The genome of an Encephalitozoon cuniculi type III strain reveals insights into the genetic diversity and mode of reproduction of a ubiquitous vertebrate pathogen.

Encephalitozoon cuniculi is a model microsporidian species with a mononucleate nucleus and a genome that has been extensively studied. To date, analyses of genome diversity have revealed the existence of four genotypes in E. cuniculi (EcI, II, III and IV). Genome sequences are available for EcI, II and III, and are all very divergent, possibly diploid and genetically homogeneous. The mechanisms that cause low genetic diversity in E. cuniculi (for example, selfing, inbreeding or a combination of both), as well as the degree of genetic variation in their natural populations, have been hard to assess because genome data have been so far gathered from laboratory-propagated strains. In this study, we aim to tackle this issue by analyzing the complete genome sequence of a natural strain of E. cuniculi isolated in 2013 from a steppe lemming. The strain belongs to the EcIII genotype and has been designated EcIII-L. The EcIII-L genome sequence harbors genomic features intermediate to known genomes of II and III lab strains, and we provide primers that differentiate the three E. cuniculi genotypes using a single PCR. Surprisingly, the EcIII-L genome is also highly homogeneous, harbors signatures of heterozygosity and also one strain-specific single-nucleotide polymorphism (SNP) that introduces a stop codon in a key meiosis gene, Spo11. Functional analyses using a heterologous system demonstrate that this SNP leads to a deficient meiosis in a model fungus. This indicates that EcIII-L meiotic machinery may be presently broken. Overall, our findings reveal previously unsuspected genome diversity in E. cuniculi, some of which appears to affect genes of primary importance for the biology of this pathogen.

Impact of sea-level rise on cross-shore sediment transport on fetch-limited barrier reef island beaches under modal and cyclonic conditions.

A one-dimensional wave model is combined with an analytical sediment transport model to investigate the likely influence of sea-level rise on net cross-shore sediment transport on fetch-limited barrier reef and lagoon island beaches. The modelling considers if changes in the nearshore wave height and wave period in the lagoon induced by different water levels over the reef flat are likely to lead to net offshore or onshore movement of sediment. The results indicate that the effects of SLR on net sediment movement are highly variable and controlled by the bathymetry of the reef and lagoon. A significant range of reef-lagoon bathymetry, and notably shallow and narrow reefs, appears to lead hydrodynamic conditions and beaches that are likely to be stable or even accrete under SLR. Loss of reef structural complexity, particularly on the reef flat, increases the chance of sediment transport away from beaches and offshore.

Impact of sea-level rise and coral mortality on the wave dynamics and wave forces on barrier reefs.

A one-dimensional wave model was used to investigate the reef top wave dynamics across a large suite of idealized reef-lagoon profiles, representing barrier coral reef systems under different sea-level rise (SLR) scenarios. The modeling shows that the impacts of SLR vary spatially and are strongly influenced by the bathymetry of the reef and coral type. A complex response occurs for the wave orbital velocity and forces on corals, such that the changes in the wave dynamics vary reef by reef. Different wave loading regimes on massive and branching corals also leads to contrasting impacts from SLR. For many reef bathymetries, wave orbital velocities increase with SLR and cyclonic wave forces are reduced for certain coral species. These changes may be beneficial to coral health and colony resilience and imply that predicting SLR impacts on coral reefs requires careful consideration of the reef bathymetry and the mix of coral species.

Determining nutrients degradation kinetics of chickpea (Cicer arietinum) straw using nylon bag technique in sheep.

Straw a by-product from grain legume crops is produced in large quantities in Iran. Straw is constant component of ruminant diets on small holder farms; however, there is little information about its nutritive value. Accordingly experiment was conducted to determine the chemical composition and ruminal organic matter (OM) and crude protein (CP) degradability of chickpea straw using nylon bags (in situ) technique. Replicated samples were incubated at 0, 2, 4, 8, 12, 24, 48 and 72 hours in three rumen canulated Ghezel rams with 50±3 kg body weight. Dry matter (DM), CP, ether extract (EE), OM, crude fiber (CF) and nitrogen free extract (NFE) content of chickpea straws were 92.2, 6.1, 5.5, 92.0, 34.3 and 46.2%, respectively. The soluble fraction (a) of the OM and CP of chickpea straw was 17.5 and 40.8% and potential degradability (a+b) of OM and CP was 56.7 and 72.0%, respectively. Effective degradability at different passage rates (2, 5 and 8% per hours) for OM was 51.0 44.9 and 40.7% and for CP were 68.4, 64.3 and 61.3%, respectively. In conclusion, based on chemical composition and degradation characteristics, chickpea straw could have moderate nutritive value for ruminants.

