Preparation of Diphosphopyridine Nucleotide from Bakers’ Yeast

A method for the preparatoin of diphosphopyridine nucleotide (DPN) from bakers’ yeast is described. This method consists of partial purification of crude extract of yeast by charcoal chromatography according to Pontis and coworkers, ion-exchange chromatography on Dowex-l acetate, and precipitation of DPN as the free acid with ethanol. 0.71~.1.1 g of DPN with a purity of 85~90% was obtained from 5 kg of fresh bakers’ yeast by this method.     

Archakebia—A New Genus of Lardizabalaceae from China

The monotypic genus Archakebia from China is described as new．The genus shares many characters with members of the genus Akebia,except sepals 6,lanceolate or linear,which are common in members of the genus Stauntonia．     

Phylogenetic positions of several amitochondriate protozoa——Evidence from phylogenetic analysis of DNA topoisomerase II

In the 1980s some special protozoa were gradually recognized: being eukaryotes, they possess some characteristics between prokaryotes and eukaryotes, for example, lack of eukaryotic organelle such as mi-tochondria, and even 70S ribosomes in some species, containing 16S, 23S rRNA like those of prokaryotes. These protozoa contain diplomonads (e.g. intestinal parasite Giardia), parabasalids (e.g. venereal disease pathogen Trichomonas), microsporidia (e.g. the human pathogen Encephalitozoon), a…     

Deletion of Genes Encoding Arginase Improves Use of “Heavy” Isotope-Labeled Arginine for Mass Spectrometry in Fission Yeast

The use of “heavy” isotope-labeled arginine for stable isotope labeling by amino acids in cell culture (SILAC) mass spectrometry in the fission yeast Schizosaccharomyces pombe is hindered by the fact that under normal conditions, arginine is extensively catabolized in vivo, resulting in the appearance of “heavy”-isotope label in several other amino acids, most notably proline, but also glutamate, glutamine and lysine. This “arginine conversion problem” significantly impairs quantification of mass spectra. Previously, we developed a method to prevent arginine conversion in fission yeast SILAC, based on deletion of genes involved in arginine catabolism. Here we show that although this method is indeed successful when ¹³C₆-arginine (Arg-6) is used for labeling, it is less successful when ¹³C₆ ¹⁵N₄-arginine (Arg-10), a theoretically preferable label, is used. In particular, we find that with this method, “heavy”-isotope label derived from Arg-10 is observed in amino acids other than arginine, indicating metabolic conversion of Arg-10. Arg-10 conversion, which severely complicates both MS and MS/MS analysis, is further confirmed by the presence of ¹³C₅ ¹⁵N₂-arginine (Arg-7) in arginine-containing peptides from Arg-10-labeled cells. We describe how all of the problems associated with the use of Arg-10 can be overcome by a simple modification of our original method. We show that simultaneous deletion of the fission yeast arginase genes car1+ and aru1+ prevents virtually all of the arginine conversion that would otherwise result from the use of Arg-10. This solution should enable a wider use of heavy isotope-labeled amino acids in fission yeast SILAC.     

Yeast cell-surface display—applications of molecular display

In a cell-surface engineering system established using the yeast Saccharomyces cerevisiae, novel, so-called arming yeasts are constructed that are armed with biocatalysts in the form of enzymes, functional proteins, antibodies, and combinatorial protein libraries. Among the many advantages of the system, in which proteins are genetically displayed on the cell surface, are easy reproduction of the displayed biocatalysts and easy separation of product from catalyst. As proteins and peptides of various kinds can be displayed on the yeast cell surface, the system is expected to allow the preparation of tailor-made functional proteins. With its ability to express many of the functional proteins necessary for post-translational modification and in a range of different sizes, the yeast-based molecular display system appears uniquely useful among the various display systems so far developed. Capable of conferring novel additional abilities upon living cells, cell-surface engineering heralds a new era of combinatorial bioengineering in the field of biotechnology. This mini-review describes molecular display using yeast and its various applications.     

