A single mutation affects L-serine deaminase, L-leucyl-, L-phenylalanyl-tRNA protein transferase, and proline oxidase activity in Escherichia coli K-12.

A mutation at a single locus, wyb, results in several phenotypic changes in Escherichia coli K-12. The Wyb- phenotype includes: (i) an increase in L-serine deaminase activity, together with a loss of inducibility by L-leucine; (ii) an absence of L-leucyl-, L-phenylalanyl-tRNA protein transferase activity; (iii) inducibility of proline oxidase by proline; and (iv) a loss of ability to use maltose as a carbon and energy source.     

Specific adsorption of starch oligosaccharides in the gel phase of starch granules

The gel phase of native starch-granules is penetrable by such low-molecular-weight solutes as oligosaccharides, amino acids, and salts [Lathe and Ruthven, Biochem. J., 62 (1956) 665]. Molecules larger than about 1000 daltons are effectively excluded. Starch oligosaccharides (maltotriose through maltoheptaose and perhaps higher) exhibit anomalous behavior in that they are taken up by the gel phase far in excess of the amount expected on the basis of their molecular size. Adsorption was measured by using radioactive starch oligosaccharides and counting weighed amounts of solution before and after equilibration with starch granules. The measurements were corrected for water sorption by the starch granules and for exclusion effects as ascertained by controls with nonstarch types of oligosaccharides. Maximum adsorption was observed with maltotetraose. The results indicate a specific binding between the starch oligosaccharides and molecular chains in the starch, presumably those chains in the gel phase. We suggest that these chains constitute interbranch regions of branched molecules, or segments of linear molecules in the gel or amorphous phase, the segments being of sufficient length to form a double helix or other association with the linear oligosaccharides.     

MaxHiC: A robust background correction model to identify biologically relevant chromatin interactions in Hi-C and capture Hi-C experiments

Hi-C is a genome-wide chromosome conformation capture technology that detects interactions between pairs of genomic regions and exploits higher order chromatin structures. Conceptually Hi-C data counts interaction frequencies between every position in the genome and every other position. Biologically functional interactions are expected to occur more frequently than transient background and artefactual interactions. To identify biologically relevant interactions, several background models that take biases such as distance, GC content and mappability into account have been proposed. Here we introduce MaxHiC, a background correction tool that deals with these complex biases and robustly identifies statistically significant interactions in both Hi-C and capture Hi-C experiments. MaxHiC uses a negative binomial distribution model and a maximum likelihood technique to correct biases in both Hi-C and capture Hi-C libraries. We systematically benchmark MaxHiC against major Hi-C background correction tools including Hi-C significant interaction callers (SIC) and Hi-C loop callers using published Hi-C, capture Hi-C, and Micro-C datasets. Our results demonstrate that 1) Interacting regions identified by MaxHiC have significantly greater levels of overlap with known regulatory features (e.g. active chromatin histone marks, CTCF binding sites, DNase sensitivity) and also disease-associated genome-wide association SNPs than those identified by currently existing models, 2) the pairs of interacting regions are more likely to be linked by eQTL pairs and 3) more likely to link known regulatory features including known functional enhancer-promoter pairs validated by CRISPRi than any of the existing methods. We also demonstrate that interactions between different genomic region types have distinct distance distributions only revealed by MaxHiC. MaxHiC is publicly available as a python package for the analysis of Hi-C, capture Hi-C and Micro-C data.     

Development of a 16S rRNA gene-based prototype microarray for the detection of selected actinomycetes genera

Actinomycetes are known for their secondary metabolites, which have been successfully used as drugs in human and veterinary medicines. However, information on the distribution of this group of Gram-positive bacteria in diverse ecosystems and a comprehension of their activities in ecosystem processes are still scarce. We have developed a 16S rRNA-based taxonomic microarray that targets key actinomycetes at the genus level. In total, 113 actinomycete 16S rRNA probes, corresponding to 55 of the 202 described genera, were designed. The microarray accuracy was evaluated by comparing signal intensities with probe/target-weighted mismatch values and the Gibbs energy of the probe/target duplex formation by hybridizing 17 non-actinomycete and 29 actinomycete strains/clones with the probe set. The validation proved that the probe set was specific, with only 1.3% of false results. The incomplete coverage of actinomycetes by a genus-specific probe was caused by the limited number of 16S rRNA gene sequences in databases or insufficient 16S rRNA gene polymorphism. The microarray enabled discrimination between actinomycete communities from three forest soil samples collected at one site. Cloning and sequencing of 16S rRNA genes from one of the soil samples confirmed the microarray results. We propose that this newly constructed microarray will be a valuable tool for genus-level comparisons of actinomycete communities in various ecological conditions.     