Global investigation of protein-protein interactions in yeast Saccharomyces cerevisiae using re-occurring short polypeptide sequences.

Protein-protein interaction (PPI) maps provide insight into cellular biology and have received considerable attention in the post-genomic era. While large-scale experimental approaches have generated large collections of experimentally determined PPIs, technical limitations preclude certain PPIs from detection. Recently, we demonstrated that yeast PPIs can be computationally predicted using re-occurring short polypeptide sequences between known interacting protein pairs. However, the computational requirements and low specificity made this method unsuitable for large-scale investigations. Here, we report an improved approach, which exhibits a specificity of approximately 99.95% and executes 16,000 times faster. Importantly, we report the first all-to-all sequence-based computational screen of PPIs in yeast, Saccharomyces cerevisiae in which we identify 29,589 high confidence interactions of approximately 2 x 10(7) possible pairs. Of these, 14,438 PPIs have not been previously reported and may represent novel interactions. In particular, these results reveal a richer set of membrane protein interactions, not readily amenable to experimental investigations. From the novel PPIs, a novel putative protein complex comprised largely of membrane proteins was revealed. In addition, two novel gene functions were predicted and experimentally confirmed to affect the efficiency of non-homologous end-joining, providing further support for the usefulness of the identified PPIs in biological investigations.

Roles of NCX and PMCA in basolateral calcium export associated with mineralization cycles and cold acclimation in crayfish.

Basolateral Na+/Ca2+ exchanger (NCX) and plasma membrane Ca2+ ATPase (PMCA) are the primary transmembrane proteins that export calcium (Ca2+) from cells. In our lab we use a nonmammalian animal model, the freshwater crayfish, to study cellular Ca2+ regulation. Two experimental conditions are employed to effect Ca2+ dyshomeostasis: (a) in the postmolt stage of the crustacean molting cycle increased unidirectional Ca2+ influx associated with cuticular mineralization is accompanied by elevated basolateral Ca2+ export (compared with intermolt Ca balance); and (b) exposure of the poikilothermic crayfish to cold acclimation (4 degrees C) causes influx of Ca2+ into cells, which is compensated by increased basolateral Ca2+ export (compared with exposure to 23 degrees C). This study compares expression of both NCX and PMCA mRNA (real-time PCR) and protein (Western) in both epithelial (kidney) and nonepithelial tissue (tail muscle) during elevated basolateral Ca2+ export. Both experimental treatments produced increases in NCX and PMCA expression (mRNA and protein) in both tissues. Mineralization produced greater upregulation of mRNA in kidney than in tail, whereas cold acclimation yielded comparable increases in both tissues. Protein expression patterns were generally confirmatory of real-time PCR data although expression changes were less pronounced. Both experimental treatments appear to increase basolateral Ca2+ export.

Molecular localization of a ribosome-dependent ATPase on Escherichia coli ribosomes.

We have previously isolated and described an Escherichia coli ribosome-bound ATPase, RbbA, that is required for protein synthesis in the presence of ATP, GTP and the elongation factors, EF-Tu and EF-G. The gene encoding RbbA, yhih, has been cloned and the deduced protein sequence harbors two ATP-motifs and one RNA-binding motif and is homologous to the fungal EF-3. Here, we describe the isolation and assay of a truncated form of the RbbA protein that is stable to overproduction and purification. Chemical protection results show that the truncated RbbA specifically protects nucleotide A937 on the 30S subunit of ribosomes, and the protected site occurs at the E-site where the tRNA is ejected upon A-site occupation. Other weakly protected bases in the region occur at or near the mRNA binding site. Using radiolabeled tRNAs, we study the stimulating effect of this truncated RbbA on the binding and release of different tRNAs bound to the (aminoacyl) A-, (peptidyl) P- and (exit) E-sites of 70S ribosomes. The combined data suggest plausible mechanisms for the function of RbbA in translation.

