An anchored restriction-mapping approach applied to the genetic analysis of the Anopheles gambiae malaria vector complex 1

We introduce here a simple approach for rapidly determining restriction maps for a number of regions of a genome; this involves "anchoring" a map with a rare restriction site (in this case the seldom-cutting EagI) followed by partial digestion of a frequent-cutting enzyme (e.g., Sau 3A). We applied this technology to five species of the Anopheles gambiae complex. In a single Southern blot we obtained about a 15-kb restriction map each for the mtDNA, rRNA gene, and a scnDNA region for each of five species. Phylogenetic analyses of these regions yield trees at odds with the more traditional chromosome inversion-based trees. The value of the approach for systematic purposes is the ease with which several large, independent regions of the genome can be quickly assayed for molecular variation.      

Glucose uptake rate as a tool to estimate hybridoma growth in a packed bed bioreactor

The immobilisation of cells in a perfusion culture allows to obtain a high cell concentration and an efficient removal of the catabolites without cell loss. A disadvantage of this system is that the cell density cannot be directly monitored. The cellular metabolism is just followed by online measurements of pH and dissolved oxygen (DO) and off-line determinations of residual metabolites. In this article, we report a high cell density achieved by the cultivation of a hybridoma in a bubble-column bioreactor filled with hollow glass cylinders. The parameters monitored during the cultivation were pH, temperature, DO, glucose, lactate and monoclonal antibody. The glucose uptake rate was used to estimate the cell concentration along the time. The maximum cell concentration calculated for the considered cultivation time was 2.7?×?10⁷ cell?·?ml^?1. The glucose concentration in the media decreased stepwise twice, causing a decrease on the specific growth rate, while maintaining high antibody productivity levels. Maximum monoclonal antibody productivity was 503?μg?·?l^?1?·?day^?1 and specific productivity, considering calculated cell density, was 0.019?ng?·?cell^?1?·?day^?1.     

A Comparison of the Use of In Vitro-Transcribed and Native rRNA for the Quantification of Microorganisms in the Environment

Abstract Nearly full-length, small subunit (SSU) rRNA was transcribed in vitro from clones of SSU rDNA genes. Comparing the use of in vitro-transcribed and native rRNA indicated that, when in vitro-transcribed rRNA was used as a standard for quantitative hybridizations with oligonucleotide probes, the population was consistently underestimated. The population abundance was expressed as a percentage of specific target SSU rRNA (determined with a specific oligonucleotide probe), relative to the total SSU rRNA (measured with a universal probe). Differences in hybridization signals could be related to specific probe target locations and rRNA denaturation conditions, suggesting that higher order structure is important in quantitative membrane hybridizations. Therefore, in vitro-transcribed rRNA cannot always be used for the absolute quantification of microbial populations, but can be employed as a standard to quantify shifts in population abundance over time, and to compare community structure in various environments.