statistical prediction model that is based on genotypes and phenotypes from thousands of individuals. IrisPlex predicts blue and brown human eye colour with, on average, >94% precision accuracy using six of the currently most eye colour informative single nucleotide polymorphisms (HERC2 rs12913832, OCA2 rs1800407, SLC24A4 rs12896399, SLC45A2 (MATP) rs16891982, TYR rs1393350, and IRF4 rs12203592) according to a previous study, while the accuracy in predicting non-blue and non-brown eye colours is considerably lower. In an effort to vigorously assess the IrisPlex system at the international level, testing was performed by 21 laboratories in the context of a collaborative exercise divided into three tasks and organised by the European DNA Profiling (EDNAP) Group of the International Society of Forensic Genetics (ISFG). Task 1 involved the assessment of 10 blood and saliva samples provided on FTA cards by the organising laboratory together with eye colour phenotypes; 99.4% of the genotypes were correctly reported and 99% of the eye colour phenotypes were correctly predicted. Task 2 involved the assessment of 5 DNA samples extracted by the host laboratory from simulated casework samples, artificially degraded, and provided to the participants in varying DNA concentrations. For this task, 98.7% of the genotypes were correctly determined and 96.2% of eye colour phenotypes were correctly inferred. For Tasks 1 and 2 together, 99.2% (1875) of the 1890 genotypes were correctly generated and of the 15 (0.8%) incorrect genotype calls, only 2 (0.1%) resulted in incorrect eye colour phenotypes. The voluntary Task 3 involved participants choosing their own test subjects for IrisPlex genotyping and eye colour phenotype inference, while eye photographs were provided to the organising laboratory and judged; 96% of the eye colour phenotypes were inferred correctly across 100 samples and 19 laboratories. The high success rates in genotyping and eye colour phenotyping clearly demonstrate the reproducibility and the robustness of the IrisPlex assay as well as the accuracy of the IrisPlex model to predict blue and brown eye colour from DNA. Additionally, this study demonstrates the ease with which the IrisPlex system is implementable and applicable across forensic laboratories around the world with varying pre-existing experiences.