Validation of short tandem repeat analysis for the investigation of cases of disputed paternity

This study details validation of two separate multiplex STR systems for use in paternity investigations. These are the Second Generation Multiplex (SGM) developed by the UK Forensic Science Service and the PowerPlex 1 multiplex commercially available from Promega Inc. (Madison, WI, USA). These multiplexes contain 12 different STR systems (two are duplicated in the two systems). Population databases from Caucasian, Asian and Afro-Caribbean populations have been compiled for all loci. In all but two of the 36 STR/ethnic group combinations, no evidence was obtained to indicate inconsistency with Hardy-Weinberg (HW) proportions. Empirical and theoretical approaches have been taken to validate these systems for paternity testing. Samples from 121 cases of disputed paternity were analysed using established Single Locus Probe (SLP) tests currently in use, and also using the two multiplex STR systems. Results of all three test systems were compared and no non-conformities in the conclusions were observed, although four examples of apparent germ line mutations in the STR systems were identified. The data was analysed to give information on expected paternity indices and exclusion rates for these STR systems. The 12 systems combined comprise a highly discriminating test suitable for paternity testing. 99.96% of non-fathers are excluded from paternity on two or more STR systems. Where no exclusion is found, Paternity Index (PI) values of > 10,000 are expected in > 96% of cases.

Short tandem repeat profiling provides an international reference standard for human cell lines

Cross-contamination between cell lines is a longstanding and frequent cause of scientific misrepresentation. Estimates from national testing services indicate that up to 36% of cell lines are of a different origin or species to that claimed. To test a standard method of cell line authentication, 253 human cell lines from banks and research institutes worldwide were analyzed by short tandem repeat profiling. The short tandem repeat profile is a simple numerical code that is reproducible between laboratories, is inexpensive, and can provide an international reference standard for every cell line. If DNA profiling of cell lines is accepted and demanded internationally, scientific misrepresentation because of cross-contamination can be largely eliminated.

Changes in race-specific virulence in pseudomonas syringae pv. phaseolicola are Associated with a chimeric transposable element and rare deletion events in a plasmid-borne pathogenicity island

Virulence for bean and soybean is determined by effector genes in a plasmid-borne pathogenicity island (PAI) in race 7 strain 1449B of Pseudomonas syringae pv. phaseolicola. One of the effector genes, avrPphF, confers either pathogenicity, virulence, or avirulence depending on the plant host and is absent from races 2, 3, 4, 6, and 8 of this pathogen. Analysis of cosmid clones and comparison of DNA sequences showed that the absence of avrPphF from strain 1448A is due to deletion of a continuous 9.5-kb fragment. The remainder of the PAI is well conserved in strains 1448A and 1449B. The left junction of the deleted region consists of a chimeric transposable element generated from the fusion of homologs of IS1492 from Pseudomonas putida and IS1090 from Ralstonia eutropha. The borders of the deletion were conserved in 66 P. syringae pv. phaseolicola strains isolated in different countries and representing the five races lacking avrPphF. However, six strains isolated in Spain had a 10.5-kb deletion that extended 1 kb further from the right junction. The perfect conservation of the 28-nucleotide right repeat of the IS1090 homolog in the two deletion types and in the other 47 insertions of the IS1090 homolog in the 1448A genome strongly suggests that the avrPphF deletions were mediated by the activity of the chimeric mobile element. Our data strongly support a clonal origin for the races of P. syringae pv. phaseolicola lacking avrPphF.

Patterns of nucleotide substitution in angiosperm cpDNA trnL (UAA)-trnF (GAA) regions

Patterns of substitution in chloroplast encoded trnL_F regions were compared between species of Actaea (Ranunculales), Digitalis (Scrophulariales), Drosera (Caryophyllales), Panicoideae (Poales), the small chromosome species clade of Pelargonium (Geraniales), each representing a different order of flowering plants, and Huperzia (Lycopodiales). In total, the study included 265 taxa, each with > 900-bp sequences, totaling 0.24 Mb. Both pairwise and phylogeny-based comparisons were used to assess nucleotide substitution patterns. In all six groups, we found that transition/transversion ratios, as estimated by maximum likelihood on most-parsimonious trees, ranged between 0.8 and 1.0 for ingroups. These values occurred both at low sequence divergences, where substitutional saturation, i.e., multiple substitutions having occurred at the same (homologous) nucleotide position, was not expected, and at higher levels of divergence. This suggests that the angiosperm trnL-F regions evolve in a pattern different from that generally observed for nuclear and animal mtDNA (transitional/transversion ratio > or = 2). Transition/transversion ratios in the intron and the spacer region differed in all alignments compared, yet base compositions between the regions were highly similar in all six groups. A>-C transversions were significantly less frequent than the other four substitution types. This correlates with results from studies on fidelity mechanisms in DNA replication that predict A<->T and G<->C transversions to be least likely to occur. It therefore strengthens confidence in the link between mutation bias at the polymerase level and the actual fixation of substitutions as recorded on evolutionary trees, and concomitantly, in the neutrality of nucleotide substitutions as phylogenetic markers.

