Estimation of Förster's distance between two ends of Dps protein from mycobacteria: distance heterogeneity as a function of oligomerization and DNA binding

Dps protein (DNA binding Protein from Starved Cells) from Mycobacterium smegmatis (Ms-Dps) is known to undergo an in vitro irreversible oligomeric transition from trimer to dodecamer. This transition helps the protein to provide for bimodal protection to the bacterial DNA from the free radical and Fenton mediated damages in the stationary state. The protein exists as a stable trimer, when purified from E. coli cells transformed with an over-expression plasmid. Both trimer as well as dodecamer are known to exhibit ferroxidation activity, thus removing toxic hydroxyl radicals in vivo, whereas iron accumulation and non-sequence specific DNA binding activity are found in dodecamer only. This seems to be aided by the positively charged long C-terminal tail of the protein. We used frequency domain phase-modulation fluorescence spectroscopy and Förster Resonance Energy Transfer (FRET) to monitor this oligomeric switch from a trimer to a dodecamer and to elucidate the structure of DNA–Dps dodecamer complex. As Ms-Dps is devoid of any Cysteine residues, a Serine is mutated to Cysteine (S169C) at a position adjacent to the putative DNA binding domain. This Cysteine is subsequently labeled with fluorescent probe and another probe is placed at the N-terminus, as crystal structure of the protein reveals several side-chain interactions between these two termini, and both are exposed towards the surface of the protein. Here, we report the Förster's distance distribution in the trimer and the dodecamer in the presence and absence of DNA. Through discrete lifetime analysis of the probes tagged at the respective regions in the macromolecule, coupled with Maximum Entropy Method (MEM) analysis, we show that the dodecamer, upon DNA binding shows conformational heterogeneity in overall structure, perhaps mediated by a non-specific DNA–protein interaction. On the other hand, the nature of DNA–Dps interaction is not known and several models exist in literature. We show here with the help of fluorescence anisotropy measurements of labeled DNA having different length and unlabeled native dodecameric protein that tandem occupation of DNA binding sites by a series of Dps molecules perhaps guide the tight packing of Dps over DNA backbone.     

Patterns of gene duplication and intron loss in the ENCODE regions suggest a confounding factor

The exon-intron structure of eukaryotic genes allows for phenomena such as alternative splicing, nonsense-mediated decay, and regulation through untranslated regions. However, the evolution of the exon structure of genes is not well elucidated because of limited and phylogenetically sparse data sets. In this study, we use the phylogenetically diverse sequencing of the ENCODE regions to study gene structure evolution in mammalian genomes. This first phylogenetically diverse study of gene structure changes offers insights into the mode and tempo of mammalian gene structure evolution. The genes undergoing structure changes appear to be moderately to highly expressed in germline cells and show levels of selection similar to those of other ENCODE genes. Patterns of gene duplication of the affected genes are more complex than expected. The number of sampled genomes is sufficiently dense to infer that certain gene duplications happened after intron loss. Thus, although gene duplication is highly correlated with intron loss, we conclude that structural changes in genes are not necessarily due to a loss of constraint following gene duplication as previously suggested.     

Recent Evolutionary History of Tigers Highlights Contrasting Roles of Genetic Drift and Selection

Species conservation can be improved by knowledge of evolutionary and genetic history. Tigers are among the most charismatic of endangered species and garner significant conservation attention. However, their evolutionary history and genomic variation remain poorly known, especially for Indian tigers. With 70% of the world’s wild tigers living in India, such knowledge is critical. We re-sequenced 65 individual tiger genomes representing most extant subspecies with a specific focus on tigers from India. As suggested by earlier studies, we found strong genetic differentiation between the putative tiger subspecies. Despite high total genomic diversity in India, individual tigers host longer runs of homozygosity, potentially suggesting recent inbreeding or founding events, possibly due to small and fragmented protected areas. We suggest the impacts of ongoing connectivity loss on inbreeding and persistence of Indian tigers be closely monitored. Surprisingly, demographic models suggest recent divergence (within the last 20,000 years) between subspecies and strong population bottlenecks. Amur tiger genomes revealed the strongest signals of selection related to metabolic adaptation to cold, whereas Sumatran tigers show evidence of weak selection for genes involved in body size regulation. We recommend detailed investigation of local adaptation in Amur and Sumatran tigers prior to initiating genetic rescue.     

Tyrosine residues 654 and 670 in beta-catenin are crucial in regulation of Met-beta-catenin interactions

beta-Catenin, a key component of the canonical Wnt pathway, is also regulated by tyrosine phosphorylation that regulates its association to E-cadherin. Previously, we reported its association with the hepatocyte growth factor (HGF) receptor Met at the membrane. HGF induced Met-beta-catenin dissociation and nuclear translocation of beta-catenin, which was tyrosine-phosphorylation-dependent. Here, we further investigate the Met-beta-catenin interaction by selectively mutating several tyrosine residues, alone or in combination, in beta-catenin. The mutants were subcloned into FLAG-CMV vector and stably transfected into rat hepatoma cells, which were treated with HGF. All single or double-mutant-transfected cells continued to show HGF-induced nuclear translocation of FLAG-beta-catenin except the mutations affecting 654 and 670 simultaneously (Y654/670F), which coincided with the lack of formation of beta-catenin-TCF complex and DNA synthesis, in response to the HGF treatment. In addition, the Y654/670F-transfected cells also showed no phosphorylation of beta-catenin or dissociation from Met in response to HGF. Thus, intact 654 and 670 tyrosine residues in beta-catenin are crucial in HGF-mediated beta-catenin translocation, activation and mitogenesis.     

Diversity in germination of seeds inCalotropis procera R.Br. population

Summary Phenotypically there appeared to exist no diversity in different individual plants in a population ofCalotropis procera. The diversity in imbibition and germination behaviour of seeds from different plants was revealed only after detailed studies were undertaken. Seeds from six individual plants were collected and stored separately for a period of two years. Ordinarily the seeds of this species did not exhibit any dormancy and were found to germinate immediately after harvest. Two years old seeds did not loose their viability in any of the six lots indicated above. It was discovered that the weight and size of seeds, the rate of imbibition of water, the amount of water imbibed, the loss of water upon drying, the rate of germination, the percentage of germination, and the germination behaviour in continuous illumination and total darkness, all exhibited diversity to some extent in the six lots studied. It was evident that there did not exist seeds exhibiting homogeneous behaviour in a population ofCalotropis procera.     

Ontogenetic allometry underlies trophic diversity in sea turtles (Chelonioidea)

Evolutionary Ecology - Despite only comprising seven species, extant sea turtles (Cheloniidae and Dermochelyidae) display great ecological diversity, with most species inhabiting a unique dietary...     

Evaluating the design of flexible manufacturing systems

Evaluating the design of flexible manufacturing systems is complex. Developing a measure of performance useful for evaluating alternate designs continues to be interesting. Here, total productivity of the system is proposed as an appropriate measure. Specification of parameters based upon strategic considerations for this measure are discussed. Finally, the usefulness of the measure is demonstrated through an example.