Moult cycle specific differential gene expression profiling of the crab Portunus pelagicus

Background: Crustacean moulting is a complex process involving many regulatory pathways. A holistic approach to examine differential gene expression profiles of transcripts relevant to the moulting process, across all moult cycle stages, was used in this study. Custom cDNA microarrays were constructed for Portunus pelagicus. The printed arrays contained 5000 transcripts derived from both the whole organism, and from individual organs such as the brain, eyestalk, mandibular organ and Y-organ from all moult cycle stages.Results: A total of 556 clones were sequenced from the cDNA libraries used to construct the arrays. These cDNAs represented 175 singletons and 62 contigs, resulting in 237 unique putative genes. The gene sequences were classified into the following biological functions: cuticular proteins associated with arthropod exoskeletons, farnesoic acid O-methyltransferase (FaMeT), proteins belonging to the hemocyanin gene family, lectins, proteins relevant to lipid metabolism, mitochondrial proteins, muscle related proteins, phenoloxidase activators and ribosomal proteins. Moult cycle-related differential expression patterns were observed for many transcripts. Of particular interest were those relating to the formation and hardening of the exoskeleton, and genes associated with cell respiration and energy metabolism.Conclusions: The expression data presented here provide a chronological depiction of the molecular events associated with the biological changes that occur during the crustacean moult cycle. Tracing the temporal expression patterns of a large variety of transcripts involved in the moult cycle of P. pelagicus can provide a greater understanding of gene function, interaction, and regulation of both known and new genes with respect to the moulting process

Ash1 and Tup1 dependent repression of the Saccharomyces cerevisiae HO promoter requires activator-dependent nucleosome eviction.

Transcriptional regulation of the Saccharomyces cerevisiae HO gene is highly complex, requiring a balance of multiple activating and repressing factors to ensure that only a few transcripts are produced in mother cells within a narrow window of the cell cycle. Here, we show that the Ash1 repressor associates with two DNA sequences that are usually concealed within nucleosomes in the HO promoter and recruits the Tup1 corepressor and the Rpd3 histone deacetylase, both of which are required for full repression in daughters. Genome-wide ChIP identified greater than 200 additional sites of co-localization of these factors, primarily within large, intergenic regions from which they could regulate adjacent genes. Most Ash1 binding sites are in nucleosome depleted regions (NDRs), while a small number overlap nucleosomes, similar to HO. We demonstrate that Ash1 binding to the HO promoter does not occur in the absence of the Swi5 transcription factor, which recruits coactivators that evict nucleosomes, including the nucleosomes obscuring the Ash1 binding sites. In the absence of Swi5, artificial nucleosome depletion allowed Ash1 to bind, demonstrating that nucleosomes are inhibitory to Ash1 binding. The location of binding sites within nucleosomes may therefore be a mechanism for limiting repressive activity to periods of nucleosome eviction that are otherwise associated with activation of the promoter. Our results illustrate that activation and repression can be intricately connected, and events set in motion by an activator may also ensure the appropriate level of repression and reset the promoter for the next activation cycle

Construction of a dimeric form of glutamate dehydrogenase from Clostridium symbiosum by site-directed mutagenesis

By using site-directed mutagenesis, Phe-187, one of the amino-acid residues involved in hydrophobic interaction between the three identical dimers comprising the hexamer of Clostridium symbiosum glutamate dehydrogenase (GDH), has been replaced by an aspartic acid residue, Over-expression in Escherichia coli led to production of large amounts of a soluble protein which, though devoid of GDH activity, showed the expected subunit M(r) on SDS-PAGE, and cross-reacted with an anti-GDH antibody preparation in Western blots, The antibody was used to monitor purification of the inactive protein, F187D GDH showed altered mobility on non-denaturing electrophoresis, consistent with changed size and/or surface charge. Gel filtration on a calibrated column indicated an M(r) of 87 000 +/- 3000. The mutant enzyme did not bind to the dye column routinely used in preparing wild-type GDH. Nevertheless suspicions of major misfolding were allayed by the results of chemical modification studies: as with wild-type GDH, NAD(+) completely protected one-SH group against modification by DTNB, implying normal coenzyme binding. A significant difference, however, is that in the mutant enzyme both cysteine groups were modified by DTNB, rather than C320 only. The CD spectrum in the far-UV region indicated no major change in secondary structure in the mutant protein. The near-UV CD spectrum, however, was less intense and showed a pronounced Phe contribution, possibly reflecting the changed environment of Phe-199, which would be buried in the hexamer. Sedimentation velocity experiments gave corrected coefficients s(20,w) of 11.08 S and 5.29 S for the wild-type and mutant proteins. Sedimentation equilibrium gave weight average molar masses M(r,app) of 280 000 +/- 5000 g/mol, consistent with the hexameric structure for the wild-type protein and 135 000 +/- 3000 g/mol for F187D. The value for the mutant is intermediate between the values expected for a dimer (98 000) and a trimer (147 000). To investigate the basis of this, sedimentation equilibrium experiments were performed over a range of protein concentrations. M(r,app) showed a linear dependence on concentration and a value of 108 118 g/mol at infinite dilution. This indicates a rapid equilibrium between dimeric and hexameric forms of the mutant protein with an equilibrium constant of 0.13 1/g. An independent analysis of the radial absorption scans with Microcal Origin software indicated a threefold association constant of 0.11 1/g, Introduction of the F187D mutation thus appears to have been successful in producing a dimeric GDH species, Since this protein is inactive it is possible that activity requires subunit interaction around the 3-fold symmetry axis. On the other hand this mutation may disrupt the structure in a way that cannot be extrapolated to other dimers. This issue can only be resolved by making alternative dimeric mutants

Use of chemical modification in the crystallization of isocitrate lyase from Escherichia coli

Two different crystal forms of isocitrate lyase (ICL) from Escherichia coli have been grown following the chemical modification of the enzyme by either 3-bromopyruvate or ethyl mercuri thiosalicylate (EMTS) contrasting strongly with difficulties in obtaining ordered crystals of the native enzyme. Both crystal forms are obtained using the hanging drop method of vapour diffusion with ammonium sulphate as the precipitant. The crystals diffract well and X-ray photographs of the crystals have established that they are in space groups C2221 and P31 (or its enantiomorph P32) respectively. Considerations of the values of Vm and measurements on the crystal density indicate that the asymmetric unit of both crystals contains four subunits

Light aerobic exercise modulates executive function and cortical excitability

Single bouts of aerobic exercise can modulate cortical excitability and executive cognitive function, but less is known about the effect of light‐intensity exercise, an intensity of exercise more achievable for certain clinical populations. Fourteen healthy adults (aged 22 to 30) completed the following study procedures twice (≥7 days apart) before and after 30 min of either light aerobic exercise (cycling) or seated rest: neurocognitive battery (multitasking performance, inhibitory control and spatial working memory), paired‐pulse TMS measures of cortical excitability. Significant improvements in response times during multitasking performance and increases in intracortical facilitation (ICF) were seen following light aerobic exercise. Light aerobic exercise can modulate cortical excitability and some executive function tasks. Populations with deficits in multitasking ability may benefit from this intervention. Light intensity aerobic exercise, suited to populations who may be unable to exercise at higher intensities can modulate multitasking performance and cortical excitability in a facilitative direction. Consistent with previous research, however, this intensity of exercise does not appear to modulate widespread executive functions