High-throughput single-nucleotide polymorphism genotyping by fluorescent competitive allele-specific polymerase chain reaction (SNiPTag)

Single nucleotide polymorphisms (SNPs) are becoming widely recognized as the new currency for gene mapping as increasing numbers are discovered. Here we outline a method for their rapid analysis based on an allele-specific polymerase chain reaction (PCR) which employs a competitive approach, whereby both allele-specific primers are present in the same reaction and carry different fluorescent labels. This procedure is simple and amenable to high-throughput genotyping using conventional automated sequencing equipment, and no post-PCR modifications are required. Verification of the procedure was carried out by comparison of results derived by this method with those from restriction enzyme digestion of the ALDH2 exon 12 functional polymorphism (Glu-487-Lys) in 109 individuals. Additionally, we have examined all combinations of nucleotide substitutions and shown them to be differentiated by this method. As proof of concept, several assays were combined and loaded on a single gel lane/capillary to substantially improve throughput. This was made possible by designing the PCR products to be of different lengths and no interference was observed between products differing in size by only six nucleotides. We outline a number of test assays for well-characterized SNPs in human candidate genes for behavioral disorders.     

Aspergillus melleus protease-catalyzed peptide synthesis using the carbamoylmethyl ester as an acyl donor in 1,1,1,3,3,3-hexafluoro- 2-propanol/N,N-dimethylformamide

The coupling between the carbamoylmethyl ester of an N-protected amino acid or dipeptide (at 25 mM) and an amino acid amide (at 100 mM) was achieved using Aspergillus melleus protease in 1,1,1,3,3,3-hexafluoro-2-propanol/N,N-dimethylformamide (1:1, v/v); the coupling efficiencies were dependent largely on the combination of amino acid residues: e.g. the dipeptide yields after 48 h were for l-Ala + Gly, 100% and for l-Leu + l-Leu, 16%.     

Affinities between Asian non-human Schistosoma species,the S. indicum group,and the African human schistosomes

Schistosoma species have traditionally been arranged in groups based on egg morphology, geographical origins, and the genus or family of snail intermediate host. One of these groups is the 'S. indicum group' comprising species from Asia that use pulmonate snails as intermediate hosts. DNA sequences were obtained from the four members of this group (S. indicum, S. spindale, S. nasale and S. incognitum) to provide information concerning their phylogenetic relationships with other Asian and African species and species groups. The sequences came from the second internal transcribed spacer (ITS2) of the ribosomal gene repeat, part of the 28S ribosomal RNA gene (28S), and part of the mitochondrial cytochrome c oxidase subunit 1 (CO1) gene. Tree analyses using both distance and parsimony methods showed the S. indicum group not to be monophyletic. Schistosoma indicum, S. spindale and S. nasale were clustered among African schistosomes, while S. incognitum was placed as sister to the African species (using ITS2 and 28S nucleotide sequences and CO1 amino acid sequences), or as sister to all other species of Schistosoma (CO1 nucleotide sequences). Based on the present molecular data, a scenario for the evolution of the S. indicum group is discussed.     

Transgenic mouse model of tauopathies with glial pathology and nervous system degeneration

Frontotemporal dementias (FTDs), including corticobasal degeneration (CBD) and progressive supranuclear palsy (PSP), are neurodegenerative tauopathies characterized by widespread CNS neuronal and glial tau pathologies, but there are no tau transgenic (Tg) mice that model neurodegeneration with glia tau lesions. Thus, we generated Tg mice overexpressing human tau in neurons and glia. No neuronal tau aggregates were detected, but old mice developed Thioflavin S- and Gallyas-positive glial tau pathology resembling CBD astrocytic plaques. Tau-immunoreactive and Gallyas-positive oligodendroglial coiled bodies (similar to CBD and PSP), glial degeneration, and motor deficits were associated with age-dependent accumulations of insoluble hyperphosphorylated human tau and tau immunopositive filaments in degenerating glial cells. Thus, tau-positive glial lesions similar to human FTDs occur in these Tg mice, and these pathologies are linked to glial and axonal degeneration.     

