Search

The marine environments surrounding the Dongsha Islands have long been considered as important fishing grounds for fishermen from China and surrounding countries. Although considerable work has been done on marine fish taxonomy of the Dongsha Islands, there is still a significant gap between our knowledge and the true fish diversity in this ocean area. Here, by analyzing the mitochondrial COI gene, we explore the fish diversity across the Dongsha Islands using a molecular taxonomic approach. In total, we identified 46 morphological species in our samples based on current taxonomical keys, yet 153 DNA barcodes using the 676 base-pair fragments of the COI gene suggested 51 putative species (belonging to 43 genera, 28 families, and 11 orders) by two DNA taxonomic approaches. The average sequence variation (K2P distance) within species was 0.33% (range 0–0.81%), while the average genetic distance between congeners was 3.43% (range 0.74–8.39%). Our results suggest that COI-based barcoding can be effectively used at a species level resolution for the marine fishes in this ocean area and that it allows a realistic idea of the fish diversity.

This study aimed to establish a multiplex PCR detection system mediated by "universal primers," which would be able to determine whether mutton meat contained nonmutton ingredients from rats, foxes, and ducks. Based on the sequence variation of specific mitochondrial genes, nine different multiplex PCR primers were designed, and four kinds of meat products were rapidly identified by electrophoresis using an optimized multiplex PCR system based on the molecular weight differences of the amplified products. Multiplex PCR applications optimized for meat food source from food samples for testing was used to verify the accuracy of the identification method. The results showed that the primers in multiple PCR system mediated by universal primers could be used for the rapid identification of rat, fox, duck, and sheep meat in mutton products, and the detection sensitivity could reach 0.05 ng/μL. The identification of food samples validated the practical value of this method. Therefore, a multiplex PCR system mediated by universal primers was established, which can be used to quickly identify the origin of animal ingredients from rats, foxes, and ducks in mutton products.

Peptide self-assembly is an effective way to construct well-ordered nanostructures for biomedical applications. Given that short peptide length always facilitates large-scale production for future applications and the easy establishment of structure-property relationship, this review highlights recent advances in the self-assembly of short amphiphilic peptides, focusing on the strategies to manipulate the involved non-covalent interactions as well as the consequent supramolecular architectures. One representative strategy is based on molecular design, in which amino acid substitution, sequence variation, terminal capping, molecular geometry, and amino acid chirality are considered. Another important strategy that varies solution conditions (pH, solvents, enzymes, and metal ions) is also discussed. A variety of self-assembled pep1tide nanostructures and nanomaterials have been achieved therefrom and more importantly, the advances make the prediction of peptide self-assembly increasingly feasible based on a certain class of peptide sequences and solution conditions. Additionally, their applications as antibiotics and templates for nanofabrication, mainly from the work of our group, demonstrate the importance of the surface properties and morphologies of self-assemblies to practical applications. Following previous studies, future endeavor in this field is suggested to focus on designing more peptide sequences with functional motifs not only to summarize general rules to predict self-assembly but also to realize practical applications.

DNA profiling the male specific region on the Y-chromosome is fundamental to forensic practise. It is recognised as a powerful tool for sexual assault investigations for resolving the male DNA components, for familial search and as a tool to aid biogeographic inferences. In this study the novel Y-chromosome Short Tandem Repeat (Y-STR) multiplex called the UniQ-Typer ™ Y-10 was utilised for population wide genotyping in South Africa. In this study we provide a significant updated to the genotyping allelic ladder for the panel and report novel variation which gave strong signals to potentially infer biogeographic ancestry.

The common cutworm (CCW, Spodopteraab litura Fabricius) is one of the pests that most severely infect soybean (Glycine max L. Merr.). In a previous report, quantitative trait loci (QTL) analysis of CCW resistance using a recombinant inbred line derived from a cross between a susceptible cultivar ‘Fukuyutaka’ and a resistant cultivar ‘Himeshirazu’, identified two antixenosis resistance QTLs, CCW-1 and CCW-2. To reveal sequence variation between the aforementioned two cultivars, whole genome resequencing was performed using Illumina HiSeq2000 (75,632,747 and 91,540,849 reads). The generated datasets can be used for fine mapping and gene isolation of CCW-1 and CCW-2 as well as for revealing more detailed genetic differences between ‘Fukuyutaka’ and ’Himeshirazu’ .