The chaperonin TRiC controls polyglutamine aggregation and toxicity through subunit-specific interactions

Misfolding and aggregation of proteins containing expanded polyglutamine repeats underlie Huntington's disease and other neurodegenerative disorders. Here, we show that the hetero-oligomeric chaperonin TRiC (also known as CCT) physically interacts with polyglutamine-expanded variants of huntingtin (Htt) and effectively inhibits their aggregation. Depletion of TRiC enhances polyglutamine aggregation in yeast and mammalian cells. Conversely, overexpression of a single TRiC subunit, CCT1, is sufficient to remodel Htt-aggregate morphology in vivo and in vitro, and reduces Htt-induced toxicity in neuronal cells. Because TRiC acts during de novo protein biogenesis, this chaperonin may have an early role preventing Htt access to pathogenic conformations. Based on the specificity of the Htt-CCT1 interaction, the CCT1 substrate-binding domain may provide a versatile scaffold for therapeutic inhibitors of neurodegenerative disease.     

Exonuclease V from Saccharomyces cerevisiae. A 5'----3'-deoxyribonuclease that produces dinucleotides in a sequential fashion

A novel deoxyribonuclease, exonuclease V, has been purified approximately 30,000-fold from Saccharomyces cerevisiae. Exonuclease V is localized in the nucleus. The nuclease degrades single-stranded, but not double-stranded, DNA from the 5'-end. The products of exonuclease action are dinucleotides, except the 3'-terminal tri- and tetranucleotides which are not degraded. Studies with synthetic oligo- and polynucleotides with specified sequence elements showed that exonuclease V cleaves off dinucleotides as primary digestion products. Thus, the polymers (pT)9pA(pT)n and (pT)10pA(pT)n yielded pTpA and pApT as digestion products, respectively. Removal of the 5'-terminal phosphate from the DNA substrate results in reduced binding of the enzyme to the substrate. In addition, the initial hydrolytic cut by exonuclease V on the dephosphorylated substrate produces a mixture of dinucleoside monophosphates and trinucleoside diphosphates. The enzyme is processive in action.     

A Bacterial Ras-Like Small GTP-Binding Protein and Its Cognate GAP Establish a Dynamic Spatial Polarity Axis to Control Directed Motility

Regulated cell polarity is central to many cellular processes. We investigated the mechanisms that govern the rapid switching of cell polarity (reversals) during motility of the bacterium Myxococcus xanthus. Cellular reversals are mediated by pole-to-pole oscillations of motility proteins and the frequency of the oscillations is under the control of the Frz chemosensory system. However, the molecular mechanism that creates dynamic polarity remained to be characterized. In this work, we establish that polarization is regulated by the GTP cycle of a Ras-like GTPase, MglA. We initially sought an MglA regulator and purified a protein, MglB, which was found to activate GTP hydrolysis by MglA. Using live fluorescence microscopy, we show that MglA and MglB localize at opposite poles and oscillate oppositely when cells reverse. In absence of MglB, MglA-YFP accumulates at the lagging cell end, leading to a strikingly aberrant reversal cycle. Spatial control of MglA is achieved through the GAP activity of MglB because an MglA mutant that cannot hydrolyze GTP accumulates at the lagging cell end, despite the presence of MglB. Genetic and cell biological studies show that the MglA-GTP cycle controls dynamic polarity and the reversal switch. The study supports a model wherein a chemosensory signal transduction system (Frz) activates reversals by relieving a spatial inhibition at the back pole of the cells: reversals are allowed by Frz-activated switching of MglB to the opposite pole, allowing MglA-GTP to accumulate at the back of the cells and create the polarity switch. In summary, our results provide insight into how bacteria regulate their polarity dynamically, revealing unsuspected conserved regulations with eukaryots.