Real-time PCR based on SYBR-Green I fluorescence: An alternative to the TaqMan assay for a relative quantification of gene rearrangements, gene amplifications and micro gene deletions

Background

Real-time PCR is increasingly being adopted for RNA quantification and genetic analysis. At present the most popular real-time PCR assay is based on the hybridisation of a dual-labelled probe to the PCR product, and the development of a signal by loss of fluorescence quenching as PCR degrades the probe. Though this so-called 'TaqMan' approach has proved easy to optimise in practice, the dual-labelled probes are relatively expensive.

Results

We have designed a new assay based on SYBR-Green I binding that is quick, reliable, easily optimised and compares well with the published assay. Here we demonstrate its general applicability by measuring copy number in three different genetic contexts; the quantification of a gene rearrangement (T-cell receptor excision circles (TREC) in peripheral blood mononuclear cells); the detection and quantification of GLI, MYC-C and MYC-N gene amplification in cell lines and cancer biopsies; and detection of deletions in the OPA1 gene in dominant optic atrophy.

Conclusion

Our assay has important clinical applications, providing accurate diagnostic results in less time, from less biopsy material and at less cost than assays currently employed such as FISH or Southern blotting.     

Visual stimuli that elicit appetitive behaviors in three morphologically distinct species of praying mantis

We assessed the differences in appetitive responses to visual stimuli by three species of praying mantis (Insecta: Mantodea), Tenodera aridifolia sinensis, Mantis religiosa, and Cilnia humeralis. Tethered, adult females watched computer generated stimuli (erratically moving disks or linearly moving rectangles) that varied along predetermined parameters. Three responses were scored: tracking, approaching, and striking. Threshold stimulus size (diameter) for tracking and striking at disks ranged from 3.5 deg (C. humeralis) to 7.8 deg (M. religiosa), and from 3.3 deg (C. humeralis) to 11.7 deg (M. religiosa), respectively. Unlike the other species which struck at disks as large as 44 deg, T. a. sinensis displayed a preference for 14 deg disks. Disks moving at 143 deg/s were preferred by all species. M. religiosa exhibited the most approaching behavior, and with T. a. sinensis distinguished between rectangular stimuli moving parallel versus perpendicular to their long axes. C. humeralis did not make this distinction. Stimulus sizes that elicited the target behaviors were not related to mantis size. However, differences in compound eye morphology may be related to species differences: C. humeralis’ eyes are farthest apart, and it has an apparently narrower binocular visual field which may affect retinal inputs to movement-sensitive visual interneurons.     

β-Dl-Methylene-aspartate,an inhibitor of aspartate aminotransferase,potently inhibitsl-glutamate uptake into astrocytes

[³H]Glutamate uptake into astrocytes in primary culture was potently inhibited by the aspartate analoguesl- andd-aspartic acid,Dl-threo--hydroxy-aspartic acid,l-aspartic acid--hydroxymate (IC50's: 136, 259, 168, and 560 M, respectively) and by -Dl-methylene-aspartate, a suicide inhibitor of asparate aminotransferase (IC50: 524 M), and by the endogenous sulphur-containing amino acidl-cysteinesulfinic acid (IC50: 114 M). [³H]Glutamate uptake was not significantly affected by either N-methyl-d-aspartate orDl-homocysteine thiolactone. These results demonstrate that other excitatory amino acids including aspartate andl-cysteinesulfinic acid (but excludingl-homocysteic acid) interact with the glutamate transport system of astrocytes. Inhibition of glutamate uptake may significantly increase the level of neuronal excitability.     