Hairs from the same donor have been found to differ in mtDNA sequence within and among themselves and from other tissues, which impacts interpretation of results obtained in a forensic setting. However, little is known on the magnitude of this phenomenon and published data on systematic studies are scarce. We addressed this issue by generating mtDNA control region (CR) profiles of >450 hair fragments from 21 donors by Sanger-type sequencing (STS). To mirror forensic scenarios, we compared hair haplotypes from the same donors to each other, to the corresponding buccal swab reference haplotypes and analyzed several fragments of individual hairs. We also investigated the effects of hair color, donor sex and age, mtDNA haplogroup and chemical treatment on mtDNA quantity, amplification success and variation. We observed a wide range of individual CR sequence variation. The reference haplotype was the only or most common (≥75%) hair haplotype for most donors. However, in two individuals, the reference haplotype was only found in about a third of the investigated hairs, mainly due to differences at highly variable positions. Similarly, most hairs revealed the reference haplotype along their entire length, however, about a fifth of the hairs contained up to 71% of segments with deviant haplotypes, independent of the longitudinal position. Variation affected numerous positions, typically restricted to the individual hair and in most cases heteroplasmic, but also fixed (i.e. homoplasmic) substitutions were observed. While existing forensic mtDNA interpretation guidelines were found still sufficient for all comparisons to reference haplotypes, some comparisons between hairs from the same donor could yield false exclusions when those guidelines are strictly followed. This study pinpoints the special care required when interpreting mtDNA results from hair in forensic casework.

We analyzed mitochondrial DNA (mtDNA) from ancient marmot teeth (∼7550–6800 cal. BP), which were recovered during archaeological excavations of two contemporary cemeteries near Lake Baikal, Russia: one archaeological site is the Shamanka II cemetery located on the southwest shoreline of Lake Baikal, and the other is the Lokomotiv-Raisovet cemetery located about 77 km to northwest and within the modern city of Irkutsk. Although the teeth had not been identified to species based on their morphology, our ancient DNA analysis revealed that all incisors from ten individuals were of the tarbagan marmot ( Marmota sibirica ), which is currently not distributed around those archaeological sites. In contrast, the black-capped marmot ( Marmota camtschatica ), which also has a dominant distribution in Eastern Siberia and whose incisors are morphologically similar to M . sibirica , was not identified from our ancient tooth samples. In addition, the mtDNA sequence variation showed that the genealogy of marmots in the Shamanka II cemetery could have been different from that of the Lokomotiv-Raisovet cemetery. These data indicate that the ancient people at the Shamanka II cemetery could have used M . sibirica from different regions than those utilized at the Lokomotiv-Raisovet site. This suggests non-overlapping marmot hunting ranges for the people buried at the two Middle Holocene cemeteries.

In short tandem repeat (STR) analysis, length polymorphisms are detected by capillary electrophoresis (CE). At most STR loci, mobility shift due to sequence variation in the repeat region was thought not to affect the typing results. In our recent population studies of 1501 Japanese individuals, off-ladder calls were observed at the D12S391 locus using PowerPlex Fusion in nine samples for allele 22, one sample for allele 25, and one sample for allele 26. However, these samples were typed as ordinary alleles within the bins using GlobalFiler. In this study, next-generation sequencing analysis using MiSeq was performed for the D12S391 locus from the 11 off-ladder samples and 33 other samples, as well as the allelic ladders of PowerPlex Fusion and GlobalFiler. All off-ladder allele 22 in the nine samples had [AGAT]11[AGAC]11 as a repeat structure, while the corresponding allele was [AGAT]15[AGAC]6[AGAT] for the PowerPlex Fusion ladder, and [AGAT]13[AGAC]9 for the GlobalFiler ladder. Overall, as the number of [AGAT] in the repeat structure decreased at the D12S391 locus, the peak migrated more slowly using PowerPlex Fusion, the reverse strand of which was labeled, and it migrated more rapidly using GlobalFiler, the forward strand of which was labeled. The allelic ladders of both STR kits were reamplified with our small amplicon D12S391 primers and their mobility was also examined. In conclusion, off-ladder observations of allele 22 at the D12S391 locus using PowerPlex Fusion were mainly attributed to a relatively large difference of the repeat structure between its allelic ladder and off-ladder allele 22.

There is little information on sequence variation of canine adenovirus type 1 (CAdV-1), the aetiological agent of infectious canine hepatitis (ICH). This study reports hexon and fibre gene sequence variants of CAdV-1 in a dog with systemic ICH and a dog with the ocular form of the disease (‘blue eye’) in Northern Italy in 2013. One of the sequence variants matched a CAdV-1 fox sequence previously detected in Italy.

The diversity of naturally occurring amino acids endows even a very short peptide with an incredible range of sequences, nanostructures and properties. Short peptide sequences have been widely exploited in molecular self-assembly, and this area of research has not only led to a wide range of ordered structures and nanomaterials but also provided insights into protein folding. Conversely, the general physical principles of protein folding accumulated over the past 55 years can be applied to the design of peptide self-assembly. De novo designed surfactant-like peptides (SLPs) are structurally akin to conventional surfactants, with several consecutive hydrophobic amino acids composing their hydrophobic tail region and one or two charged residues making up the hydrophilic head group. Due to their short length, well-defined hydrophobic and hydrophilic regions, innate molecular amphiphilicity, and excellent water solubility, their self-assembly and applications can be orchestrated through molecular design and varying solution conditions. With the canonical 20 amino acids as a starting point, this review introduces the physical principles dictating peptide self-assembly. Then, the major design rules for SLPs, including amino acid substitution, sequence variation, peptide length, amino acid chirality, and incorporation of specific amino acids or sequences, are described, followed by recent advances in their applications for cell culture, antibacterial and anticancer treatments, drug delivery and controlled release, biomimetic mineralization and nanofabrication, hemostasis, and membrane protein stabilization. Finally, a brief outlook to future challenges and opportunities is provided. We hope this review provides a panoramic sketch of SLPs and will inspire more effort into their fundamental research and exploitation.