Cloning of the Fatty Acid Synthetase β Subunit from Fission Yeast, Coexpression with the α Subunit, and Purification of the Intact Multifunctional Enzyme Complex

We have cloned and sequenced the fission yeast (Schizosaccharomyces pombe)fas1⁺gene, which encodes the fatty acid synthetase (FAS) β subunit, by applying a PCR technique to conserved regions in the β subunit of the α₆β₆types of FAS among different organisms. The deduced amino acid sequence of the Fas1 polypeptide, consisting of 2073 amino acids (M_r= 230,616), exhibits the 48.1% identity with the β subunit from the budding yeast (Saccharomyces cerevisiae). This subunit, with five different catalytic activities, bears four distinct domains, while the α subunit, the sequence of which was previously reported by Saitohet al.(S. Saitohet al.,1996,J. Cell Biol.134, 949–961), carries three domains. We have developed a co-expression system of the FAS α and β subunits by cotransformation of two expression vectors, containing thelsd1⁺/fas2⁺gene and thefas1⁺gene, into fission yeast cells. The isolated FAS complex showed quite high specific activity, of more than 4000 mU/mg, suggesting complete purification. Its molecular weight was determined by dynamic light scattering and ultracentrifugation analysis to be 2.1–2.4 × 10⁶, and one molecule of the FAS complex was found to contain approximately six FMN molecules. These results indicate that the FAS complex fromS. pombeforms a heterododecameric α₆β₆structure. Electron micrographs of the negatively stained molecule suggest that the complex adopts a unique barrel-shaped cage architecture.     

Origin of Chrysanthemum cultivars — Evidence from nuclear low-copy LFY gene sequences

The chrysanthemums (Chrysanthemum × morifolium Ramat.) are a well-known group of traditional ornamental flowers in China and have been cultivated all over the world. Yet the origin of chrysanthemum cultivars has been highly debated. In this study we employ the nuclear low-copy LFY gene to study the evolutionary history of chrysanthemum cultivars. The structure of the LFY gene in all Chrysanthemum species examined is highly conserved with three exons and two introns. The length of the LFY gene in Chrysanthemum varied from 2, 887 to 3, 348 bp. The two introns exhibited high levels of variation in length and sequence composition at the intraspecific and interspecific levels. Phylogenetic analysis of the whole LFY sequences of Chrysanthemum resulted in topologies that contained three major clades. The LFY sequences from the same cultivars are present in two or three clades, supporting that hybridization and allopolyploidy were important mechanisms in the origins of different chrysanthemums. Our results suggest that different cultivars had different ancestors. Chrysanthemum indicum, C. zawadskii and C. nankingense were likely the direct ancestors of most chrysanthemum cultivars examined. Chrysanthemum vestitum is a putative ancestor for some cultivars, and may have indirectly involved in the development of the chrysanthemum cultivars. Sequences of the LFY gene are informative to shed insights into the origin of chrysanthemum cultivars and show great potential as a phylogenetic marker to decipher the phylogeny of Chrysanthemum and its close relatives.     

Origins of cultivars of Chrysanthemum—Evidence from the chloroplast genome and nuclear LFY gene

The origins of cultivated chrysanthemums have attracted considerable attention, but they remain poorly known. Here, we reconstructed the phylogeny of representative well‐known cultivars and wild species of the genus Chrysanthemum using chloroplast genomes and the nuclear LEAFY gene. Our results suggest that geographic and ecological factors may determine the opportunities for wild species to be involved in the origin of the cultivars. The wild species C. indicum, C. zawadskii, C. dichrum, C. nankingense, C. argyrophyllum, and C. vestitum were likely directly or indirectly involved as paternal species of most of the chrysanthemum cultivars examined in this study. Yet, the maternal species is supported to be a lineage of an extinct wild Chrysanthemum species and its subsequent cultivars, as all accessions of chrysanthemum cultivars sampled formed a strongly supported clade, distinct from all other species of Chrysanthemum in the plastome tree. Thus, the cultivated chrysanthemums originated from multiple hybridizations involving several paternal species rather than only two or a few wild species, with an extinct species and its subsequent cultivars serving as the maternal parents. This finding is consistent with Chrysanthemum having high rates of hybridization and gene flow, which has been demonstrated within previous studies; nevertheless, it is important to unravel the role of an extinct wild Chrysanthemum species as the ultimate maternal parent species for all the chrysanthemum cultivars. Our results also suggest that C. vestitum from Tianzhu and Funiu Mountains in Anhui and Henan Provinces of China represent two distinct cryptic species.     

