The evolution of the vertebrate mitochondrial genome has been the focus of numerous genetic and evolutionary studies over the last several decades. Initially, sampling was heavily biased toward taxonomic orders of greatest economic or health importance, but recent advances in DNA sequencing technology have facilitated a much broader phylogenetic sampling from which we can clarify general evolutionary trends such as patterns of gene rearrangement. Toward this end, we performed a comparative genomic analysis of the 2,831 vertebrate mitochondrial genomes representing 12 classes that are available in the NCBI database. Using a combination of bioinformatics methods, we determined that there is a great variation in the proportion of rearrangement by gene and by taxonomic class, with higher rates being observed in Reptilia, Amphibia, Petromyzonti, Mammalia, and Actinopteri. Further, within each class, there is large variation in proportion of reorganization among different orders or even taxonomic families. Eleven events of convergence in the genic order among different taxonomic orders were determined, most of them not previously reported.
Early-stage antibody discovery and engineering typically require the cloning, expression, and screening of large numbers of proteins. Normally, DNA fragments encoding proteins of interest are cloned into extra-chromosomal plasmids that are amplified in Escherichia coli . Following purification from the bacteria, the plasmids are introduced into appropriate cells, and the expressed recombinant proteins screened for desired binding or function in a high-throughput manner. Even in a 96-well plate format, plasmid purification from E. coli is typically a labor intensive and time-consuming process. To further accelerate our existing biotherapeutic discovery workflows we designed, qualified, and enabled a fully integrated high-throughput plasmid purification and quantification workstation which we have termed AMPS (Automated Miniprep Plasmid Station). Using components from a commercially available kit, AMPS can purify plasmid preparations from twenty 96deep-well plates of E. coli cultures, measure DNA absorbance at 260 nm, calculate plasmid concentrations, and prepare 96-deep-well plates for mammalian expression in an operator-independent manner. Plasmid yields and concentrations are equivalent to those obtained off-line. Furthermore, the quality of the DNA purified on the AMPS is equivalent to that obtained off-line in terms of DNA topology, and absence of contaminating bacterial chromosomal DNA and RNA. Most importantly, plasmids purified on the AMPS provide similar antibody titers following transfection in CHO cells as plasmids purified off-line. The AMPS bridges high-throughput E. coli colony picking capabilities typically available in an automation lab with downstream CHO expression needs and will facilitate screening of large numbers of biotherapeutics in binding and cell assay screens.
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