The sequencing of STR markers provides additional information present in the underlying sequence variation that is typically masked by traditional fragment-based genotyping. However, the interpretation of STR profiles generated by targeted sequencing methods are susceptible to the same factors encountered in profiles processed through capillary gel electrophoresis. These factors include stochastic variation, noise, stutter artifacts, heterozygote imbalance, and allelic drop-out/in. Our goal is to characterize and understand how these behave in targeted sequence datasets. Here, we developed a framework using statistical tools to systematically interpret the characteristics of single-source DNA profiles generated by targeted sequencing. Sensitivity studies were performed using known single-source samples amplified with the PowerSeq 46GY System Prototype with varying DNA target masses ranging from 15 pg to 500 pg. The STR loci were subjected to DNA library preparation using two commercially available library kits and sequenced on the Illumina MiSeq platform. Raw FASTQ data files were analyzed in STRait Razor v2.0 without applying any thresholds (at a coverage ≥ 1). We investigated the effect of library normalization on average locus coverage and studied methods for setting analytical and zygosity thresholds. All the data were analyzed per DNA quantity as well as investigated per method. Analyses presented can be applied to sequence data generated by similar targeted sequencing panels and/or NGS platforms.

Mitochondrial DNA (mtDNA) sequence variation and maternal inheritance are valuable tools in assessing ancestry of different human populations and for clinical practice. A new study (Wei et al., 2019) reports that the fate of new mtDNA variants in the female germline is non-random as they report functional selection and matching to nuclear ancestry to shape human mtDNA variation.

Enhancer biology is emerging as a critical area of research that informs studies of evolution, development, and disease. From early experiments that defined and mapped the first enhancers to recent enhancer models of human disease, functional experiments in the mouse have played a central role in revealing enhancer biology. Three decades of in vivo enhancer studies in mouse have laid the groundwork for the current understanding of mammalian enhancers, demonstrating the developmental and tissue-specific activity of enhancers and illuminating general features of enhancer evolution and function. Recent studies offer an emerging perspective on the importance of chromosomal context, combinatorial enhancer activity, and the functional consequences of enhancer sequence variation. This review describes the basic principles of functional testing in mouse, summarizes the contributions these studies have made to our understanding of enhancer biology, and describes limitations and future outlook of in vivo mouse enhancer studies.

Schmid-type metaphyseal chondrodysplasia (MCDS) is characterized by short stature with short legs, bowing of the long bones, coxa vara, and waddling gait. MCDS is a relatively common form of MCD. Most mutations that cause MCDS occur within the carboxyl-terminal non-collagenous domain (NC1) of the COL10A1 gene. We performed mutational analysis of the COL10A1 genes in 4 unrelated Korean patients with diagnosed MCDS. Mutational analysis of COL10A1 identified c.1904_1915delinsT (p.Gln635LeufsX10) and c.1969dupG (p.Ala657GlyfsX10), 2 novel frameshift mutations, and c.2030T>A (p.Val677Glu) and c.862G>C (p.Gly288Arg) at unusual mutational sites, which could be pathogenic. We present the first report of the molecular characteristics of MCDS in 4 Korean patients. Our findings suggest that a novel sequence variation involving an unusual mutational site of the COL10A1 gene can cause mild MCDS.

In the last few years the cost of massively parallel sequencing has reduced dramatically to the point that it can now be practically considered as a tool in forensic casework. An important consideration for the implementation of any new approach is the ability to remain compatible with previous technology. With this is mind we conducted two sets of experiments to evaluate sequencing the previously amplified products of two commercial forensic STR multiplexes. Samples amplified with AmpFlSTR ® Identifiler ® and PowerPlex ® Y were sequenced on the Illumina © MiSeq and Ion PGM™ Sequencer (Life Technologies). We found it is possible to sequence such amplified DNA and to accurately determine the STR genotype of forensic samples using both platforms. Sequencing these STR loci provided extra information in the form of sequence variation, something that is not possible measuring amplicon length alone. And from these results we begin to characterise the sequence data from a forensic perspective, by looking at sequence variation within the repeats, stutter and heterozygote imbalance.