Geraniol-inducible glutathione S-transferase in cultured soybean cells

When the cultured cells of Glycine max (soybean) were treated with 5 mM geraniol as a chemical stress, an mRNA level was elevated in a rapid but transient increase. The mRNA was cloned and sequenced, and found to correspond to the mRNA encoding glutathione S-transferase (GST). The GST mRNA level and GST activity were elevated to maxima at 4-6 h and 8 h, respectively, after treatment of the cultures with geraniol. These indicate that GST is one of the geraniol-responsive factors in soybean cells.     

Preparation of the addition products of alpha-tocopherol with cholesteryl linoleate-peroxyl radicals

a-Tocopherol was reacted with cholesteryl linoleate hydroperoxides (Ch18:2-OOH) in the presence of an iron-chelate, Fe(III) acetylacetonate, at 37 degrees C in benzene. The reaction products were isolated and identified as four positional isomers of cholesteryl (8a-dioxy-alpha-tocopherone)-epoxyoctadecenoates and two positional isomers of cholesteryl (8a-dioxy-alpha-tocopherone)-octadecadienoates. The result indicates that the peroxyl radicals from Ch18:2-OOH react with the 8a-carbon radical of alpha-tocopherol to form the addition products.     

Mice that lack astrotactin have slowed neuronal migration

The cortical regions of the brain are laminated as a result of directed migration of precursor cells along glia during development. Previously, we have used an assay system to identify astrotactin as a neuronal ligand for migration on glial fibers. To examine the function of astrotactin in vivo, we generated a null mutation by targeted gene disruption. The cerebella of astrotactin null mice are approximately 10% smaller than wild type. In vitro and in vivo cerebellar granule cell assays show a decrease in neuron-glial binding, a reduction in migration rates and abnormal development of Purkinje cells. Consequences of this are poorer balance and coordination. Thus, astrotactin functions in migration along glial processes in vivo, a process required for generating laminar structures and for the development of synaptic partner systems.     

Controlling pore assembly of staphylococcal gamma-haemolysin by low temperature and by disulphide bond formation in double-cysteine LukF mutants

Staphylococcal LukF and Hlg2 are water-soluble monomers of gamma-haemolysin that assemble into oligomeric pores on the erythrocyte membranes. Here, we have created double-cysteine LukF mutants, in which single disulphide bonds connect either the prestem domain and the cap domain (V12C-T136C, Cap-Stem), or two beta-strands within the prestem domain (T117C-T136C, Stem-Stem) to control pore assembly of gamma-haemolysin at intermediate stages. The disulphide-trapped mutants were inactive in erythrocyte lysis, but gained full haemolytic activity if the disulphide bonds were reduced. The disulphide bonds blocked neither the membrane binding ability nor the intermediate prepore oligomerization, but efficiently inhibited the transition from prepores to pores. The prepores of Cap-Stem were dissociated into monomers in 1% SDS. In contrast, the prepores of Stem-Stem were stable in SDS and had ring-shaped structures similar to those of wild-type LukF, as observed by transmission electron microscopy. The transition of both mutants from prepores to pores could even be achieved by reducing disulphide bonds at low temperature (2 degrees C), whereas prepore oligomerization was effectively inhibited by low temperature. Finally, real-time transition of Stem-Stem from prepores to pores on ghost cells, visualized using a Ca2+-sensitive fluorescent indicator (Rhod2), was shown by the sequential appearance of fluorescence spots, indicating pore-opening events. Taken together, these data indicate that the prepores are legitimate intermediates during gamma-haemolysin pore assembly, and that conformational changes around residues 117 and 136 of the prestem domain are essential for pore formation, but not for membrane binding or prepore oligomerization. We propose a mechanism for gamma-haemolysin pore assembly based on the demonstrated intermediates.