Advantages of q-PCR as a method of screening for gene targeting in mammalian cells using conventional and whole BAC-based constructs

We evaluate here the use of real-time quantitative PCR (q-PCR) as a method for screening for homologous recombinants generated in mammalian cells from either conventional gene-targeting constructs or whole BAC-based constructs. Using gene-targeted events at different loci, we show that q-PCR is a highly sensitive and accurate method for screening for conventional gene targeting that can reduce the number of clones requiring follow-up screening by Southern blotting. We further compared q-PCR to fluorescent in situ hybridization (FISH) for the detection of gene-targeting events using full-length BAC-based constructs designed to introduce mutations either into one gene or simultaneously into two adjacent genes. We find that although BAC-based constructs appeared to have high rates of homologous recombination when evaluated by FISH, screening by FISH was prone to false positives that were detected by q-PCR. Our results demonstrate the utility of q-PCR as a screening tool for gene targeting and further highlight potential problems with the use of whole BAC-based constructs for homologous recombination.     

Rheology and gelation of water-insoluble dextran from Leuconostoc mesenteroides NRRL B-523

Dynamic oscillatory testing has been used to study the rheology of water-insoluble dextran. The rheological properties (storage and loss moduli) of dextran gel were measured and dextran was found to be neither a strong gel nor a weak gel, but an entanglement network at a concentration of 250 mg/ml. The extent of gelation, illustrated by the gel elastic modulus G′, is found to decrease with increasing concentration of calcium ions. This was confirmed by shift of crossover frequencies towards higher values on the dynamic spectra and lower yield stress τ values obtained from stress ramp experiments. Finally, a comparison between gelation of dextran and alginate (a similar biopolymer) was made for clear understanding of effect of calcium ions on the dextran gelation.     

Aerobic reduction of manganese oxide by Salmonella sp. strain MR4

A new isolate of Salmonella, strain MR4, reduced Mn(IV)O₂ at 2.3 mM under aerobic conditions by about 83% over 24 h. Direct contact of cells to MnO₂ was not necessary as the cell-free spent medium produced a similar amount of Mn(II). Pyruvate (1.6 mM) and oxalate (0.8 mM) were identified in the culture medium and presumed to have a role in Mn(II) production in this microorganism.     

Properties of photoreceptor-specific phospholipase C encoded by the norpA gene of Drosophila melanogaster.

Mutations in the norpA gene drastically affect the phototransduction process in Drosophila. To study the biochemical characteristics of the norpA protein and its cellular and subcellular distributions, we have generated antisera against the major gene product of norpA. The antisera recognize an eye-specific protein of 130-kDa relative molecular mass that is present in wild-type head extracts but not in those of strong norpA mutants. The protein is associated with membranes and can be extracted with high salt. Immunohistochemical analysis at the light and electron microscopic levels indicates that the protein is expressed in all adult photoreceptor cells and specifically localized within the rhabdomeres, preferentially adjacent to, but not within, the rhabdomeric membranes. The results of the present study strongly support the previous suggestion that the norpA gene encodes the major phosphoinositol-specific phospholipase C in the photoreceptors. Moreover, insofar as the rhabdomeres are specialized structures for photoreception and phototransduction, specific localization of the norpA protein within these structures, in close association with the membranes, is consistent with the proposal that it has an important role in phototransduction.     

Behavioural variations among strains of Trichogramma spp.: Adaptability to field-temperature conditions

In order to select a candidate strain of Trichogramma sp. for inundative releases against lepidopteran pests in cabbage field-crops in the Netherlands, the parasitization activity of a collection of 60 different Trichogramma spp. strains was studied during 2 h and 24 h exposures at 12°C. Activity and parasitism varied significantly among strains and the two characteristics were not correlated, suggesting the action of two differentially temperature-dependent mechanisms influencing both characteristics. Native strains were characterized by a low activity at 12°C, making their usefulness for inundative releases doubtful. In three strains tested at 12, 17, 20, 25 and 30°C, activity increased linearly with temperature, until nearly all females parasitized at 20 or 25°C. Parasitism increased with increasing temperature to a maximum at 20–25°C and declined at 30°C. Handling time decreased asymptotically with increasing temperature. The results of this study suggest that Trichogramma strains vary in adaptability to low temperature, making this characteristic a useful criterion for evaluation of candidate strains.

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Résumé Afin de sélectionner une souche de Trichogramma sp. (Hymenoptera, Trichogrammatidae) candidate pour des lâchés inondatifs contre les Lépidoptères pestes de chou aux Pays-Bas, l'activité de parasitisation d'une collection de 60 souches différentes de Trichogramma spp. a été étudiée à 12°C pendant des durées d'exposition de 2 h et 24 h. L'activité proportionnelle et le degré de parasitisation des femelles varient significativement selon des souches et ne sont par corrélés. Ceci suggère l'action des deux mécanismes distincts, dépendant de la température et contrôlant independamment chacun de ces processus. Les souches indigènes sont caractérisées par une faible activité à 12°C, mettant en doute leur utilisation pour les lâchés inondatifs. Dans 3 souches testées à 12, 17, 20, 25 et 30°C, l'activité augmente linéairement avec la température, jusqu'a ce que quasi toutes les femelles parasitent les oeufs hôtes à 20°C et 25°C. Le taux de parasitisation augmente avec la température atteignant un maximum à 20–25°C puis diminue à 30°C. Le temps de manipulation d'un oeuf hôte diminue asymptotiquement lorsque la température croît. Les résultats de cette étude suggèrent que la capacité d'adaptation aux températures basses varie selon les souches de Trichogramma. Cette caractéristique constitue un critère utile pour l'évaluation de souches candidates potentielles.