Background Current HIV-1 sequencing-based methods for detecting drug resistance-associated mutations are open and susceptible to contamination. Informatic identification of clinical sequences that are nearly identical to one another may indicate specimen-to-specimen contamination or another laboratory-associated issue. Objectives To design an informatic tool to rapidly identify potential contamination in the clinical laboratory using sequence analysis and to establish reference ranges for sequence variation in the HIV-1 protease and reverse transcriptase regions among a U.S. patient population. Study design We developed an open-source tool named HIV Contamination Detection (HIVCD). HIVCD was utilized to make pairwise comparisons of nearly 8000 partial HIV-1 pol gene sequences from patients across the United States and to calculate percent identities (PIDs) for each pair. ROC analysis and standard deviations of PID data were used to determine reference ranges for between-patient and within-patient comparisons and to guide selection of a threshold for identifying abnormally high PID between two unrelated sequences. Results The PID reference range for between-patient comparisons ranged from 83.8 to 95.7% while within-patient comparisons ranged from 96 to 100%. Interestingly, 48% of between-patient sequence pairs with a PID > 96.5 were geographically related. The selected threshold for abnormally high PIDs was 96 (AUC = 0.993, sensitivity = 0.980, specificity = 0.999). During routine use, HIVCD identified a specimen mix-up and the source of contamination of a negative control. Conclusions In our experience, HIVCD is easily incorporated into laboratory workflow, useful for identifying potential laboratory errors, and contributes to quality testing. This type of analysis should be incorporated into routine laboratory practice.

Understanding the structural and dynamic information encoded in the primary sequence of a protein is one of the most fundamental challenges in modern biology. Most studies of protein design and ligand binding are extremely native-state centric, ignoring the role of a protein's energy landscape. The amino acid sequence of a protein, however, encodes more than the native three-dimensional structure; it encodes the entire energy landscape - an ensemble of conformations whose energetics and dynamics are finely tuned for folding, binding and activity. I will present our results demonstrating that for proteins without a well-folded native state, small sequence variation can have dramatic effects on ligand binding and specificity. I will also present single-molecule studies demonstrating that small changes in the sequence and environment can alter the folding trajectory of a protein.

The mechanical failure of mature amyloid fibers produces fragments that act as seeds for the growth of new fibrils. Fragmentation may also be correlated with cytotoxicity. We have used steered atomistic molecular dynamics simulations to study the mechanical failure of fibrils formed by the amyloidogenic fragment of human amylin hIAPP20-29 subjected to force applied in a variety of directions. By introducing systematic variations to this peptide sequence in silico, we have also investigated the role of the amino-acid sequence in determining the mechanical stability of amyloid fibrils. Our calculations show that the force required to induce mechanical failure depends on the direction of the applied stress and upon the degree of structural order present in the β -sheet assemblies, which in turn depends on the peptide sequence. The results have implications for the importance of sequence-dependent mechanical properties on seeding the growth of new fibrils and the role of breakage events in cytotoxicity.

The next decade will focus on identifying the missing heritability of coronary artery disease (CAD). This process will involve a more comprehensive interrogation of common single nucleotide polymorphisms (SNPs) that impart modest biologic effect and an interrogation of rare SNPs that impart profound biologic effect. In parallel, an investigation of the underlying biology of the described association will likely yield novel pathways that provide therapeutic targets. Once we obtain a more complete inventory of sequence variation that predisposes to CAD, a more realistic assessment of the role of genetic risk scoring allied with standard risk algorithms will be possible.

Near-complete collagen (I) sequences are proposed for elephantid and mammutid taxa, based upon available African elephant genomic data and supported with LC–MALDI-MS/MS and LC–ESI-MS/MS analyses of collagen digests from proboscidean bone. Collagen sequence coverage was investigated from several specimens of two extinct mammoths (Mammuthus trogontherii and Mammuthus primigenius), the extinct American mastodon (Mammut americanum), the extinct straight-tusked elephant (Elephas (Palaeoloxodon) antiquus) and extant Asian (Elephas maximus) and African (Loxodonta africana) elephants and compared between the two ionization techniques used. Two suspected mammoth fossils from the British Middle Pleistocene (Cromerian) deposits of the West Runton Forest Bed were analysed to investigate the potential use of peptide mass spectrometry for fossil identification. Despite the age of the fossils, sufficient peptides were obtained to identify these as elephantid, and sufficient sequence variation to discriminate elephantid and mammutid collagen (I). In-depth LC–MS analyses further failed to identify a peptide that could be used to reliably distinguish between the three genera of elephantids (Elephas, Loxodonta and Mammuthus), an observation consistent with predicted amino acid substitution rates between these species.

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) often produce Panton–Valentine leukocin (PVL), which is encoded by two co-transcribed genes located on lysogenized bacteriophages. Six PVL-encoding temperate phages have been described and single nucleotide polymorphisms (SNPs) in the PVL genes have been reported. In the present study, 22 PVL-positive CA-MRSA isolates were chosen to reflect the diversity of multilocus sequence type (MLST) clonal complexes (CC) identified in our hospital. Isolates were characterized by antimicrobial resistance profile, staphylococcal cassette chromosome mec (SCCmec) and spa type, pulsed-field gel electrophoresis profile and MLST. Primers were designed to sequence the lukSF-PV genes. PVL-encoding phages were characterized using a PCR-based assay. SNPs were identified at seven locations in the lukSF-PV genes, which varied with S. aureus MLST lineage. One SNP was nonsynonymous. All CC80 and some CC1 isolates carried FSa2mw; CC8, CC88 and CC154 isolates harboured PVL-encoding elongated head-type phages; and some CC59 isolates harboured a FSa2958-like phage. Novel or variant phages were present in CC5 and some CC1 and CC59 isolates. The PVL gene sequence and the PVL-encoding phage varied with lineage. Further work is required to determine whether PVL sequence and/or phage variations result in biological differences.

