Mapping the idiotopes of a monoclonal anti-myoglobin antibody with syngeneic monoclonal anti-idiotypic antibodies: detection of a common idiotope

A panel of syngeneic monoclonal anti-idiotypic antibodies was prepared by immunizing A.SW mice with keyhole limpet hemocyanin-coupled A.SW monoclonal anti-myoglobin (HAL 19, IgG1) and screening the cloned hybridomas for production of IgG2 binding to idiotype but not to certain other anti-myoglobin antibodies of the same subclass in an ELISA. With these antibodies, we identified three nonoverlapping idiotopes, based on three clusters of monoclonal anti-idiotopes that mutually inhibit within each cluster, but not between clusters (Cluster I: S2, S6, S8; Cluster II: S5, S7; Cluster III: S9). Only Cluster II antibodies block the binding of myoglobin to HAL 19 and so identify a binding site-related idiotope(s). Binding of both Cluster II monoclonals (S5 and S7) to Hal 19 is inhibited by a rabbit anti-idiotype that we previously reported detects a common cross-reactive anti-myoglobin idiotope in immune sera. However, only one of these, S7, and not S5, identifies an idiotope that is present on 20 to 30% of A.SW anti-myoglobin antibodies in immune sera and ascites. The panel of syngeneic monoclonal anti-idiotype antibodies also detects new idiotopes not detected by the rabbit anti-idiotype. The development of a panel of syngeneic monoclonal anti-idiotypic antibodies to different clusters of idiotopes on the same antibody molecule, including one that identifies a major common idiotope in immune sera, should allow the analysis of possible idiotype network regulation in vivo and in vitro in a completely syngeneic system.     

Population genetics and phylogenetics of DNA sequence variation at multiple loci within the Drosophila melanogaster species complex

Two regions of the genome, a 1-kbp portion of the zeste locus and a 1.1- kbp portion of the yolk protein 2 locus, were sequenced in six individuals from each of four species: Drosophila melanogaster, D. simulans, D. mauritiana, and D. sechellia. The species and strains were the same as those of a previous study of a 1.9-kbp region of the period locus. No evidence was found for recent balancing or directional selection or for the accumulation of selected differences between species. Yolk protein 2 has a high level of amino acid replacement variation and a low level of synonymous variation, while zeste has the opposite pattern. This contrast is consistent with information on gene function and patterns of codon bias. Polymorphism levels are consistent with a ranking of effective population sizes, from low to high, in the following order: D. sechellia, D. melanogaster, D.mauritiana, and D. simulans. The apparent species relationships are very similar to those suggested by the period locus study. In particular, D. simulans appears to be a large population that is still segregating variation that arose before the separation of D. mauritiana and D. sechellia. It is estimated that the separation of ancestral D. melanogaster from the other species occurred 2.5-3.4 Mya. The separations of D. sechellia and D. mauritiana from ancestral D. simulans appear to have occurred 0.58- 0.86 Mya, with D. mauritiana having diverged from ancestral D. simulans 0.1 Myr more recently than D. sechellia.      

Homeotic duplication of the pelvic body segment in regenerating tadpole tails induced by retinoic acid

 Homeosis, the ectopic formation of a body part, is one of the key phenomena that prompted the identification of the essential selector genes controlling body organization. Shared elements of such homeotic genes exist in all studied animal classes, but homeotic transformations of the same order of magnitude as in insects, such as the duplication of the thorax in Drosophila mutants, have not been described in vertebrates. Here we investigate the capacity of retinoic acid to modify tail regeneration in amphibians. We show that retinoic acid causes the formation of an additional body segment in regenerating tails of Rana temporaria tadpoles. A second pelvic section, including vertebral elements, pelvic girdle elements and limb buds, forms at the mid-tail level. This is the first report of a homeotic duplication of a whole body segment in vertebrate axial regeneration. Received: 16 August 1996 / Accepted: 20 September 1996     

Purification of glutamine synthetase from a variety of bacteria

We have developed two procedures which allow the very rapid purification of glutamine synthetase (GS) from a diverse variety of bacteria. The first procedure, based upon differential sedimentation, depends upon the association of GS with deoxyribonucleic acid in cell extracts. The second procedure, derived from the method of C. Gross et al (J. Bacteriol. 128:382-389, 1976) for purifying ribonucleic acid polymerase by polyethylene glycol (PEG) precipitation, enabled us to obtain high yields of GS from either small or large quantities of cells. We used the PEG procedure to purify GS from Klebsiella aerogenes, K. pneumoniae, Escherichia coli, Salmonella typhimurium, Rhizobium sp. strain 32H1, R. meliloti, Azotobacter vinelandii, Pseudomonas putida, Caulobacter crescentus, and Rhodopseudomonas capsulata. The purity of the GS obtained, judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was high, and in many instances only a single protein band was detected.     