Single nucleotide polymorphisms at the ADIPOQ gene locus interact with age and dietary intake of fat to determine serum adiponectin in subjects at risk of the metabolic syndrome

Background: Adiponectin gene expression is modulated by peroxisome proliferator–activated receptor γ, which is a transcription factor activated by unsaturated fatty acids. Objective: We investigated the effect of the interaction between variants at the ADIPOQ gene locus, age, sex, body mass index (BMI), ethnicity, and the replacement of dietary saturated fatty acids (SFAs) with monounsaturated fatty acids (MUFAs) or carbohydrates on serum adiponectin concentrations. Design: The RISCK (Reading, Imperial, Surrey, Cambridge, and Kings) study is a parallel-design, randomized controlled trial. Serum adiponectin concentrations were measured after a 4-wk high-SFA (HS) diet and a 24-wk intervention with reference (HS), high-MUFA (HM), and low-fat (LF) diets. Single nucleotide polymorphisms at the ADIPOQ locus −11391 G/A (rs17300539), −10066 G/A (rs182052), −7734 A/C (rs16861209), and +276 G/T (rs1501299) were genotyped in 448 participants. Results: In white Europeans, +276 T was associated with higher serum adiponectin concentrations (n = 340; P = 0.006) and −10066 A was associated with lower serum adiponectin concentrations (n = 360; P = 0.03), after adjustment for age, BMI, and sex. After the HM diet, −10066 G/G subjects showed a 3.8% increase (95% CI: −0.1%, 7.7%) and G/A+A/A subjects a 2.6% decrease (95% CI: −5.6%, 0.4%) in serum adiponectin (P = 0.006 for difference after adjustment for the change in BMI, age, and sex). In −10066 G/G homozygotes, serum adiponectin increased with age after the HM diet and decreased after the LF diet. Conclusion: In white −10066 G/G homozygotes, an HM diet may help to increase adiponectin concentrations with advancing age. This trial was registered at clinicaltrials.gov as ISRCTN29111298.

Identification and characterisation of five novel miniature inverted-repeat transposable elements (MITEs) in amphioxus (Branchiostoma floridae)

As the sister group to vertebrates, amphioxus is consistently used as a model of genome evolution for understanding the invertebrate/vertebrate transition. The amphioxus genome has not undergone massive duplications like those in the vertebrates or disruptive rearrangements like in the genome of Ciona, a urochordate, making it an ideal evolutionary model. Transposable elements have been linked to many genomic evolutionary changes including increased genome size, modified gene expression, massive gene rearrangements, and possibly intron evolution. Despite their importance in genome evolution, few previous examples of transposable elements have been identified in amphioxus. We report five novel Miniature Inverted-repeat Transposable Elements (MITEs) identified by an analysis of amphioxus DNA sequence, which we have named LanceleTn-1, LanceleTn-2, LanceleTn-3a, LanceleTn-3b and LanceleTn-4. Several of the LanceleTn elements were identified in the amphioxus ParaHox cluster, and we suggest these have had important implications for the evolution of this highly conserved gene cluster. The estimated high copy numbers of these elements implies that MITEs are probably the most abundant type of mobile element in amphioxus, and are thus likely to have been of fundamental importance in shaping the evolution of the amphioxus genome.

PPARγ2 gene Pro12Ala and PPARα gene Leu162Val single nucleotide polymorphisms interact with dietary intake of fat in determination of plasma lipid concentrations

Background/Aims: The peroxisome proliferator-activated receptors (PPARs) are transcriptional regulators of lipid metabolism, activated by unsaturated fatty acids. We investigated independent and interactive effects of PPARγ2 gene PPARG Pro12Ala (rs1801282) andPPARαgene PPARA Leu162Val (rs1800206) genotypes with dietary intake of fatty acids on concentrations of plasma lipids in subjects of whom 47.5% had metabolic syndrome. Methods: The RISCK study is a parallel design, randomised controlled trial. Plasma lipids were quantified at baseline after a 4-week high saturated fatty acids diet and after three parallel 24-week interventions with reference (high saturated fatty acids), high monounsaturated fatty acids and low-fat diets. Single nucleotide polymorphisms were genotyped in 466 subjects. Results: At baseline, the PPARG Ala12allele was associated with increased plasma total cholesterol (n = 378; p = 0.04), LDL cholesterol (p = 0.05) and apoB (p =0.05) after adjustment for age, gender and ethnicity. At baseline, PPARA Leu162Val × PPARG Pro12Ala genotype interaction did not significantly influence plasma lipid concentrations. After dietary intervention, gene-gene interaction significantly influenced LDL cholesterol (p =0.0002) and small dense LDL as a proportion of LDL (p = 0.005) after adjustments. Conclusions: Interaction between PPARG Pro12Ala and PPARA Leu162Valgenotypes may influence plasma LDL cholesterol concentration and the proportion as small dense LDL after a high monounsaturated fatty acids diet.