We analyzed mitochondrial DNA sequence variation across 69 Amethyst-throated Hummingbirds (Lampornis amethystinus), comparing with samples of related taxa. Although this group shows discrete phenotypic variation in throat color among populations in Oaxaca and Guerrero (Mexico), the only phylogeographic structure observed was between phenotypically similar populations north and south of the Isthmus of Tehuantepec. As such, it appears that throat color variation is of recent origin and likely based only on minor genetic differences.

In order to understand the genomic changes during the evolution of hexaploid wheat, two sets of synthetic hexaploid wheat from hybridization between maternal tetraploid wheat (AABB) and paternal diploid goat grass (DD) were used for DNA-AFLP and single strand conformation polymorphism (SSCP) analysis to determine the genomic and genic variation in the synthetic hexaploid wheat. Results indicated that more DNA sequences from paternal diploid species were eliminated in the synthetic hexaploid wheat than from maternal tetraploid wheat, suggesting that genome from parental species of lower ploidity tends to be eliminated preferentially. However, sequence variation detected by SSCP procedure was much lower than those detected by DNA-AFLP, which indicated that much less variation in the genic regions occurred in the synthetic hexaploid wheat, and sequence variations detected by DNA-AFLP could be derived mostly from non-coding regions and repetitive sequences. Our results also indicated that sequence variation in 4 genes can be detected in hybrid F 1 , which suggested that this type of sequence variation could be resulted from distant hybridization. It was interesting to note that 3 out of the 4 genes were mapped and clustered on the long arm of chromosome 2D, which indicated that variation in genic sequences in synthetic hexaploid wheat might not be a randomized process.

ACMG previously developed recommendations for standards for interpretation of sequence variations. We now present the updated revised recommendations. Here, we describe six interpretative categories of sequence variations: (1) sequence variation is previously reported and is a recognized cause of the disorder; (2) sequence variation is previously unreported and is of the type which is expected to cause the disorder; (3) sequence variation is previously unreported and is of the type which may or may not be causative of the disorder; (4) sequence variation is previously unreported and is probably not causative of disease; (5) sequence variation is previously reported and is a recognized neutral variant; and (6) sequence variation is previously not known or expected to be causative of disease, but is found to be associated with a clinical presentation. We emphasize the importance of appropriate reporting of sequence variations using standardized terminology and established databases, and of clearly reporting the limitations of sequence-based testing. We discuss follow-up studies that may be used to ascertain the clinical significance of sequence variations, including the use of additional tools (such as predictive software programs) that may be useful in variant classification. As more information becomes available allowing the interpretation of a new sequence variant, it is recommended that the laboratory amend previous reports and provide updated results to the physician. The ACMG strongly recommends that the clinical and technical validation of sequence variation detection be performed in a CLIA-approved laboratory and interpreted by a board-certified clinical molecular geneticist or equivalent.

Levels of oxygen can vary dramatically in aquatic environments. Aquatic organisms, including fishes, have adapted accordingly to survive. As there are both phylogenetically closely related fish species with differing oxygen requirements and distantly related species with similar oxygen requirements, fishes are good candidates for examining oxygen-related functions in vertebrates. We set out to investigate if sequence variation in the hypoxia-inducible factor-1 alpha (HIF-1alpha) gene is associated with variations in oxygen requirements. Since the teleost HIF-1alpha sequences available in databases represent a very limited dataset both phylogenetically and with regard to oxygen requirements, we have sequenced the protein coding sequence for HIF-1alpha from an additional 9 fish species. Our results indicate that the deduced HIF-1alpha proteins of teleost fishes are somewhat shorter than those of tetrapods. Additionally, the results suggest that tetrapod sequences more closely resemble the ancestral form of the protein than do teleost sequences. No clear signatures which could be associated with the oxygen requirements of the species were found. This study suggests that if species-specific differences in HIF-1alpha function with regards to oxygen dependence have evolved, they do not occur in the protein coding sequence but at other levels of the HIF-1alpha pathway.

Nucleotide sequences of a broad range of Peach Latent Mosaic Viroid (PLMVd) variants were determined. The variants were isolated from peach, pear, and almond tree samples collected in Tunisia. Sequence analysis confirmed the high variability of PLMVd, as no less than 119 new variants were identified. Variations included new polymorphic positions, insertions of 11 to 14 nucleotides, and new mutations within the hammerhead self-cleavage motifs. We provide the first covariation-based evidence for certain stems within the proposed secondary structure. Our covariation analysis also strengthens the view that a pseudoknot closes the replication domain. On the basis of phylogenetic tree studies and informative positions, PLMVd variants are proposed to cluster into groups and subgroups likely to have resulted from recombination events. PLMVd thus emerges as a suitable viroid for retracing the evolution of an RNA genome.