X-ray interference studies of crossbridge action in muscle contraction: evidence from muscles during steady shortening

During normal muscle shortening, the myosin heads must undergo many cycles of interaction with the actin filaments sliding past them. It is important to determine what range of configurations is found under these circumstances, and, in terms of the tilting lever arm model, what range of orientations the lever arms undergo. We have studied this using the X-ray interference technique described in the previous article, focusing mainly on the changes in the first order meridional reflection (M3) as compared to isometric. The change in ratio of the heights of the interference peaks indicates how far the mean lever arm angle has moved towards the end of the working stroke; the total intensity change depends on the angle change, on the number of heads now attached at any one time, and on the dispersion of lever arm angles. The latter provides a measure of the distance over which myosin heads remain attached to actin as they go through their working strokes. Surprisingly, the mean position of the attached heads moves only about 1 nm inwards (towards the center of the A-band) at low velocity shortening (around 0.9 T0): their dispersion changes very little. This shows that they must be detaching very early in the working stroke. However, at loads around 0.5 T0, the mean lever arm angle is about half way towards the end of the working stroke, and the dispersion of lever arm angles (with a uniform dispersion) is such as to distribute the heads throughout the whole of the working stroke. At higher velocities of shortening (at 0.3 T0), the mean position shifts further towards the end of the stroke, and the dispersion increases further. The details of the measurements, together with other data on muscle indicate that the force-generating mechanism within the myosin heads must have some unexpected properties.     

RAPD divergence caused by microsite edaphic selection in wild barley

Random amplified polymorphic DNA polymerase chain reaction (RAPDPCR) was used to assess genetic diversity in four subpopulations (86 individuals) of wild barley, Hordeum spontaneum, sampled from Tabigha microsite near the Sea of Galilee, Israel. The microsite consists of two 100 m transects that are topographically separated by 100 m, each equally subdivided into 50 m of basalt and terra rossa soil types. Despite the same macroclimate characterizing the area around the Sea of Galilee, the microsite offers two edaphically different microhabitats, with basalt being a more ecologically heterogeneous and broader-niche than the relatively drier but more homogeneous and narrow-niche terra rossa. Analysis of 118 putative loci revealed significant (P<0.05) genetic differentiation in polymorphism (P0.05) between the two soils across the transects with P being higher in the more heterogeneous basalt (mean P0.05 = 0.902), than in terra rossa (mean P0.05 = 0.820). Gene diversity (He) was higher in basalt (mean He=0.371), than in terra rossa (mean He=0.259). Furthermore, unique alleles were confined to one soil type, either in one or both transects. Rare alleles were observed more frequently in terra rossa than basalt, and in transect II only. Gametic phase disequilibria showed a larger multilocus association of alleles in basalt than terra rossa, and in transect I than II. Spearman rank correlation (r_s) revealed a strong association between specific loci and soil types, and transects. Also, analysis of multilocus organization revealed soil-specific multilocus-genotypes. Therefore, our results suggest an edaphically differentiated genetic structure, which corroborates the niche width-variation hypothesis, and can be explained, in part, by natural selection. This pattern of RAPD diversity is in agreement with allozyme and hordein protein diversities in the same subpopulations studied previously. This revised version was published online in July 2006 with corrections to the Cover Date.     

Identification of a sialate O-acetyltransferase from Campylobacter jejuni: demonstration of direct transfer to the C-9 position of terminalalpha-2, 8-linked sialic acid

We have identified a sialate O-acetyltransferase in the lipo-oligosaccharide biosynthesis locus of Campylobacter jejuni. Strains possessing this locus are known to produce sialylated outer core structures that mimic host gangliosides, and have been implicated in triggering the onset of Guillain-Barré syndrome. The acetyltransferase, which was cloned and expressed as a fusion construct in Escherichia coli, is soluble and homologous with members of the NodL-LacA-CysE family of O-acetyltransferases. This enzyme catalyzes the transfer of O-acetyl groups onto oligosaccharide-bound sialic acid, with a high specificity for terminal alpha2,8-linked residues. The modification is directed to C-9 and not C-7 as is believed to occur more commonly in other organisms. Despite their wide prevalence and importance in both eukaryotes and prokaryotes, this is the first report to describe the characterization of a purified sialate O-acetyltransferase.     

Recombination and the evolution of mutational robustness

Mutational robustness is the degree to which a phenotype, such as fitness, is resistant to mutational perturbations. Since most of these perturbations will tend to reduce fitness, robustness provides an immediate benefit for the mutated individual. However, robust systems decay due to the accumulation of deleterious mutations that would otherwise have been cleared by selection. This decay has received very little theoretical attention. At equilibrium, a population or asexual lineage is expected to have a mutation load that is invariant with respect to the selection coefficient of deleterious alleles, so the benefit of robustness (at the level of the population or asexual lineage) is temporary. However, previous work has shown that robustness can be favoured when robustness loci segregate independently of the mutating loci they act upon. We examine a simple two-locus model that allows for intermediate rates of recombination and inbreeding to show that increasing the effective recombination rate allows for the evolution of greater mutational robustness.     