Analysis of 16S rDNA sequences from pathogenic Leptospira serovars and use of single nucleotide polymorphisms for rapid speciation by D-HPLC

Leptospira have a worldwide distribution and include important zoonotic pathogens yet diagnosis and differentiation still tend to rely on traditional bacteriological and serological approaches. In this study a 1.3 kb fragment of the rrs gene (16S rDNA) was sequenced from a panel of 22 control strains, representing serovars within the pathogenic species Leptospira interrogans, Leptospira borgpetersenii, and Leptospira kirschneri, to identify single nucleotide polymorphisms (SNPs). These were identified in the 5' variable region of the 16S sequence and a 181 bp PCR fragment encompassing this region was used for speciation by Denaturing High Performance Liquid Chromatography (D-HPLC). This method was applied to eleven additional species, representing pathogenic, non-pathogenic and intermediate species and was demonstrated to rapidly differentiate all but 2 of the non-pathogenic Leptospira species. The method was applied successfully to infected tissues from field samples proving its value for diagnosing leptospiral infections found in animals in the UK. Crown Copyright (C) 2010 Published by Elsevier Ltd. All rights reserved.

The Glu298Asp single nucleotide polymorphism in the endothelial nitric oxide synthase gene differentially effects the vascular response to acute consumption of fruit and vegetable puree-based drinks

Scope Diets low in fruits and vegetables (FV) are responsible for 2.7 million deaths from cardiovascular diseases (CVD) and certain cancers annually. Many FV and their juices contain flavonoids, some of which increase endothelial nitric oxide synthase (eNOS) activity. A single nucleotide polymorphism in the eNOS gene, where thymine (T) replaces guanine (G) at position 894 predicting substitution of glutamate for aspartate at codon 298 (Glu298Asp), has been associated with increased CVD risk due to effects on nitric oxide synthesis and subsequently vascular reactivity. Individuals can be homozygous for guanine (GG), thymine (TT) or heterozygous (GT). Methods and results We investigated the effects of acute ingestion of a FV-puree-based-drink (FVPD) on vasodilation and antioxidant status in subjects retrospectively genotyped for this polymorphism. Healthy volunteers (n = 24; 11 GG, 11 GT, 2 TT) aged 30–70 were recruited to a randomized, controlled, crossover, acute study. We showed that acute consumption of 400 mL FVPD differentially affected individuals depending on their genotype. There was a significant genotype interaction for endothelium-dependent vasodilation measured by laser Doppler imaging with iontophoresis (P < 0.05) and ex vivo low-density lipoproteins (LDL) oxidation (P = 0.002). GG subjects had increased endothelium-dependent vasodilation 180 min (P = 0.028) and reduced ex vivo LDL oxidation (P = 0.013) after 60 min after FVPD compared with control, no differences were observed in GT subjects. Conclusion eNOS Glu298Asp genotype differentially affects vasodilation and ex vivo LDL oxidation after consumption of FV in the form of a puree-based drink.

The CtsR regulator of Listeria monocytogenes contains a variant glycine repeat region that affects piezotolerance, stress resistance, motility and virulence

A spontaneous high hydrostatic pressure (HHP)-tolerant mutant of Listeria monocytogenes ScottA, named AK01, was isolated previously. This mutant was immotile and showed increased resistance to heat, acid and H2O2 compared with the wild type (wt) (Karatzas, K.A.G. and Bennik, M.H.J. 2002 Appl Environ Microbiol 68: 3183–3189). In this study, we conclusively linked the increased HHP and stress tolerance of strain AK01 to a single codon deletion in ctsR (class three stress gene repressor) in a region encoding a highly conserved glycine repeat. CtsR negatively regulates the expression of the clp genes, including clpP, clpE and the clpC operon (encompassing ctsR itself), which belong to the class III heat shock genes. Allelic replacement of the ctsR gene in the wt background with the mutant ctsR gene, designated ctsRΔGly, rendered mutants with phenotypes and protein expression profiles identical to those of strain AK01. The expression levels of CtsR, ClpC and ClpP proteins were significantly higher in ctsRΔGly mutants than in the wt strain, indicative of the CtsRΔGly protein being inactive. Further evidence that the CtsRΔGly protein lacks its repressor function came from the finding that the Clp proteins in the mutant were not further induced upon heat shock, and that HHP tolerance of a ctsR deletion strain was as high as that of a ctsRΔGly mutant. The high HHP tolerance possibly results from the increased expression of the clp genes in the absence of (active) CtsR repressor. Importantly, the strains expressing CtsRΔGly show significantly attenuated virulence compared with the wt strain; however, no indication of disregulation of PrfA in the mutant strains was found. Our data highlight an important regulatory role of the glycine-rich region of CtsR in stress resistance and virulence.