Parapoxvirus of red deer in New Zealand (PVNZ), a species of the Parapoxvirus genus, causes scabby lesions on the skin and the velvet of red deer. The three other species of the genus have each been shown to encode homologs of vascular endothelial growth factor (VEGF). We report here that PVNZ strain RD86 also encodes a VEGF and that the predicted PVNZ protein shows only 37–54% amino acid identity to VEGFs encoded by the other species of the genus. Despite this extensive sequence divergence, assays of purified PVNZ VEGF (PVNZ RD86 VEGF) demonstrated that it shares the unique VEGF receptor (VEGFR) binding profile of the other parapoxvirus VEGFs, in that it binds VEGFR-2 and induces VEGFR-2-mediated proliferation of Ba/F3-derived cells, but does not bind VEGFR-1 or VEGFR-3. In contrast to some other viral VEGFs, it does not bind neuropilin-1. Our results indicate that PVNZ RD86 VEGF is a biologically active member of the VEGF family and is likely to contribute to the proliferative and highly vascularized nature of PVNZ lesions. Our data also reveal that all members of the genus encode a VEGF and that an extraordinary degree of inter-species sequence variation is a general feature of the parapoxvirus VEGFs.

While mutations in genes that function in the core molecular clock may disrupt circadian periodicity , their relevance to diurnal variation in metabolic, cardiovascular, and respiratory function is unknown. The circadian Clock gene product is an essential regulator of central and peripheral circadian rhythms in mammals. We have elucidated the complete exon–intron organization of the Clock gene in rat and have carried out an extensive search for single nucleotide polymorphisms (SNPs) in a panel of 12 inbred rat strains that exhibit diversity in studies of central and peripheral organ function and disease. The rat Clock gene consists of 23 exons spanning approximately 75 kb. Comparative sequence analysis identified 33 novel SNPs, including 32 that distinguish the Brown Norway (BN) rat from the other strains studied. Most notable were two novel mutations in the BN sequence at exon 8, Ile131Val and Ile132Val, occurring in a segment of the highly conserved PAS-A domain of the protein. These results afford the opportunity to assess the impact of genetic variation in Clock on central and peripheral functions subject to the core molecular clock and to test the importance of Clock variants in explaining diversity among rat strains in the expression of phenotypes, such as blood pressure, subject to circadian oscillation.

Though meningococcal conjugate vaccines are effective against serogroup C, there is currently no vaccine solution for serogroup B disease. PorA outer membrane protein (OMP) is a potential serogroup B vaccine candidate. A hexavalent PorA outer membrane vesicle (OMV) vaccine has been evaluated in phase I and II trials with promising results. However, considerable sequence variation occurs in the variable regions (VRs) encoding these serosubtypes. By using five wild type P1.19,15 variant strains we examined the serum bactericidal antibody (SBA) titres from sera collected from toddlers and school children pre- and post-vaccination. The numbers of subjects with SBA titres of4, 4 andor = 8 varied greatly between the different strains. This was also reflected whenor = 4-fold rises in SBA titres were examined. This finding in sera from toddlers and school children may have implications for PorA based vaccines.

The aim of the present work was to determine the identity and molecular relationships between 127 strains of dark septate (DS) fungi isolated from healthy root tips, decayed coarse roots, live healthy-looking stems, coarse (stumps, snags and logs) and fine (tree branches and tops) woody debris in temperate-boreal forests in Sweden and Lithuania. Sequence analysis of ITS rDNA was used to identify the fungi. In a neighbour-joining similarity tree, all sequences were grouped into five distinct clusters. Within each of these, ITS rDNA sequence variation consisted of 2-18 nucleotides, corresponding to 1-3% of their total length. The four least variable clusters were supported with high bootstrap values of 86-100%. Comparisons with the sequences in the GenBank database showed that all our strains had a 95-100% homology with identified Phialocephala species, and they were thus assigned to this genus. The representatives of two clusters were identified, as P. fortinii and P. dimorphospora. The representatives of three remaining clusters were defined as Phialocephala sp. 35, Phialocephala sp. 6 and Phialocephala sp. 18. Within each of these clusters, ITS rDNA sequence uniformity was higher than that observed within P. fortinii and P. dimorphospora. Consequently, their clusters were most discrete, supported with bootstrap values of 100%. Genetic variation in the five distinguished Phialocephala species and their possible ecological roles are discussed. Phialocephala sp. 6 was confined to healthy root tips of conifers. P. dimorphospora was only associated with dead woody tissue of P. abies. P. fortinii, Phialocephala sp. 18 and sp. 35 were isolated from both dead and living conifers and Betula pendula. In conclusion, the present study revealed the ability of fungi from the genus Phialocephala to colonise and persist in live and dead trees under strikingly different ecological conditions.

