Brood cell size of <Emphasis Type="Italic">Apis</Emphasis> <Emphasis Type="Italic">mellifera</Emphasis> modifies the reproductive behavior of <Emphasis Type="Italic">Varroa</Emphasis> <Emphasis Type="Italic">destructor</Emphasis>

We undertook a field study to determine whether comb cell size affects the reproductive behavior of Varroa destructor under natural conditions. We examined the effect of brood cell width on the reproductive behavior of V. destructor in honey bee colonies, under natural conditions. Drone and worker brood combs were sampled from 11 colonies of Apis mellifera. A Pearson correlation test and a Tukey test were used to determine whether mite reproduction rate varied with brood cell width. Generalized additive model analysis showed that infestation rate increased positively and linearly with the width of worker and drone cells. The reproduction rate for viable mother mites was 0.96 viable female descendants per original invading female. No significant correlation was observed between brood cell width and number of offspring of V. destructor. Infertile mother mites were more frequent in narrower brood cells.     

Heterologous production of secondary metabolites as pharmaceuticals in <Emphasis Type="Italic">Saccharomyces</Emphasis> <Emphasis Type="Italic">cerevisiae</Emphasis>

Heterologous expression of genes involved in the biosynthesis of various products is of increasing interest in biotechnology and in drug research and development. Microbial cells are most appropriate for this purpose. Availability of more microbial genomic sequences in recent years has greatly facilitated the elucidation of metabolic and regulatory networks and helped gain overproduction of desired metabolites or create novel production of commercially important compounds. Saccharomyces cerevisiae, as one of the most intensely studied eukaryotic model organisms with a rich density of knowledge detailing its genetics, biochemistry, physiology, and large-scale fermentation performance, can be capitalized upon to enable a substantial increase in the industrial application of this yeast. In this review, we describe recent efforts made to produce commercial secondary metabolites in Saccharomyces cerevisiae as pharmaceuticals. As natural products are increasingly becoming the center of attention of the pharmaceutical and nutraceutical industries, such as naringenin, coumarate, artemisinin, taxol, amorphadiene and vitamin C, the use of S. cerevisiae for their production is only expected to expand in the future, further allowing the biosynthesis of novel molecular structures with unique properties.     

Overexpressing <Emphasis Type="Italic">GLT1</Emphasis> in <Emphasis Type="Italic">gpd1</Emphasis>Δ mutant to improve the production of ethanol of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

To improve ethanol production in Saccharomyces cerevisiae, two yeast strains were constructed. In the mutant, KAM-4, the GPD1 gene, which encodes a glycerol 3-phosphate dehydrogenase of S. cerevisiae to synthesize glycerol, was deleted. The mutant KAM-12 had the GLT1 gene (encodes glutamate synthase) placed under the PGK1 promoter while harboring the GPD1 deletion. Notably, overexpression of GLT1 by the PGK1 promoter along with GPD1 deletion resulted in a 10.8% higher ethanol production and a 25.0% lower glycerol formation compared to the wild type in anaerobic fermentations. The growth rate of KAM-4 was slightly lower than that of the wild type under the exponential phase whereas KAM-12 and the wild type were indistinguishable in the biomass concentration at the end of growth period. Meanwhile, dramatic reduction of formation of acetate and pyruvic acid was observed in all the mutants compared to the wild type.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of <Emphasis Type="Italic">Codonopsis lanceolata</Emphasis> using the <Emphasis Type="Italic">γ-TMT</Emphasis> gene

Efficient transformation of leaf disc-derived callus of Codonopsis lanceolata was obtained using Agrobacterium tumefaciens strain LBA4404 harboring a binary vector, pYBI121, that carries the neomycin phosphotransferase (npt II) gene as a selectable marker. The green shoots recovered from agroinfected explants on selection medium (containing 0.1 mg/l α-naphthaleneacetic acid (NAA), 1 mg/l 6-benzylaminopurine (BAP), 100 mg/l kanamycin, and 250 mg/l cefotaxime) were rooted on Murashige and Skoog (MS) medium supplemented with 2 mg/l IBA and 10 mg/l kanamycin. To optimize the transformation conditions, several factors were assessed, including the co-cultivation period, the duration of pre- and post-culture in darkness and light, the kanamycin concentration, and the Agrobacterium densities. We produced transgenic Codonopsis lanceolata overexpressing γ-tocopherol methyltransferase (γ-TMT) by this protocol. Moreover, the α-tocopherol content of the plants was enhanced by the overexpression of this gene. Bimal Kumar Ghimire and Eun Soo Seong contributed equally to this work.     