The Presence of (+)-S-Adenosyl-l-Methionine in the Rat Brain and Its Lack of Effect on Phenylethanolamine N-Methyltransferase Activity

Abstract: (+)-S-Adenosyl- l -methionine [(+)-SAM] was isolated from rat brain and was quantified by HPLC followed by UV spectrophotometric measurements and by ¹H-NMR. Its estimated ratio in brain is 3% of total SAM. Because of its commercial unavailability, (+)-SAM was also prepared from chemically synthesized SAM by separation of the two diastereoisomers on a preparative reverse-phase Nucleosil C8 column. The (+) diastereoisomer thus obtained was then assayed in vitro both as an inhibitor and a substrate of phenylethanolamine N -methyltransferase. Enzymatic activity was measured by HPLC analysis. It was shown that (+)-SAM has no effect on phenylethanolamine N -methyltransferase activity; therefore, it is unlikely that (+)-SAM plays any possible role in regulation of adrenaline synthesis in the brain.     

Computational Estimates of Membrane Flow and Tension Gradient in Motile Cells

All parts of motile cells, including the plasma membrane, have to translocate in the direction of locomotion. Both directed intracellular membrane transport coupled with polarized endo- and exocytosis and fluid flow in the plane of the plasma membrane can contribute to this overall plasma membrane translocation. It remains unclear how strong a force is required to generate this flow. We numerically solve Stokes equations for the viscous membrane flow across a flat plasma membrane surface in the presence of transmembrane proteins attached to the cytoskeleton and find the membrane tension gradient associated with this flow. This gradient is sensitive to the size and density of the transmembrane proteins attached to the cytoskeleton and can become significant enough to slow down cell movement. We estimate the influence of intracellular membrane transport and actin growth and contraction on the tension gradient, and discuss possible ‘tank tread’ flow at ventral and dorsal surfaces.     

Functional and Molecular Surveillance of Helicobacter pylori Antibiotic Resistance in Kuala Lumpur

Background

Helicobacter pylori is the etiological agent for diseases ranging from chronic gastritis and peptic ulcer disease to gastric adenocarcinoma and primary gastric B-cell lymphoma. Emergence of resistance to antibiotics possesses a challenge to the effort to eradicate H. pylori using conventional antibiotic-based therapies. The molecular mechanisms that contribute to the resistance of these strains have yet to be identified and are important for understanding the evolutional pattern and selective pressure imposed by the environment.

Methods and Findings

H. pylori was isolated from 102 patients diagnosed with gastrointestinal diseases, who underwent endoscopy at University Malaya Medical Centre (UMMC). The isolates were tested for their susceptibility on eleven antibiotics using Etest. Based on susceptibility test, 32.3% of the isolates were found to have primary metronidazole resistance; followed by clarithromycin (6.8%) and fluoroquinolones (6.8%). To further investigate the resistant strains, mutational patterns of gene rdxA, frxA, gyrA, gyrB, and 23S rRNA were studied. Consistent with the previous reports, metronidazole resistance was prevalent in the local population. However, clarithromycin, fluoroquinolone and multi-drug resistance were shown to be emerging. Molecular patterns correlated well with phenotypic data. Interestingly, multi-drug resistant (MDR) strains were found to be associated with higher minimum inhibitory concentration (MIC) than their single-drug resistant (SDR) counterparts. Most importantly, clarithromycin-resistant strains were suggested to have a higher incidence for developing multi-drug resistance.

Conclusion

Data from this study highlighted the urgency to monitor closely the prevalence of antibiotic resistance in the Malaysian population; especially that of clarithromycin and multi-drug resistance. Further study is needed to understand the molecular association between clarithromycin resistance and multi-drug resistance in H. pylori. The report serves a reminder that a strict antibiotic usage policy is needed in Malaysia and other developing countries (especially those where H. pylori prevalence remained high).