Streamlined Purification of Plasmid DNA From Prokaryotic Cultures

We describe the complete process of AcroPrep Advance Filter Plates for 96 plasmid preparations, starting from prokaryotic culture and ending with high purity DNA. Based on multi-well filtration for bacterial lysate clearance and DNA purification, this method creates a streamlined process for plasmid preparation. Filter plates containing silica-based media can easily be processed by vacuum filtration or centrifuge to yield appreciable quantities of plasmid DNA. Quantitative analyses determine the purified plasmid DNA is consistently of high quality with average OD_260/280 ratios of 1.97. Overall, plasmid yields offer more pure DNA for downstream applications, such as sequencing and cloning. This streamlined method of using AcroPrep Advance Filter Plates allows for manual, semi-automated or fully-automated processing.     

CLAVATA2 forms a distinct CLE‐binding receptor complex regulating Arabidopsis stem cell specification

CLAVATA1 (CLV1), CLV2, CLV3, CORYNE (CRN), BAM1 and BAM2 are key regulators that function at the shoot apical meristem (SAM) of plants to promote differentiation by limiting the size of the organizing center that maintains stem cell identity in neighboring cells. Previous results have indicated that the extracellular domain of the receptor kinase CLV1 binds to the CLV3‐derived CLE ligand. The biochemical role of the receptor‐like protein CLV2 has remained largely unknown. Although genetic analysis suggested that CLV2, together with the membrane kinase CRN, acts in parallel with CLV1, recent studies using transient expression indicated that CLV2 and CRN from a complex with CLV1. Here, we report detection of distinct CLV2‐CRN heteromultimeric and CLV1‐BAM multimeric complexes in transient expression in tobacco and in Arabidopsis meristems. Weaker interactions between the two complexes were detectable in transient expression. We also find that CLV2 alone generates a membrane‐localized CLE binding activity independent of CLV1. CLV2, CLV1 and the CLV1 homologs BAM1 and BAM2 all bind to the CLV3‐derived CLE peptide with similar kinetics, but BAM receptors show a broader range of interactions with different CLE peptides. Finally, we show that BAM and CLV1 overexpression can compensate for the loss of CLV2 function in vivo. These results suggest two parallel ligand‐binding receptor complexes controlling stem cell specification in Arabidopsis.     

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Immunoglobulin carbohydrate requirement for formation of an IgG-IgG complex.

In addition to their Fc oligosaccharides, some immunoglobulin molecules have oligosaccharides linked to variable segments of H or L chains. These Fab oligosaccharides are potential determinants of antibody specificity. This possibility was considered in a study of the IgG antiglobulin from a patient with IgG-IgG complexes. F(ab')2 fragments of the antiglobulin retained the ability to form complexes with normal IgG as detected by analytical ultracentrifugation. Removal of F(ab')2 sialic acids by neuraminidase abolished complex formation. Recombination experiments further localized antiglobulin activity to the L chains. Antiglobulin activity of the recombinant molecules was shown by analytical ultracentrifugation and by column chromatography with molecules containing 125I-labeled L chains. L chains from the subject's IgG were enriched in sialic acids. Thus, a sialic acid-containing oligosaccharide on the L chain of this antiglobulin is required for its binding action.