Is the <Emphasis Type="Italic">Mnr</Emphasis> locus of Triticeae species the same as the <Emphasis Type="Italic">Ndh</Emphasis> and <Emphasis Type="Italic">Dia</Emphasis> loci?

The menadione reductase (MNR), the nicotinamide adenine dinucleotide dehydrogenase (NDH) and diaphorase (DIA) isozymes were studied in the allohexaploid Triticum aestivum cv ”Chinese Spring” and in five diploid Triticeae species. The Mnr1, Ndh3 and Dia1 loci were located on the chromosome arms 3AL, 3BL and 3DL of T. aestivum, respectively. These loci were also located on the 3H chromosome of Hordeum vulgare cv ”Betzes”, the 3L chromosome of Aegilops longissima and the 6RL chromosome arm of Secale cereale cv ”Imperial”. The chromosomal location results together with the segregation studies support a tetrameric behaviour of the MNR1, NDH3 and DIA1 isozymes. The Ndh1 and Dia3 loci were located on homoeologous group 4 showing a monomeric behaviour. The chromosomal locations and linkage data of the Mnr, Ndh and Dia loci suggest that Mnr1=Ndh3=Dia1; Ndh1=Dia3 and Ndh2=Dia2. Received: 3 June 2001 / Accepted: 11 July 2001     

Production of a Soluble Disulfide Bond-Linked TCR in the Cytoplasm of <Emphasis Type="Italic">Escherichia</Emphasis> <Emphasis Type="Italic">coli</Emphasis><Emphasis Type="Italic">trx</Emphasis>B <Emphasis Type="Italic">gor</Emphasis> Mutants

Previously, we have described the use of phage display to generate high affinity disulfide bond-linked T cell receptors (TCRs). The affinities of the mutant TCRs were analysed after refolding of separately expressed α and β chains from Escherichia coli inclusion bodies. This approach is only suitable for the analysis of small numbers of TCR variants. An attractive alternative would be soluble expression within the bacterial periplasm, but the generic production of TCRs within the E. coli periplasm has so far not proved successful. Here we show that functional, soluble TCR can be produced within the cytoplasm of trxB gor mutant E. coli strains, with maximum yields of 3.4 mg/l. We also investigated the effect of coexpressing the folding modulators Skp and DsbC finding that the TCR expression levels were largely unaffected by these chaperones. Importantly, we demonstrated that the amount of protein purified from 50 ml starter cultures was sufficient to show functionality of the TCR by specific antigen binding in both ELISA and surface plasmon resonance (SPR) assays. This TCR production method has the potential to allow rapid and medium throughput analysis of affinity-matured TCRs selected from TCR phage display libraries.     

Sequence Analysis of the <Emphasis Type="Italic">Lactoferrin</Emphasis> Gene and Variation of <Emphasis Type="Italic">g</Emphasis>.<Emphasis Type="Italic">7605C</Emphasis>→<Emphasis Type="Italic">T</Emphasis> in 10 Chinese Indigenous Goat Breeds

Much attention has been focused on the study of lactoferrin at the protein or nucleotide level in mice, humans, and cattle, but little is known about it in goats. The goat LF gene from 5' UTR to exon 17 was amplified, and the variation of g.7605C→T in 10 Chinese indigenous goat breeds was analyzed. Among the three ruminant species (cattle, sheep, and goats), the intron-exon distribution pattern was similar, and all the exons had the same length, but the length of introns varied greatly due to insertions or deletions. The frequency of allele T at g.7605C→T (50.12%) was a little higher than that of allele C (49.88%), and the genotype distribution differed greatly between goat populations. The g.7605C→T site showed higher genetic diversity in goat populations. The genetic differentiation was 0.0783, and gene flow was 2.9433 among the 10 Chinese indigenous goat populations.     

