Cloning of Paracoccus cytochrome c oxidase subunit II

Cytochrome c oxidase from Paracoccus denitrificans is composed of two subunits, yet is active in both electron transport and proton translocation. A cloning approach and immunologic screening protocol is described for the isolation of the subunit II gene expressed in E. coli. DNA sequencing should establish the extent of homology to eukaryotic oxidase.     

Affinity purification of HIV-1 and HIV-2 proteases from recombinant E. coli strains using pepstatin-agarose

A procedure is described which employs pepstatin-agarose for the affinity purification of either HIV-1 or HIV-2 protease from two similar recombinant E. coli constructs that were developed for the expression of these enzymes. HIV-2 protease was routinely expressed at much higher levels than the HIV-1 enzyme and pepstatin-agarose was the only chromatography step required to isolate pure HIV-2 protease from crude bacterial lysates. A Mono S ionic exchange step following pepstatin-agarose chromatography was sufficient to bring the HIV-1 protease to homogeneity. Purification of either enzyme can be completed in several days yielding homogeneous preparations suitable for crystallization and other physical characterization.     

Statistical aspects of design and validation of microtitre-plate-based linear and non-linear parallel in vitro bioassays

Assay validation was performed using four consecutive experiments with the related statistical evaluation. A cell-based assay on microtitre plates measured repeatedly within 1 day and on consecutive days was chosen as the model. The following problems were addressed: (i) choosing an appropriate design on a plate to avoid heterogeneities, (ii) quantification of all sources of variability and (iii) selecting between linear and non-linear parallel line assays. A mixed model was used with the random factors: rows, columns and plates and fixed effect factors with either linear or non-linear parallel line models.     

Inference by Exclusion in Goffin Cockatoos (Cacatua goffini)

Inference by exclusion, the ability to base choices on the systematic exclusion of alternatives, has been studied in many nonhuman species over the past decade. However, the majority of methodologies employed so far are hard to integrate into a comparative framework as they rarely use controls for the effect of neophilia. Here, we present an improved approach that takes neophilia into account, using an abstract two-choice task on a touch screen, which is equally feasible for a large variety of species. To test this approach we chose Goffin cockatoos (Cacatua goffini), a highly explorative Indonesian parrot species, which have recently been reported to have sophisticated cognitive skills in the technical domain. Our results indicate that Goffin cockatoos are able to solve such abstract two-choice tasks employing inference by exclusion but also highlight the importance of other response strategies.     

Variable outcrossing and the genetic structure of predominantly self-pollinated species

Several studies of highly inbreeding plant species have revealed appreciable levels of heterozygosity, exceeding those anticipated from the mating system. Hence net heterozygote advantage has been postulated. An alternative proposal for the observed heterozygous excess is that it arises from fluctuating outcrossing rates. This study analyses the effects of variable outcrossing on heterozygosity and on the expected inbreeding, using simple models and simulations. Generally, estimates of observed heterozygosity are not seriously biased. However, estimates of the predicted fixation index, estimated from the current outcrossing rate, were biased upward, especially when both over-dominant selection operates and the distribution of outcrossing is markedly skewed. Such an upward bias would increase the difference between observed and expected heterozygosity. Even so, only a small portion of the observed excess heterozygosity in inbreeders is likely to be due to variable outcrossing. The bias can be effectively reduced by a few independent measurements of outcrossing in different generations.