Derived Polymorphic Amplified Cleaved Sequence (dPACS): A Novel PCR-RFLP Procedure for Detecting Known Single Nucleotide and Deletion-Insertion Polymorphisms.

Most methods developed for detecting known single nucleotide polymorphisms (SNP) and deletion-insertion polymorphisms (DIP) are dependent on sequence conservation around the SNP/DIP and are therefore not suitable for application to heterogeneous organisms. Here we describe a novel, versatile and simple PCR-RFLP procedure baptised 'derived Polymorphic Amplified Cleaved Sequence' (dPACS) for genotyping individual samples. The notable advantage of the method is that it employs a pair of primers that cover the entire fragment to be amplified except for one or few diagnostic bases around the SNP/DIP being investigated. As such, it provides greater opportunities to introduce mismatches in one or both of the 35-55 bp primers for creating a restriction site that unambiguously differentiates wild from mutant sequences following PCR-RFLP and horizontal MetaPhorTM gel electrophoresis. Selection of effective restriction enzymes and primers is aided by the newly developed dPACS 1.0 software. The highly transferable dPACS procedure is exemplified here with the positive detection (in up to 24 grass and broadleaf species tested) of wild type proline106 of 5-enolpyruvylshikimate-3-phosphate synthase and its serine, threonine and alanine variants that confer resistance to glyphosate, and serine264 and isoleucine2041 which are key target-site determinants for weed sensitivities to some photosystem II and acetyl-CoA carboxylase inhibiting herbicides, respectively.

Derived Polymorphic Amplified Cleaved Sequence (dPACS) Assay.

The derived polymorphic amplified cleaved sequence (dPACS) assay is a simple polymerase chain reaction/restriction fragment length polymorphism (PCR-RFLP)-based procedure for detecting known single-nucleotide polymorphisms (SNPs) and deletion-insertion polymorphisms (DIPs). It is relatively straightforward to carry out using basic and commonly available molecular biology kits. The method differs from other PCR-RFLP assays in that it employs 35-55 bp primer pairs that encompass the entire targeted DNA region except for a few diagnostic nucleotides being examined. In so doing, it allows for the introduction of nucleotide mismatches in one or both primers for differentiating wild from mutant sequences following polymerase chain reaction, restriction digestion and MetaPhor gel electrophoresis. Primer design and the selection of discriminating enzymes are achieved with the help of the dPACS 1.0 program. The method is exemplified here with the positive detection of serine 264-psbA, a key determinant for the effective binding of some photosystem II inhibitors to their target. A serine-to-glycine mutation at codon 264 of psbA causes resistance to serine-binding photosystem II herbicides in several grasses and broad-leaf weeds, including Amaranthus retroflexus, which is employed in this study.

A feasibility study of optical coherence tomography for guiding deep brain probes.

Deep brain simulation (DBS) is effective for the treatment of various diseases including Parkinson's disease and essential tremor. However, anatomical targeting combined with microelectrode mapping of the region requires significant surgical time. Also, the fine-tipped microelectrode imposes a risk of hemorrhage in the event that the trajectory intersects subcortical vessels. To reduce the operation time and the risk of hemorrhage, we propose to use optical coherence tomography (OCT) to guide the insertion of the DBS probe. We conducted in vitro experiments in the rat brain to study the feasibility of this application. The result shows that OCT is able to differentiate structures in the rat brain. White matter tends to have higher peak reflectivity and steeper attenuation rate compared to gray matter. This structural information may help guide DBS probe advance and electrical measurements.

Plasmodium berghei: plasmodium perforin-like protein 5 is required for mosquito midgut invasion in Anopheles stephensi.

During its life cycle the malarial parasite Plasmodium forms three invasive stages which have to invade different and specific cells for replication to ensue. Invasion is vital to parasite survival and consequently proteins responsible for invasion are considered to be candidate vaccine/drug targets. Plasmodium perforin-like proteins (PPLPs) have been implicated in invasion because they contain a predicted pore-forming domain. Ookinetes express three PPLPs, and one of them (PPLP3) has previously been shown to be essential for mosquito midgut invasion. In this study we show through phenotypic analysis of loss-of-function mutants that PPLP5 is equally essential for mosquito infection. Deltapplp5 ookinetes cannot invade midgut epithelial cells, but subsequent parasite development is rescued if the midgut is bypassed by injection of ookinetes into the hemocoel. The indistinguishable phenotypes of Deltapplp5 and Deltapplp3 ookinetes strongly suggest that these two proteins contribute to a common process.

Different evolutionary histories of the two classical class I genes BF1 and BF2 illustrate drift and selection within the stable MHC haplotypes of chickens.

Compared with the MHC of typical mammals, the chicken MHC (BF/BL region) of the B12 haplotype is smaller, simpler, and rearranged, with two classical class I genes of which only one is highly expressed. In this study, we describe the development of long-distance PCR to amplify some or all of each class I gene separately, allowing us to make the following points. First, six other haplotypes have the same genomic organization as B12, with a poorly expressed (minor) BF1 gene between DMB2 and TAP2 and a well-expressed (major) BF2 gene between TAP2 and C4. Second, the expression of the BF1 gene is crippled in three different ways in these haplotypes: enhancer A deletion (B12, B19), enhancer A divergence and transcription start site deletion (B2, B4, B21), and insertion/rearrangement leading to pseudogenes (B14, B15). Third, the three kinds of alterations in the BF1 gene correspond to dendrograms of the BF1 and poorly expressed class II B (BLB1) genes reflecting mostly neutral changes, while the dendrograms of the BF2 and well-expressed class II (BLB2) genes each have completely different topologies reflecting selection. The common pattern for the poorly expressed genes reflects the fact the BF/BL region undergoes little recombination and allows us to propose a pattern of descent for these chicken MHC haplotypes from a common ancestor. Taken together, these data explain how stable MHC haplotypes predominantly express a single class I molecule, which in turn leads to striking associations of the chicken MHC with resistance to infectious pathogens and response to vaccines.