Enhancing functional expression of heterologous lipase in the periplasm of <Emphasis Type="Italic">Escherichia</Emphasis><Emphasis Type="Bold"> </Emphasis><Emphasis Type="Italic">coli</Emphasis>

Functional expression of heterologous Pseudozyma antarctica lipase B (PalB) in the periplasm of Escherichia coli was explored using four fusion tags, i.e. DsbC, DsbA, maltose-binding protein (MBP), and FLAG in the sequence of increasing expression efficacy. Amongst these fusion tags, FLAG and MBP appear to be the most effective ones in terms of boosting enzyme activity and enhancing solubility of PalB, respectively. Overexpression of these PalB fusions often resulted in concomitant formation of insoluble inclusion bodies. Coexpression of a selection of periplasmic folding factors, including DegP (and its mutant variant of DegP_S210A), FkpA, DsbA, DsbC, and a cocktail of SurA, FkpA, DsbA, and DsbC, could improve the expression performance. Coexpression of DsbA appeared to be the most effective in reducing the formation of inclusion bodies for all the four PalB fusions, implying that functional expression of PalB could be limited by initial bridging of disulfide bonds. Culture performance was optimized by overexpressing FLAG-PalB with DsbA coexpression, resulting in a high volumetric PalB activity of 360 U/L.     

Rapid induction of <Emphasis Type="Italic">Agrobacterium</Emphasis> <Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transgenic roots directly from adventitious roots in <Emphasis Type="Italic">Panax</Emphasis> <Emphasis Type="Italic">ginseng</Emphasis>

Root segments from seedlings of Panax ginseng produced adventitious roots directly when cultured on 1/2 MS solid medium lacking NH₄NO₃ and containing 3.0 mg l⁻¹ IBA. Using this adventitious root formation, we developed rapid and efficient transgenic root formation directly from adventitious root segments in P. ginseng. Root segments were co-cultivated with Agrobacterium tumefaciens (GV3101) caring β-glucuronidase (GUS) gene. Putative transgenic adventitious roots were formed directly from root segments on medium with 400 mg l⁻¹ cefotaxime and 50 mg l⁻¹ kanamycin. Kanamycin resistant adventitious roots were selected and proliferated as individual lines by subculturing on medium with 300 mg l⁻¹ cefotaxime and 50 mg l⁻¹ kanamycin at two weeks subculture interval. Frequency of transient and stable expression of GUS gene was enhanced by acetosyringon (50 mg l⁻¹) treatment. Integration of transgene into the plants was confirmed by the X-gluc reaction, PCR and Southern analysis. Production of transgenic plants was achieved via somatic embryogenesis from the embryogenic callus derived from independent lines of adventitious roots. The protocol for rapid induction of transgenic adventitious roots directly from adventitious roots can be applied for a new Agrobacterium tumefaciens-mediated genetic transformation protocol in P. ginseng.     

The ectomycorrhiza <Emphasis Type="Italic">Piceirhiza internicrassihyphis</Emphasis>: A weak competitor of <Emphasis Type="Italic">Cortinarius obtusus</Emphasis>?