Transferase-deficient galactosaemia is an inherited disorder of carbohydrate metabolism, caused by mutation at the galactose-1-phosphate uridyl transferase ( GALT ) locus. A denaturing high performance liquid chromatography (dHPLC) method was developed for variant scanning of the GALT gene. The method unequivocally identified the Duarte D1, D2, Q188R, and K285N GALT alleles and associated polymorphisms. Length polymorphism in an intronic Alu repeat was characterised and a novel Single Nucleotide Polymorphism (IVS10nt-322g → t) associated with the D1 allele was identified.

Fil: Castro de Guerra, Dinorah Tibisay. Instituto Venezolano de Investigaciones Científicas; Venezuela Fil: Figuera Pérez, C.. Instituto Venezolano de Investigaciones Científicas; Venezuela Fil: Bravi, Claudio Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; Argentina Fil: Saunier, J.. Armed Forces DNA Identification Laboratory; Estados Unidos Fil: Scheible, M.. Armed Forces DNA Identification Laboratory; Estados Unidos Fil: Irwin, J.. Armed Forces DNA Identification Laboratory; Estados Unidos Fil: Coble, M. D.. Armed Forces DNA Identification Laboratory; Estados Unidos Fil: Rodríguez Larralde, A.. Instituto Venezolano de Investigaciones Científicas; Venezuela

While nature exploits folded biopolymers to achieve molecular recognition and catalysis, comparable abiological heteropolymer systems have been difficult to create. We synthesized and identified abiological peptoid heteroploymers capable of binding a dye. Using combinatorial synthesis, we constructed a library of 3400 amphiphilic 15-mer peptoids on an ultra-high-capacity beaded support. Individual macrobeads, each containing a single peptoid sequence, were arrayed into plates, cleaved, and screened in aqueous solution to locate dye binding heteropolymer assemblies. Resynthesis and characterization demonstrated the formation of defined helical assemblies as judged by size-exclusion chromatography, circular dichroism, and analytical ultracentrifugation. Inspired by nature's process of sequence variation and natural selection, we identified rare abiological sequence-specific heteropolymers that begin to mimic the structure and functional properties of their biological counterparts.

Current efforts to find disease-causing genes depend on patterns of linkage disequilibrium in human populations. Recent work has shown that linkage disequilibrium can extend over much larger genomic regions than expected, and that the patterns of linkage disequilibrium can differ markedly among populations.

We report the integrated proviral LTR sequence variation in four patients on highly active antiretroviral therapy (HAART). Integrated proviral fragments of LTR taken from four time points were PCR amplified from PBMCs and 10 to 12 individual clones were sequenced for each time point. Intrasample genetic distances and phylogenetic reconstruction of all LTR sequences demonstrated that 1–2 years of successful HAART did not significantly reduce the genetic repertoire of the integrated reservoir of HIV-1.

Purpose: To report on the use of fluorescence in situ hybridization (FISH) and dosage-sensitive Southern blot analysis in the molecular diagnosis of patients with Saethre-Chotzen syndrome. Methods: FISH and dosage-sensitive Southern blot analysis utilizing TWIST gene probes were performed on patients with Saethre-Chotzen syndrome but without an identifiable TWIST sequence variation. Results: Four unrelated patients with a deletion of the TWIST gene were identified by Southern blot; one of them had a complex chromosomal rearrangement involving 7p21 and no apparent deletion by FISH, suggesting a smaller deletion in the region including the TWIST gene. A fifth patient had an abnormal TWIST gene fragment on Southern blot analysis that segregated with the disease in the family; FISH was normal in this patient, suggesting a partial deletion or rearrangement in or near the gene. Conclusion: FISH and dosage-sensitive Southern blot analysis are useful diagnostic tools in Saethre-Chotzen syndrome without TWIST sequence variation.

Studies of nuclear sequence variation are accumulating, such that we can expect a good description of the structure of human variation across populations and genomic regions in the near future. This description will help to elucidate the evolutionary forces that shape patterns of variability. Such an understanding will be of general biological interest, but could also facilitate the design and interpretation of disease-mapping studies. Here, we integrate the results from surveys of nuclear sequence variation. When nuclear sequences are considered together with mtDNA and microsatellites, it becomes clear that neither the standard neutral model, nor a simple long-term exponential growth model, can account for all the available human variation data. A possible explanation is that a subset of loci are not evolving neutrally; even so, more-complex models of effective population size and structure might be necessary to explain the data.

Differential or genetic sequencing requires searching sample DNA for variations with respect to a reference sequence. Conventional detection techniques are too labor and cost expensive for use in diagnostic applications, therefore new technologies will be required. Measurement techniques based on mass spectrometry (MS) possess the potential for high-throughput, high fidelity measurement of sequence variation. Unambiguous detection of polymorphic sequences has been demonstrated, even in heterozygous samples. Automated reproducible measurements of microscopic arrays of samples will enable the high-throughput detection required for large-scale applications. Computational simulation and analysis of experimental parameters prior to experimentation will provide the optimization necessary for development of robust, reproducible measurements.

