Flux Analysis of Glucose Metabolism in Rhizopus oryzae for the Purpose of Increasing Lactate Yields

A flux analysis of glucose metabolism in the filamentous fungus Rhizopus oryzae was achieved using a specific radioactivity curve-matching program, TFLUX. Glycolytic and tricarboxylic acid cycle intermediates labeled through the addition of extracellular [U-14C]glucose were isolated and purified for specific radioactivity determinations. This information, together with pool sizes and the rates of glucose utilization and end product production, provided input for flux maps of the metabolic network under two different experimental conditions. Based upon the flux analysis of this system, a mutant of R. oryzae with higher lactate and lower ethanol yields than the parent was sought for and found.     

The Effect of Coumarin on Growth, Production of Dry Matter, Protein and Nucleic Acids in Roots of Maize and Wheat and the Interaction of Coumarin with Metabolic Inhibitors

Effects of coumarin on fresh weight, dry matter, protein and nucleic acid content per cell in attached roots of maize and wheat and in whole excised elongation zones of maize were determined. The inhibition in cell length exerted by coumarin did not correspond to an inhibition of the net synthetic capacity. Coumarin treatment increased the cell surface, the production of dry matter and the protein content per cell. The dry matter and the protein content per unit surface was slightly increased or unaffected. The effect of coumann on cell shape seemed to be independent of that on dry matter production and net protein synthesis. The same was found in excised elongation zones. —The net DNA-synthesis per cell was slightly increased in attached roots by coumann treatment, but this effect was probably not correlated with the morphogenetic changes. Inhibition of DNA-synthesis with hydroxyurea did not alter the coumarin induced changes in cell shape. —The net RNA-synthesis per cell was slightly decreased after coumarin treatment, but the net RNA-synthesis per cell and the morphogenetic effects exerted by coumarin were not related with each other. Inhibition of m-RNA-synthesis with actinomycin D did not prevent the effects of coumarin on cell division, cell expansion, dry matter production and net protein synthesis. The same was true for inhibitors of protein synthesis, puromycin and p-fluorophenyl-alanine. The findings are in support of the view that coumarin affects already existing structures or enzymes. —Comparisons between coumarin and the uncouplers, DNP and dicoumarol, showed that the effects of coumarin were not, solely, due to uncoupling. SH-protecting agents, BAL, DTE and glutathione, did, with few exceptions, not reduce the morphogenetic effects of coumarin.     

Phylogenomic species delimitation and host‐symbiont coevolution in the fungus‐farming ant genus Sericomyrmex Mayr (Hymenoptera: Formicidae): ultraconserved elements (UCEs) resolve a recent radiation

Ants in the Neotropical genus Sericomyrmex Mayr cultivate fungi for food. Both ants and fungi are obligate, coevolved symbionts. The taxonomy of Sericomyrmex is problematic because the morphology of the worker caste is generally homogeneous across all of the species within the genus, species limits are vague, and the relationships between them are unknown. We used ultraconserved elements (UCEs) as genome‐scale markers to reconstruct evolutionary history and to infer species boundaries in Sericomyrmex. We recovered an average of ～990 UCE loci for 88 Sericomyrmex samples from across the geographical range of the genus as well as for five outgroup taxa. Using maximum likelihood and species‐tree approaches, we recovered nearly identical topologies across datasets with 50–95% matrix completeness. We identify nine species‐level lineages in Sericomyrmex, including two new species. This is less than the previously described 19 species, even accounting for two species for which we had no UCE samples, which brings the total number of Sericomyrmex species to 11. Divergence‐dating analyses recovered 4.3 Ma as the crown‐group age estimates for Sericomyrmex, indicating a recent, rapid radiation. We also sequenced mitochondrial cytochrome oxidase subunit I (COI) for 125 specimens. Resolution and support for clades in our COI phylogeny are weak, indicating that COI is not an appropriate species‐delimitation tool. However, taxa within species consistently cluster together, suggesting that COI is useful as a species identification (‘DNA barcoding’) tool. We also sequenced internal transcribed spacer (ITS) and large subunit (LSU) for 32 Sericomyrmex fungal cultivars. The fungal phylogeny confirms that Sericomyrmex fungi are generalized higher‐attine cultivars, interspersed with Trachymyrmex‐associated fungal species, indicating cultivar sharing and horizontal transfer between these two genera. Our results indicate that UCEs offer immense potential for delimiting and resolving relationships of problematic, recently diverged species.     

Multiple histone modifications in euchromatin promote heterochromatin formation by redundant mechanisms in Saccharomyces cerevisiae

Background  

Methylation of lysine 79 on histone H3 by Dot1 is required for maintenance of heterochromatin structure in yeast and humans. However, this histone modification occurs predominantly in euchromatin. Thus, Dot1 affects silencing by indirect mechanisms and does not act by the recruitment model commonly proposed for histone modifications. To better understand the role of H3K79 methylation gene silencing, we investigated the silencing function of Dot1 by genetic suppressor and enhancer analysis and examined the relationship between Dot1 and other global euchromatic histone modifiers.     

Evolutionary dynamics of the kinetochore network in eukaryotes as revealed by comparative genomics

During eukaryotic cell division, the sister chromatids of duplicated chromosomes are pulled apart by microtubules, which connect via kinetochores. The kinetochore is a multiprotein structure that links centromeres to microtubules, and that emits molecular signals in order to safeguard the equal distribution of duplicated chromosomes over daughter cells. Although microtubule‐mediated chromosome segregation is evolutionary conserved, kinetochore compositions seem to have diverged. To systematically inventory kinetochore diversity and to reconstruct its evolution, we determined orthologs of 70 kinetochore proteins in 90 phylogenetically diverse eukaryotes. The resulting ortholog sets imply that the last eukaryotic common ancestor (LECA) possessed a complex kinetochore and highlight that current‐day kinetochores differ substantially. These kinetochores diverged through gene loss, duplication, and, less frequently, invention and displacement. Various kinetochore components co‐evolved with one another, albeit in different manners. These co‐evolutionary patterns improve our understanding of kinetochore function and evolution, which we illustrated with the RZZ complex, TRIP13, the MCC, and some nuclear pore proteins. The extensive diversity of kinetochore compositions in eukaryotes poses numerous questions regarding evolutionary flexibility of essential cellular functions.     