Epsilon as an initiator of translation of CAT mRNA in Escherichia coli.

Epsilon sequence (UUAACUUUA) has originally been found in the bacteriophage T7 gene 10 leader region. It enhances translation in Escherichia coli via base pairing with nucleotides 458-466 located in the helical domain #17 of 16S rRNA. We have recently reported that when the complementarity to 16S rRNA is extended, the epsilon is converted from an enhancer to an independent initiator of translation. Here we report the effect of two other structural parameters, positioning in mRNA and the degree of complementarity to 16S rRNA on the translation initiation activity of epsilon in E. coli cells. Our results show that epsilon displays maximal activity as a translational initiator at its natural 9-nucleotide-long complementarity to 16S rRNA and at a 16-nucleotide-long distance to the initiation codon. Under these conditions its efficiency is comparable with that of the consensus Shine-Dalgarno sequence.

Enhancing activity of epsilon in Escherichia coli and Agrobacterium tumefaciens cells.

Epsilon (epsilon) sequence is a bacterial enhancer of translation found in the bacteriophage T7 gene 10. It is believed that its enhancing effect of epsilon is due to a base-pairing with the nucleotides 458-467 from the helical domain 17 of Escherichia coli 16S rRNA. To prove this we have taken advantage of the difference of this domain in Agrobacterium tumefaciens and E. coli. To evaluate the significance of nucleotide complementarity for the enhancing activity of epsilon, a series of nucleotide sequences matching either E. coli or A. tumefaciens domain 17 are cloned in a binary expression vector in front of the chloramphenicol acetyltransferase (CAT) gene. The CAT assay shows that: (i) the epsilon in combination with an SD consensus sequence increases the yield of CAT in both microorganisms over that obtained with the SD alone; (ii) the epsilon sequence complementary to the A. tumefaciens domain 17 leads to a 2.71-fold increase in the yield of CAT in homologous cells but not in E. coli cells; (iii) the yield of CAT correlates with the free energy of base-pairing with the helical domain 17 in both microorganisms.

The low level expression of chloramphenicol acetyltransferase (CAT) mRNA in Escherichia coli is not dependent on either Shine-Dalgarno or the downstream boxes in the CAT gene.

Recent studies have shown that the canonical Shine-Dalgarno (SD)-anti-SD interaction is dispensable for the initiation of translation of certain mRNAs in Escherichia coli. Alternative non-SD sequences (located upstream from the initiation codon) and also downstream sequences ("downstream boxes") complementary to 16S rRNA were found to be involved in the initiation of translation of mRNAs devoid of either SD or any leader sequences. In this study the chloramphenicol acetyltransferase (CAT) gene was modified to remove the 5' terminal non-translated region and/or the two potential downstream boxes in the CAT gene. Thus a series of ten CAT gene constructs was created and expressed in E. coli under a strong constitutive promoter. The results showed that CAT mRNAs devoid of both leader sequence nucleotides and the two downstream boxes in the CAT gene remained active in vivo and produced CAT protein in sufficient amounts for survival of the transformed cells at chloramphenicol concentrations up to 20-30 micrograms/ml.

Does the epsilon sequence of phage T7 function as an initiator for the translation of CAT mRNA in Escherichia coli?

Epsilon (epsilon) sequence [UUAACUUUA, complementary to nucleotides 458-466 of the 16S ribosomal RNA (rRNA)] which is naturally occurring at the 5'-untranslated leader of phage T7 gene 10 mRNA was originally described as a powerful translational enhancer in Escherichia coli. Recent studies with this sequence led to controversial conclusions about its translational initiation and enhancing capability. In this study different sequence derivatives of epsilon were constructed to evaluate its efficiency not only to enhance translation of the chloramphenicol acetyltransferase (CAT) mRNA in E. coli, but also to function as an independent initiator of translation. It was observed that the epsilon sequence in combination with the CAT natural Shine-Dalgarno (SDn) or the SD consensus sequences enhanced, as expected, the translation of CAT mRNA. The natural epsilon sequence without an SD sequence failed to initiate or enhance the translation of CAT mRNA. However, when the complementarity of epsilon to 16S rRNA was increased from 9 to 16 nucleotides, epsilon alone (without the SD sequence) became an independent translational initiator with an efficiency of about 80% that obtained with the SD consensus sequence.