Detection of cyanobacterial sxt genes and paralytic shellfish toxins in freshwater lakes and brackish waters on Åland Islands,Finland

Harmful cyanobacteria are a globally growing concern. They produce a large variety of toxic compounds, including saxitoxin and its many structural variants, a group of potent neurotoxins collectively called paralytic shellfish toxins or PST. Nucleic acid based detection methods, such as qPCR, have been proposed as potential screening and monitoring tools for toxic cyanobacteria, but it is not clear how well the presence and quantity of saxitoxin biosynthesis (sxt) genes can be used to predict the production of PST in the environment. In this study, the prevalence of three sxt genes and their co-occurrence with paralytic shellfish toxins in the environment was investigated. The sxtA, sxtG and sxtB genes were present on average in 31% of the samples collected from lakes and brackish coastal waters on Åland Islands, Finland, during the three-year monitoring period. PST detection frequency varied from 13% to 59% from year to year, and concentrations were generally low. On average higher sxtB copy numbers were associated with PST detection, and although a positive correlation between gene copy numbers and toxin concentrations was observed (Spearman rank correlation, ρ = 0.53, P = 0.012), sxt gene presence or quantity didn’t reliably predict PST production. Sequencing of sxtA fragments and identification of main cyanobacteria indicated that the likely candidate responsible for PST production in the samples belonged to the genus Anabaena.     

Characterization of tetranucleotide microsatellite loci and development and validation of multiplex reactions for the study of right whale species (genus Eubalaena)

A North Atlantic right whale (Eubalaena glacialis) genomic library was developed and screened with a (GATA)₈ probe to identify tetranucleotide microsatellite loci. Sixteen characterized loci were polymorphic in North Atlantic and/or South Atlantic (Eubalaena australis) right whales, 12 being polymorphic in E. glacialis, and 15 in E. australis. Fourteen of these were combined with 21 other previously identified loci for a suite of 35 loci which can be used to increase resolution of genetic analyses of these species. Multiplex reactions were developed for genotyping samples at these loci, providing a method that is rapid, reliable and cost‐effective.     

Strategies for signal amplification in nucleic acid detection

Many aspects of molecular genetics necessitate the detection of nucleic acid sequences. Current approaches involving target amplification (in situ PCR, Primed in situ Labeling, Self-Sustained Sequence Replication, Strand Displacement Amplification), probe amplification (Ligase Chain Reaction, Padlock Probes, Rolling Circle Amplification) and signal amplification (Tyramide Signal Amplification, Branched DNA Amplification) are summarized in the present review, together with their advantages and limitations.     

Primers for the amplification of sequence‐characterized loci in Cryphonectria cubensis populations

We describe the development of DNA markers for the fungal pathogen of Eucalyptus, Cryphonectria cubensis. These markers originated from cloned intershort sequence repeat polymerase chain reactions, which enrich for medium to highly repetitive DNA sequences. In total, 10 markers were isolated, eight of which were polymorphic, and these can subsequently be applied to study populations of C. cubensis.     

Lysine Succinylation of VBS Contributes to Sclerotia Development and Aflatoxin Biosynthesis in Aspergillus flavus

Aspergillus flavus is a common saprophytic and pathogenic fungus, and its secondary metabolic pathways are one of the most highly characterized owing to its aflatoxin (AF) metabolite affecting global economic crops and human health. Different natural environments can cause significant variations in AF synthesis. Succinylation was recently identified as one of the most critical regulatory post-translational modifications affecting metabolic pathways. It is primarily reported in human cells and bacteria with few studies on fungi. Proteomic quantification of lysine succinylation (Ksuc) exploring its potential involvement in secondary metabolism regulation (including AF production) has not been performed under natural conditions in A. flavus. In this study, a quantification method was performed based on tandem mass tag labeling and antibody-based affinity enrichment of succinylated peptides via high accuracy nano-liquid chromatography with tandem mass spectrometry to explore the succinylation mechanism affecting the pathogenicity of naturally isolated A. flavus strains with varying toxin production. Altogether, 1240 Ksuc sites in 768 proteins were identified with 1103 sites in 685 proteins quantified. Comparing succinylated protein levels between high and low AF-producing A. flavus strains, bioinformatics analysis indicated that most succinylated proteins located in the AF biosynthetic pathway were downregulated, which directly affected AF synthesis. Versicolorin B synthase is a key catalytic enzyme for heterochrome B synthesis during AF synthesis. Site-directed mutagenesis and biochemical studies revealed that versicolorin B synthase succinylation is an important regulatory mechanism affecting sclerotia development and AF biosynthesis in A. flavus. In summary, our quantitative study of the lysine succinylome in high/low AF-producing strains revealed the role of Ksuc in regulating AF biosynthesis. We revealed novel insights into the metabolism of AF biosynthesis using naturally isolated A. flavus strains and identified a rich source of metabolism-related enzymes regulated by succinylation.     