Yeast Immobilization in LentiKats®: A New Strategy for Xylitol Bioproduction from Sugarcane Bagasse

Summary A new PVA-hydrogel matrix for yeast cell immobilization for xylitol bioproduction from sugarcane bagasse was studied. Five repeated-batch fermentation runs were carried out in medium based on sugarcane bagasse hemicellulosic hydrolysate with reuse of the entrapped biocatalyst. The system performance as well as the metabolic behaviour of cells entrapped into the matrix were evaluated. The biocatalyst remained stable and exhibited a similar fermentative profile in all the successive batches, demonstrating the viability of the system. At the end of the run, an average xylitol production was observed of 35.1 g l⁻¹ and average xylitol yield and productivity of 0.58 g g⁻¹ and 0.49 g l⁻¹ h⁻¹, respectively.     

Asymmetric Segregation of Aged Spindle Pole Bodies During Cell Division: Mechanisms and Relevance Beyond Budding Yeast?

Asymmetric cell division generates cell diversity and contributes to cellular aging and rejuvenation. Here, we review the molecular mechanisms enabling budding yeast to recognize spindle pole bodies (SPB, centrosome equivalent) based on their age, and guide their non‐random mitotic segregation: SPB inheritance requires the distinction of old from new SPBs and is regulated by the SPB‐inheritance network (SPIN) and the mitotic exit network (MEN). The SPIN marks the pre‐existing SPB as old and the MEN recognizes these marks translating them into spindle orientation. We next revisit other molecules and structures that partition depending on their age rather than their abundance at mitosis as, for example, DNA, centrosomes, mitochondria, and histones in yeast and other systems. The recurrence of this differential behavior suggests a functional significance for numerous cell types, which we then discuss. We conclude that non‐random segregation may facilitate asymmetric cell fate determination and thereby indirectly aging and rejuvenation. Also see the video abstract here: https://youtu.be/1sQ4rAomnWY .     

Spatial Niche Segregation of Sympatric Stone Marten and Pine Marten – Avoidance of Competition or Selection of Optimal Habitat?

Coexistence of ecologically similar species relies on differences in one or more dimensions of their ecological niches, such as space, time and resources in diel and/or seasonal scales. However, niche differentiation may result from other mechanisms such as avoidance of high predation pressure, different adaptations or requirements of ecologically similar species. Stone marten (Martes foina) and pine marten (Martes martes) occur sympatrically over a large area in Central Europe and utilize similar habitats and food, therefore it is expected that their coexistence requires differentiation in at least one of their niche dimensions or the mechanisms through which these dimensions are used. To test this hypothesis, we used differences in the species activity patterns and habitat selection, estimated with a resource selection function (RSF), to predict the relative probability of occurrence of the two species within a large forest complex in the northern geographic range of the stone marten. Stone martens were significantly heavier, have a longer body and a better body condition than pine martens. We found weak evidence for temporal niche segregation between the species. Stone and pine martens were both primarily nocturnal, but pine martens were active more frequently during the day and significantly reduced the duration of activity during autumn-winter. Stone and pine martens utilized different habitats and almost completely separated their habitat niches. Stone marten strongly preferred developed areas and avoided meadows and coniferous or deciduous forests. Pine marten preferred deciduous forest and small patches covered by trees, and avoided developed areas and meadows. We conclude that complete habitat segregation of the two marten species facilitates sympatric coexistence in this area. However, spatial niche segregation between these species was more likely due to differences in adaptation to cold climate, avoidance of high predator pressure and/or food preferences by both species than competitive interaction between them.     

New Approaches for Calculating Moran’s Index of Spatial Autocorrelation

Spatial autocorrelation plays an important role in geographical analysis; however, there is still room for improvement of this method. The formula for Moran’s index is complicated, and several basic problems remain to be solved. Therefore, I will reconstruct its mathematical framework using mathematical derivation based on linear algebra and present four simple approaches to calculating Moran’s index. Moran’s scatterplot will be ameliorated, and new test methods will be proposed. The relationship between the global Moran’s index and Geary’s coefficient will be discussed from two different vantage points: spatial population and spatial sample. The sphere of applications for both Moran’s index and Geary’s coefficient will be clarified and defined. One of theoretical findings is that Moran’s index is a characteristic parameter of spatial weight matrices, so the selection of weight functions is very significant for autocorrelation analysis of geographical systems. A case study of 29 Chinese cities in 2000 will be employed to validate the innovatory models and methods. This work is a methodological study, which will simplify the process of autocorrelation analysis. The results of this study will lay the foundation for the scaling analysis of spatial autocorrelation.