Host diversity and behavior determine patterns of interspecies transmission and geographic diffusion of avian influenza A subtypes among North American wild reservoir species

Wild birds can carry avian influenza viruses (AIV), including those with pandemic or panzootic potential, long distances. Even though AIV has a broad host range, few studies account for host diversity when estimating AIV spread. We analyzed AIV genomic sequences from North American wild birds, including 303 newly sequenced isolates, to estimate interspecies and geographic viral transition patterns among multiple co-circulating subtypes. Our results show high transition rates within Anseriformes and Charadriiformes, but limited transitions between these orders. Patterns of transition between species were positively associated with breeding habitat range overlap, and negatively associated with host genetic distance. Distance between regions (negative correlation) and summer temperature at origin (positive correlation) were strong predictors of transition between locations. Taken together, this study demonstrates that host diversity and ecology can determine evolutionary processes that underlie AIV natural history and spread. Understanding these processes can provide important insights for effective control of AIV.     

Covalent cross-linking of tRNAGly1 to the ribosomal P site via the dihydrouridine loop

The dihydrouracil residue at position 20 of Escherichia coli tRNAGly1 has been replaced by the photoaffinity reagent, N-(4-azido-2-nitrophenyl)glycyl hydrazide (AGH). The location of the substituent was confirmed by the susceptibility of the modified tRNA to cleavage with aniline. When N-acetylglycyl-tRNAGly1 derivatized with AGH was bound noncovalently to the P site of E. coli 70 S ribosomes, 5-6% on average was photochemically cross-linked to the ribosomal particles in a reaction requiring poly(G,U), irradiation and the presence of the AGH label in the tRNA. Approximately two-thirds of the covalently attached tRNA was associated with 16 S RNA in the 30 S subunit. This material was judged to be in the P site by the criterion of puromycin reactivity. As partial RNAase digestion of the tRNA-16 S RNA complex produced labeled fragments from both 5' and 3' segments of the rRNA, there appeared to be more than one site of cross-linking in the 30 S subunit. The small amount of N-acetylglycyl-tRNAGly1 associated with the 50 S subunit was also linked mainly to rRNA, but it was not puromycin-reactive.     

Identification of dipeptidyl peptidase IV as a protein shared by the plasma membrane of hepatocytes and liver biomatrix

The histotypic organization of liver parenchyma involves specific intercellular contacts and interaction of hepatocytes with supporting biomatrix. Evidence from this laboratory identified a peptide (Hep105, apparent Mr 105 000) that is shared by the plasma membrane of rat hepatocytes and rat liver biomatrix. This report identifies Hep105 as a peptide component of dipeptidyl peptidase IV (DPPIV; EC 3.4.14.-). A monoclonal antibody (MAb 236.3) was shown to immunoprecipitate DPPIV from non-ionic detergent extracts of surface-labeled 125I hepatocytes. The immunoprecipitate contained two 125I-labeled peptides: Hep105 and a peptide of apparent Mr 150000 (Hep150). Proteolysis of 125I-labeled Hep105 and Hep150 by Staphylococcus aureus V8 protease yielded essentially identical patterns of 125I-labeled peptide degradation products, indicating that Hep105 and Hep150 are structurally related. Only Hep150 exhibited DPPIV activity on transblot analysis, an observation that is consistent with the interpretation that it is the monomeric form of the enzyme. Heating (100 degrees C, 5 min) of purified Hep150 in the presence of sodium dodecylsulfate (SDS) resulted in its conversion to Hep105 and the disappearance of any demonstrable enzymatic activity. 3H-labeled diisopropyl fluorophosphate was incorporated into Hep105, indicating that Hep105 contains the active site for DPPIV. Purified rat liver biomatrix was shown to possess significant DPPIV activity. Taken together, these data indicate that Hep105 s a peptide component of DPPIV.     

Hepatocyte cell surface polarity as demonstrated by lectin binding

We performed an investigation at the ultrastructural level of the differential distribution of lectin-binding sites among sinusoidal, lateral, and bile canalicular domains of adult rat hepatocytes. Lectin binding to hepatocyte glycocalices was studied in situ or after cellular dissociation by enzymatic (collagenase), chemical (EDTA), and mechanical methods, as well as during cell culture. Using thirteen biotinylated lectins and an avidin-biotin-peroxidase complex (ABC), we have identified lectin-binding sites that are predominantly localized in the bile canalicular [Ricinus communis agglutinin (RCA)] or sinusoidal [Phaseolus vulgaris (PHA)] domains in situ and in mechanically dissociated cells. Lens culinaris (LCA) staining was prominent on sinusoidal surfaces, slight along lateral surfaces, and completely absent in the bile canalicular domain. Concanavalin A (ConA) was unique in binding equally to all domains. Triticum vulgaris [wheat germ agglutinin (WGA)] was also bound to all domains, but most intensely to the bile canalicular region. Cells dissociated via collagenase or EDTA treatment exhibited a spherical morphology characterized by many surface microvilli and absence of morphological domains. Lectin binding to dissociated cells was uniformly distributed over the entire cell surface, suggesting a redistribution of lectin receptors that was independent of the separation procedure. Hepatocytes in culture exhibited a partial restoration of morphological domains, but lectin binding polarity was not re-established.