Structure of the ROC domain from the Parkinson's disease-associated leucine-rich repeat kinase 2 reveals a dimeric GTPase

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of Parkinson's disease (PD). LRRK2 contains a Ras of complex proteins (ROC) domain that may act as a GTPase to regulate its protein kinase activity. The structure of ROC and the mechanism(s) by which it regulates kinase activity are not known. Here, we report the crystal structure of the LRRK2 ROC domain in complex with GDP-Mg2+ at 2.0-Å resolution. The structure displays a dimeric fold generated by extensive domain-swapping, resulting in a pair of active sites constructed with essential functional groups contributed from both monomers. Two PD-associated pathogenic residues, R1441 and I1371, are located at the interface of two monomers and provide exquisite interactions to stabilize the ROC dimer. The structure demonstrates that loss of stabilizing forces in the ROC dimer is likely related to decreased GTPase activity resulting from mutations at these sites. Our data suggest that the ROC domain may regulate LRRK2 kinase activity as a dimer, possibly via the C-terminal of ROC (COR) domain as a molecular hinge. The structure of the LRRK2 ROC domain also represents a signature from a previously undescribed class of GTPases from complex proteins and results may provide a unique molecular target for therapeutics in PD.

Sequence analysis and distribution in Salmonella enterica serovars of IS3-like elements

The genome of Salmonella enterica serovar Enteritidis was shown to possess three IS3-like insertion elements, designated IS1230A, B and C, and each was cloned and their respective deoxynucleotide sequences determined. Mutations in elements IS1230A and B resulted in frameshifts in the open reading frames that encoded a putative transposase to be inactive. IS1230C was truncated at nucleotide 774 relative to IS1230B and therefore did not possess the 3' terminal inverted repeat. The three IS1230 derivatives were closely related to each other based on nucleotide sequence similarity. IS1230A was located adjacent to the sef operon encoding SEF14 fimbriae located at minute 97 of the genome of S. Enteritidis. IS1230B was located adjacent to the umuDC operon at minute 42.5 on the genome, itself located near to one terminus of an 815-kb genome inversion of S. Enteritidis relative to S. Typhimurium. IS1230C was located next to attB, the bacteriophage P22 attachment site, and proB, encoding gamma-glutamyl phosphate reductase. A truncated 3' remnant of IS1230, designated IS1230T, was identified in a clinical isolate of S. Typhimurium DT193 strain 2391. This element was located next to attB adjacent to which were bacteriophage P22-like sequences. Southern hybridisation of total genomic DNA from eighteen phage types of S. Enteritidis and eighteen definitive types of S. Typhimurium showed similar, if not identical, restriction fragment profiles in the respective serovars when probed with IS1230A.

The impact of obesity-related single nucleotide polymorphisms on appetite and energy intake: a pilot study

An increasing number of studies have reported a heritable component for the regulation of energy intake and eating behaviour, although the individual polymorphisms and their ‘effect size’ are not fully elucidated. The aim of the present study was to examine the relationship between specific SNP and appetite responses and energy intake in overweight men. In a randomised cross-over trial, forty overweight men (age 32 (sd 09) years; BMI 27 (sd 2) kg/m2) attended four sessions 1 week apart and received three isoenergetic and isovolumetric servings of dairy snacks or water (control) in random order. Appetite ratings were determined using visual analogue scales and energy intake at an ad libitum lunch was assessed 90 min after the dairy snacks. Individuals were genotyped for SNP in the fat mass and obesity-associated (FTO), leptin (LEP), leptin receptor (LEPR) genes and a variant near the melanocortin-4 receptor (MC4R) locus. The postprandial fullness rating over the full experiment following intake of the different snacks was 17·2 % (P= 0·026) lower in A carriers compared with TT homozygotes for rs9939609 (FTO, dominant) and 18·6 % (P= 0·020) lower in G carriers compared with AA homozygotes for rs7799039 (LEP, dominant). These observations indicate that FTO and LEP polymorphisms are related to the variation in the feeling of fullness and may play a role in the regulation of food intake. Further studies are required to confirm these initial observations and investigate the ‘penetrance’ of these genotypes in additional population subgroups.