Replication stress at microsatellites causes DNA double-strand breaks and break-induced replication

Short tandemly repeated DNA sequences, termed microsatellites, are abundant in the human genome. These microsatellites exhibit length instability and susceptibility to DNA double-strand breaks (DSBs) due to their tendency to form stable non-B DNA structures. Replication-dependent microsatellite DSBs are linked to genome instability signatures in human developmental diseases and cancers. To probe the causes and consequences of microsatellite DSBs, we designed a dual-fluorescence reporter system to detect DSBs at expanded (CTG/CAG)n and polypurine/polypyrimidine (Pu/Py) mirror repeat structures alongside the c-myc replication origin integrated at a single ectopic chromosomal site. Restriction cleavage near the (CTG/CAG)100 microsatellite leads to homology-directed single-strand annealing between flanking AluY elements and reporter gene deletion that can be detected by flow cytometry. However, in the absence of restriction cleavage, endogenous and exogenous replication stressors induce DSBs at the (CTG/CAG)100 and Pu/Py microsatellites. DSBs map to a narrow region at the downstream edge of the (CTG)100 lagging-strand template. (CTG/CAG)n chromosome fragility is repeat length–dependent, whereas instability at the (Pu/Py) microsatellites depends on replication polarity. Strikingly, restriction-generated DSBs and replication-dependent DSBs are not repaired by the same mechanism. Knockdown of DNA damage response proteins increases (Rad18, polymerase (Pol) η, Pol κ) or decreases (Mus81) the sensitivity of the (CTG/CAG)100 microsatellites to replication stress. Replication stress and DSBs at the ectopic (CTG/CAG)100 microsatellite lead to break-induced replication and high-frequency mutagenesis at a flanking thymidine kinase gene. Our results show that non-B structure–prone microsatellites are susceptible to replication-dependent DSBs that cause genome instability.

Identification of Smallanthus sonchifolius in herbal tea mixtures by PCR and DART/TOF-MS methods

The identification of yacon, a medicinal plant, in tea mixtures by rapid Polymerase Chain Reaction (PCR) and the Direct Analysis in Real Time coupled with Time-of-Flight Mass Spectrometry (DART/TOF-MS) method were evaluated. Three tea products and a pure yacon tea were analysed using the molecular method PCR, concretely the intraspecific variation of the internal transcribed spacer (ITS) regions of rDNA and the DART method coupled with TOF-MS. The results show the reliability of PCR and restriction cleavage of the ITS as a combined approach to confirm the presence of yacon in herbal tea mixtures. Three fragments of approximately 700, 408, and 235 bp in length are present when yacon is a part of the herbal tea mixture. The Principal Component Analysis (PCA) based on the fingerprints of the complete Total Ion Current (TIC) mass spectra shows sufficient separation of herbal teas with and without yacon leaves. The reported methods are technically rapid and can be used as an effective tool for the purposes of yacon identification or authentication.

Comparative morphological and molecular identification of Haemonchus species in sheep

A combined approach in the determination of Haemonchus nematodes from sheep was applied in this trial. Using selected morphological characters 90.2 % females and 84.2 % males of Haemonchus contortus and 9.8 % females and 15.8 % males of Haemonchus placei were identified. Although cluster analysis based on morphological identification clearly separated two Haemonchus species, H. contortus was exclusively detected in all specimens using restriction cleavage of the ITS-2 region with FspBI endonuclease as well as through the sequencing analysis. Because sheep from both farms have never had contact with other ruminants, and the farmers apply only closed flock turnover, we assume that only H. contortus mono-infection occurred on both farms. This opinion is also supported by molecular data. The most striking result of our study was the finding which indicates that the discriminant function is not able to accurately identify Haemonchus males at the species level.

Phenotypic and molecular characterization of Salmonella enterica serovar Sofia, an avirulent species in Australian poultry

Salmonella enterica serovar Sofia (S. Sofia) is often isolated from chickens in Australia. However, despite its high frequency of isolation from chicken and chicken meat products, S. Sofia is rarely associated with animal or human salmonellosis, presumably because this serovar is avirulent in nature. The objective of this work was to investigate the phenotypic and molecular properties of S. Sofia in order to assess its pathogenic potential. Our in vivo studies support the observation that this serovar can colonize tissues, but does not cause disease in chickens. This was further confirmed with tissue culture assays, which showed that the ability of S. Sofia to adhere, invade and survive intracellularly is significantly diminished compared with the pathogenic Salmonella enterica serovar Typhimurium (S. Typhimurium) 82/6915. Molecular analysis of Salmonella pathogenicity islands (SPIs) showed that most of the differences observed in SPI1 to SPI5 of S. Sofia could be attributed to minor changes in the sequences, as indicated by a loss or gain of restriction cleavage sites within these regions. Sequence analysis demonstrated that the majority of virulence genes identified were predicted to encode proteins sharing a high identity (75–100 %) with corresponding proteins from S. Typhimurium. However, a number of virulence genes in S. Sofia have accumulated mutations predicted to affect transcription and/or translation. The avirulence of this serovar is probably not the result of a single genetic change but rather of a series of alterations in a large number of virulence-associated genes. The acquisition of any single virulence gene will almost certainly not be sufficient to restore S. Sofia virulence.

A New Pathotype of Pepper mild mottle virus (PMMoV) Overcomes the L4 Resistance Genotype of Pepper Cultivars

The biological, serological, and molecular characteristics of a newly isolated L4 resistance-breaking isolate of Pepper mild mottle virus (PMMoV) were studied. The new pathotype of PMMoV is closely related to the Israeli pathotypes P1,2 and P1,2,3 of the virus; however, the mosaic symptoms caused by this new pathotype on pepper plants with an L4 genotype were more severe than the mild mosaic symptoms caused by other common pathotypes of the virus in susceptible plants. The predicted amino acid sequence of the putative coat protein (CP) of the newly described pathotype has two amino acid mismatches when compared with the CP of pathotype P1,2, leucine to glutamine at position 47, and alanine to glycine at position 87. The CP of the new pathotype has one amino acid mismatch when compared with P1,2,3, having alanine instead of glycine at position 87. Based on its biological characteristics, the new pathotype was designated P1,2,3,4 of PMMoV-Is. A method is described for the differentiation among the three PMMoV pathotypes using restriction cleavage analysis of reverse-transcription polymerase chain reaction products made from virus-infected plants. An additional unique MnlI site in the CP gene of the newly isolated P1,2,3,4 allows its distinction from the other two isolates, while BglI cleaved only products of the P1,2 pathotype.