Sequence variation for the X chromosome short tandem repeats (X-STRs) DXS9898, DXS6789 and GATA172D05 was studied in two major population groups, namely Caucasians and Africans. DXS6789 revealed two different subtype sequence polymorphisms: for shorter alleles, with less than 17 repeats, results showed a simple composition with the following structure: (TATG) m –(TATC) n . For longer alleles, a constant TATC insertion was observed at the beginning of the variable repeat unit. Additionally, alleles identical in size revealed structural variations regarding the TATG/TATC proportion. Africans showed a higher intra-allelic variation at this locus than the Caucasian population group. For all three loci, DXS9898, DXS6789 and GATA172D05, no unique structure was found among Africans and Caucasians.

Autosomal STR polymorphisms at 2 loci (D12S392 and D17S906) are presented. Samples from current paternity analysis ( n = 909) were analysed. The observed heterozygosities were 0.878 (D12S392) and 0.915 (D17S906). No significant deviation from Hardy–Weinberg equilibrium was observed. 17 different alleles were observed at D12S392 whereas a total of 57 different alleles were observed at D17S906. The repeat numbers ranged from 15 to 27 at D12S392. For D17S906 the allele sizes ranged from 331 to 441 bases. Sequence variation at both loci is presented as well as frequency databases and other relevant forensic genetic parameters.

A minor groove binder (MGB) TaqMan real-time PCR assay was developed for the detection of respiratory syncytial virus (RSV) in clinical specimens. Upon evaluation of the assay, notable differences were observed in the overall fluorescent response obtained from RSV positive specimens, with some linear amplification curves deviating only slightly from baseline fluorescence. Sequencing of the probes targets in these RSV strains revealed single base mismatches with the MGB TaqMan probe. Overall, these results highlight the usefulness of MGB TaqMan probes for the detection of mismatches, but suggest that MGB Taqman probes have limitations for routine screening for uncharacterised viral strains.

The majority of condylomata acuminata (anogenital warts) are caused by infection with Human Papilloma Virus type 6 (HPV-6). We have sequenced the HPV-6 early genes, E1^E4, E6 and E7 from wart biopsy DNA samples sourced from the UK and USA and derived a consensus sequence for these genes and the proteins they encode. When compared to the prototype HPV-6b sequence, published over 12 years ago, the E1^E4 consensus sequence showed 3/91 (3.3%) amino acid changes, the E6 consensus sequence showed 1/150 (0.7%) changes and the E7 consensus sequence showed 1/98 (1.0%) changes. Since many of the early gene sequences from biopsy material were more similar to the HPV-6a subtype than HPV-6b, this data supports the use of HPV-6a as the HPV-6 prototype.

By inter-repeat PCR, multiple polymorphic loci can be targeted in parallel. To improve resolution and extend the number of detectable polymorphisms, Alu -directed inter-repeat PCR products from two large pedigrees of the Centre d»Etude du Polymorphisme Humain (CEPH) were electrophoretically resolved in non-denaturing polyacrylamide gels and, separately, on the basis of sequence content by denaturing gradient gel electrophoresis (DGGE). The resolution in DGGE gels was found to be superior to that in non-denaturing gels and a higher number of fragments was detected separately. The number of polymorphic bands detected by DGGE alone, however, was lower than that after size separation. This is ascribed to the fact that because of complete melting, small polymorphic fragments can run off the gel. With three Alu -specific primers, 18 and 16 polymorphic bands per individual were detected by size separation in pedigrees 1200 and 6600, respectively. In the same two pedigrees, seven and 15 polymorphic bands, respectively, were detected by DGGE. Segregation analysis of polymorphisms in the CEPH pedigrees indicated that most polymorphisms detected by DGGE were different from those detected by size separation.

Although there is a tendency for rDNA genes within a species to maintain sequence homogeneity, there can be significant levels of variation among rDNA repeat sequences within populations or individuals of a species as a consequence of mutation mechanisms. To date, there have been no practical techniques available in molecular parasitology that allow the extent of sequence variation among the repeats (ie, number of sequence types) to be displayed visually. In this report, we describe the use of the denaturing gradient gel electrophoresis (DGGE) technique for the rapid screening of parasite rDNA for sequence variation without the need for exhaustive cloning or DNA sequencing. The resolution of this variation by DGGE provides a diagnostic fingerprint for a species.

On the occasion of a study on mutation rates of human STR loci, numerous alleles have been sequenced. Several new sequence or length variant alleles were found.

The analysis of correlated sequence variation in evolutionarily related proteins is beginning to provide useful information regarding allosteric coupling between different functional sites. Such an analysis has been carried out for the nuclear hormone receptors, and the conclusions tested by making mutations that switch the allosteric response to ligands of RXR heterodimers.