The toad’s warts: Discordance creates bumpy expectations of mitochondrial‐nuclear evolution between species

Discordance between the mitochondrial and nuclear genomes is a prevalent phenomenon in nature, in which the underlying processes responsible are considered to be important in shaping genetic variation in natural populations. Among the evolutionary processes that best explain such genomic mismatches incomplete lineage sorting and introgression are commonly identified, however, many studies are unable to distinguish between these hypotheses, which has become a major challenge in the field. In this issue of Molecular Ecology, Firneno et al. (2020) present an elegant exploration of mitochondrial‐nuclear discordance in Mesoamerican toads. Integrating genome‐scale and spatial data to test between these hypotheses within an empirical model testing framework, they find strong support that incomplete lineage sorting explains the observed discordance. Their work, along with many previous articles in Molecular Ecology, highlights the commonality of mito‐nuclear discordance among species despite the expectations of tightly concerted mitochondrial and nuclear genome evolution. It is increasingly clear that the nuclear genomes of many species are (at least for short periods of evolutionary time) functionally compatible with multiple, divergent mitochondrial haplotypes. As such, we suggest future research not only seeks to understand the processes causing spatial mito‐nuclear discordance (e.g. incomplete lineage sorting, introgression), but also explores those that maintain discordance through time and space (e.g. relaxed selection on mito‐nuclear interactions, heterozygosity, population demographics). We also discuss the vital role that taxonomy plays in interpreting patterns of mito‐nuclear discordance when data‐consistent yet differing taxonomies are used, such as treating allopatrically distributed taxa as multiple isolated populations versus multiple micro‐endemic species.     

Self‐assembly of designed coiled coil peptides studied by small‐angle X‐ray scattering and analytical ultracentrifugation

α‐Helical coiled coil structures, which are noncovalently associated heptad repeat peptide sequences, are ubiquitous in nature. Similar amphipathic repeat sequences have also been found in helix‐containing proteins and have played a central role in de novo design of proteins. In addition, they are promising tools for the construction of nanomaterials. Small‐angle X‐ray scattering (SAXS) has emerged as a new biophysical technique for elucidation of protein topology. Here, we describe a systematic study of the self‐assembly of a small ensemble of coiled coil sequences using SAXS and analytical ultracentrifugation (AUC), which was correlated with molecular dynamics simulations. Our results show that even minor sequence changes have an effect on the folding topology and the self‐assembly and that these differences can be observed by a combination of AUC, SAXS, and circular dichroism spectroscopy. A small difference in these methods was observed, as SAXS for one peptide and revealed the presence of a population of longer aggregates, which was not observed by AUC. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Conformation-specific anti-Mad2 monoclonal antibodies for the dissection of checkpoint signaling

The spindle assembly checkpoint (SAC) ensures accurate chromosome segregation during mitosis by delaying the activation of the anaphase-promoting complex/cyclosome (APC/C) in response to unattached kinetochores. The Mad2 protein is essential for a functional checkpoint because it binds directly to Cdc20, the mitotic co-activator of the APC/C, thereby inhibiting progression into anaphase. Mad2 exists in at least 2 different conformations, open-Mad2 (O-Mad2) and closed-Mad2 (C-Mad2), with the latter representing the active form that is able to bind Cdc20. Our ability to dissect Mad2 biology in vivo is limited by the absence of monoclonal antibodies (mAbs) useful for recognizing the different conformations of Mad2. Here, we describe and extensively characterize mAbs specific for either O-Mad2 or C-Mad2, as well as a pan-Mad2 antibody, and use these to investigate the different Mad2 complexes present in mitotic cells. Our antibodies validate current Mad2 models but also suggest that O-Mad2 can associate with checkpoint complexes, most likely through dimerization with C-Mad2. Furthermore, we investigate the makeup of checkpoint complexes bound to the APC/C, which indicate the presence of both Cdc20-BubR1-Bub3 and Mad2-Cdc20-BubR1-Bub3 complexes, with Cdc20 being ubiquitinated in both. Thus, our defined mAbs provide insight into checkpoint signaling and provide useful tools for future research on Mad2 function and regulation.