A comparison of different lysis buffers to assess allele dropout from single cells for preimplantation genetic diagnosis

Single cell polymerase chain reaction (PCR) for preimplantation genetic diagnosis (PGD) requires high efficiency and accuracy. Allele dropout (ADO), the random amplification failure of one of the two parental alleles, remains the most significant problem in PCR-based PGD testing since it can result in serious misdiagnosis for compound heterozygous or autosomal dominant conditions. A number of different strategies (including the use of lysis buffers to break down the cell and make the DNA accessible) have been employed to combat ADO with varying degrees of success, yet there is still no consensus among PGD centres over which lysis buffer should be used (ESHRE PGD Consortium, 1999 ). To address this issue, PCR amplification of three genes (CFTR, LAMA3 and PKP1) at different chromosomal loci was investigated. Single lymphocytes from individuals heterozygous for mutations within each of the three genes were collected and lysed in either alkaline lysis buffer (ALB) or proteinase K/SDS lysis buffer (PK). PCR amplification efficiencies were comparable between alkaline lysis and proteinase K lysis for PCR products spanning each of the three mutated loci (ΔF508 in CFTR 90% vs 88%; R650X in LAMA3 82% vs 78%; and Y71X in PKP1 91% vs 87%). While there was no appreciable difference between ADO rates between the two lysis buffers for the LAMA3 PCR product (25% vs 26%), there were significant differences in ADO rates between ALB and PK for the CFTR PCR product (0% vs 23%) and the PKP1 PCR product (8% vs 56%). Based on these results, we are currently using ALB in preference to PK/SDS buffer for the lysis of cells in clinical PGD. Copyright © 2001 John Wiley & Sons, Ltd.     

THE INFLUENCE OF SETTINGS AND ENVIRONMENTAL EVENTS ON METAMOTIVATIONAL STATE AND AROUSAL: FURTHER TESTS OF REVERSAL THEORY ON ASIAN SAMPLES

The two studies reported further test reversal theory hypotheses concerning human psychological responses to different types of settings and environmental events on an Asian sample. In study 1, individuals in four settings representing different combinations of metamotivational state and arousal level (telic and paratelic/low arousal; telic and paratelic/high arousal) completed the telic state measure (TSM; Svebak & Murgatroyd, 1985). Significant differences between participants' mean scores across the four settings were obtained for TSM items serious–playful, planning–spontaneous, felt arousal, preferred arousal, effort and arousal discrepancy. In study 2 different environmental events in the same setting (changes in reading tasks), used in a crossover design, were used to induce reversals in individuals' operative metamotivational states. The results obtained from TSM scores pretask 1, between tasks and posttask 2. indicated significant time-of-testing interaction effects on the TSM serious–playful, planning–spontaneous and effort items. In addition, a main effect for TSM felt arousal was found. These results showed that reversals in metamotivational states were induced by the environmental event and that felt arousal scores increased across the three administrations of the TSM.     

Environmental contaminants in prey and tissues of the peregrine falcon in the Big Bend Region, Texas, USA.

Peregrine falcons (Falco peregrinus) have been recorded nesting in Big Bend National Park, Texas, USA and other areas of the Chihuahuan Desert since the early 1900s. From 1993 to 1996, peregrine falcon productivity rates were very low and coincided with periods of low rainfall. However, low productivity also was suspected to be caused by environmental contaminants. To evaluate potential impacts of contaminants on peregrine falcon populations, likely avian and bat prey species were collected during 1994 and 1997 breeding seasons in selected regions of western Texas, primarily in Big Bend National Park. Tissues of three peregrine falcons found injured or dead and feathers of one live fledgling also were analyzed. Overall, mean concentrations of DDE [1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene], a metabolite of DDT [1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane], were low in all prey species except for northern rough-winged swallows (Stelgidopteryx serripennis, mean = 5.1 microg/g ww). Concentrations of mercury and selenium were elevated in some species, up to 2.5 microg/g dw, and 15 microg/g dw, respectively, which upon consumption could seriously affect reproduction of top predators. DDE levels near 5 microg/g ww were detected in carcass of one peregrine falcon found dead but the cause of death was unknown. Mercury, selenium, and DDE to some extent, may be contributing to low reproductive rates of peregrine falcons in the Big Bend region.     

Structure of a Mediterranean cryptobenthic fish community and its relationships with habitat characteristics

In the last 10 years, several studies have been carried out on the fish fauna of the Ustica Island marine reserve, yet no investigation was specifically addressed to the cryptobenthic fish assemblage. The first task of this study, conducted along the shallow rocky reefs of Ustica, was to determine the species composition, diversity and relative density of the resident cryptobenthic fishes. Furthermore, we aimed to assess the effects of some macro- and microscale habitat characteristics on the distribution pattern of fishes. In particular, the effect of predator density was indirectly evaluated by comparing density data collected within and outside the integral reserve zone. Overall, 20 species belonging to Blenniidae, Gobiidae, Tripterygiidae, Scorpaenidae and Gobiesocidae were recorded. Gobius bucchichi, Scorpaena maderensis, Tripterygion delaisi, T. melanurus and T. tripteronotus were the numerically dominant and most common species. The effects of zone (i.e. of predator density), bottom type and depth on species richness, diversity and evenness were not significant. A greater total fish density was observed on stones compared with rocky cliff and plateau, but only in the shallowest depth range. At level of single species, G. bucchichi was more abundant inside than outside the integral reserve, but only on stones and at 0–2 m depth range. Density of G. bucchichi was generally higher on stones than on rocky cliffs or plateau and between 0 and 5 m depth, although these differences were not always significant. T. delaisi was conversely more abundant in the deepest stratum (7–10 m). Canonical analyses demonstrated that bottom type and depth influenced significantly the fish assemblage structure. The observed differences in the assemblage structure relied mainly upon the dominant species. T. tripteronotus was mainly associated with rocky plateau and the intermediate depth range (3–5 m), whereas S. maderensis, T. melanurus and Lipophrys trigloides inhabited preferentially the rocky cliffs. At microscale level, the habitat choice of the investigated species was almost entirely based on whether the substrate was either vegetated or composed of bare rock. T. delaisi and T. tripteronotus were associated with substrata covered by algae, whilst G. bucchichi, S. maderensis and T. melanurus preferred bare rock bottoms. In some species, the electivity indices for the less abundant type of cover, measured at different spatial scale, changed accordingly. For instance, the smaller the size of the sampled area, the higher was the intensity of the association between G. bucchichi and Anemonia viridis.