Mitochondrial DNA haplogrouping of the brown bear,Ursus arctos (Carnivora: Ursidae) in Asia,based on a newly developed APLP analysis

Sequence analyses of the complete brown bear, Ursus arctos, mitochondrial DNA (mtDNA) genome have detected scattered single nucleotide polymorphisms (SNPs) that define distinct mtDNA haplogroups in phylogeographical studies. The degraded DNA in historical samples, such as stuffed or excavated specimens, however, is often not suitable for sequence analyses. To address this problem, we developed an amplified product length polymorphism (APLP) analysis for mtDNA‐haplogrouping U. arctos specimens by detecting haplogroup‐specific SNPs. We verified the validity and utility of this method by analysing up to 170‐year‐old skin samples from U. arctos specimens collected widely across continental Eurasia. We detected some of the same haplogroups as those occurring in eastern Hokkaido (Japan) and eastern Alaska in continental Eurasia (the Altai and the Caucasus). Our results show that U. arctos in eastern Hokkaido and eastern Alaska descended from a common ancestor in continental Eurasia, and suggest that U. arctos occupied several refugia in southern Asia during the Last Glacial Maximum. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 111 , 627–635.     

DNA hybridization analysis of mycobacterial DNA using the 18-kDa protein gene of Mycobacterium leprae

Abstract DNA hybridization studies using a 611-base pair (bp) probe, encoding the entire 18-kDa protein of Mycobacterium leprae , demonstrated that M. simiae, M. intracellulare, M. kansasii, M. terrae , ADM-2, M. avium, M. scrofulaceum, M. gordonae and M. chelonei appear to posses DNA sequences homologous to the 18-kDa protein gene of M. leprae . RFLP analysis revealed that the restriction sites in the M. leprae 18-kDa gene were not conserved in the putative gene homologs of M. simiae and M. intracellulare . The restriction patterns observed with the 611-bp probe were useful in differentiating M. intracellulare, M. simiae , and M. leprae from each other, as well as in distinguishing strains of M. simiae serovar 1. Finally, the presence of homologous sequences in various mycobacteria did not affect the specificity of a previously described PCR test for detection of M. leprae , based on the M. leprae 18-kDa protein gene.     

马铃薯Y病毒外壳蛋白基因的克隆及序列分析

本文报道应用聚合酶链式反应(PCR)技术,在体外扩增马铃薯 Y 病毒外壳蛋白基因及其克隆和序列分析的结果。病毒 RNA 从马铃薯 Y 病毒感染的烟草叶片中提取,用合成的PCR 3引物及 AMV 逆转录酶合成了单链的 cDNA。利用 PCR 技术,经30个循玎的扩增。得到了一特异的0.8kb 片段。克隆后对此片段进行了限制性内切酶物理图谱分析,并测定了其全序列。实验结果证明,我们克隆到的是完整的马铃薯 Y 病毒的外壳蛋白基因。与国外报道的马铃薯 Y 病毒 N 株相比,其核苷酸序列及推测的氨基酸序列的同源率分别为97.8%和97%。将该基因导入马铃薯以期获得抗 Y 病毒马铃薯的工作正在进行。本文还对 PCR 技术用于扩增植物 RNA 病毒的方法以及用基因工程方法培育抗病毒作物新品种的可行性等进行了讨论。     

Polymerase chain reaction (PCR) techniques for site-directed mutagenesis

Polymerase chain reaction (PCR) technology has revolutionized the process of isolating and amplifying segments of DNA. One powerful application of PCR is its use in precise site-directed mutagenesis (SDM). SDM provides an elegant tool for scientists and engineers to explore biocatalytic mechanisms and processes to understand the structural-functional relationships of enzymes and other proteins. This article reviews techniques and methodology used in site-directed mutagenesis of genes by PCR.