The ectomycorrhiza (ECM) Piceirhiza internicrassihyphis on Picea abies is described in detail. It is yellowish brown to brown with a mantle of a transitional type between plectenchymatous and pseudoparenchymatous. The inner mantle layers show some thick-walled hyphae, often with a few of them growing in parallel. These thick-walled hyphae contain particles which turn brownish in Melzer’s reagent, and their septa and walls are partially amyloid. Rhizomorphs occur infrequently and are undifferentiated, and are composed of rather loosely woven hyphae of uniform diameter. This combination of characteristics has not yet been identified in any other ECM. The lack of cystidia and the presence of amyloidy suggest that this ECM is formed by a species of the family Thelephoraceae. Piceirhiza internicrassihyphis can be found associated with ECM of Cortinarius obtusus, and the hyphae of the latter species often grow on the surface of P. internicrassihyphis and can even cover the whole tip. Although there is a close association and possibly a penetration between these two fungi, it can be excluded that the thick-walled hyphae of the inner mantle layers originate from C. obtusus. Some emanating hyphae of C. obtusus ECM seem to interact with those of P. internicrassihyphis, as indicated by short, hyphal outgrowths which were found attached to the foreign hyphae.     

Factors influencing the production of stilbenes by the knotweed, <Emphasis Type="Italic">Reynoutria</Emphasis> × <Emphasis Type="Italic">bohemica</Emphasis>

Background  

Japanese knotweed, Reynoutria japonica, is known for its high growth rate, even on adverse substrates, and for containing organic substances that are beneficial to human health. Its hybrid, Reynoutria × bohemica, was described in the Czech Republic in 1983 and has been widespread ever since. We examined whether Reynoutria × bohemica as a medicinal plant providing stilbenes and emodin, can be cultivated in spoil bank substrates and hence in the coalmine spoil banks changed into arable fields. We designed a pot experiment and a field experiment to assess the effects of various factors on the growth efficiency of Reynoutria × bohemica on clayish substrates and on the production of stilbenes and emodin in this plant.     

On the algebraic representation of RNA secondary structures with <Emphasis Type="Italic">G</Emphasis>⋅<Emphasis Type="Italic">U</Emphasis> pairs

 Magarshak et al. represented an RNA molecule as a complex vector and an RNA secondary structure Γ as a complex matrix S _Γ in such a way that the molecule represented by was compatible with the secondary structure Γ if and only if . They only considered Watson-Crick base pairs and their representation cannot be extended to allow for G⋅U pairs. In this paper we study a generalization of Magarshak's representation that allows for these pairs, and in particular we provide a family of algebraic structures where that generalization can be carried out. We also show that this representation can be used to compare secondary structures, through transfer matrices which transform the representation of one secondary structure into the representation of the other. Received: 10 December 2001 / Revised version: 7 May 2002 / Published online: 28 February 2003 Key words or phrases: RNA secondary structure – Algebra – Finite field     

How promising is <Emphasis Type="Italic">Lasioseius floridensis</Emphasis> as a control agent of <Emphasis Type="Italic">Polyphagotarsonemus</Emphasis><Emphasis Type="Italic">latus</Emphasis>?

The blattisociid mite Lasioseius floridensis Berlese was found associated with the broad mite, Polyphagotarsonemus latus (Banks), on gerbera leaves in Mogi das Cruzes, State of Sao Paulo, Brazil. Blattisociid mites are not common on aerial plant parts, except under high air humidity levels. Some Lasioseius species have been mentioned as effective control agents of rice pest mites, but nothing is known about the biology of L. floridensis. The objective of this study was to evaluate whether the observed co-occurrence of L. floridensis and P. latus was just occasional or whether the latter could be important as food source for the former, assumed by laboratory evaluation of the ability of the predator to maintain itself, reproduce and develop on that prey. Biological parameters of L. floridensis were compared when exposed to P. latus and to other items as food. The study showed that mating is a pre-requisite for L. floridensis to oviposit and that oviposition rate was much higher on the soil nematode Rhabditella axei (Cobbold) (Rhabditidae) than on P. latus. Ovipositon on the acarid mite Tyrophagus putrescentiae (Schrank) was about the same as on P. latus, but it was nearly zero when the predator was fed the fungi Aspergillus flavus Link or Penicillium sp., or cattail (Typha sp.) pollen. Survivorship was higher in the presence of pollen and lower in the presence of A. flavus or Penicillium sp. than in the absence of those types of food. Life table parameters indicated that the predator performed much better on R. axei than on P. latus. To evaluate the potential effect of L. floridensis as predator of P. latus, complementary studies are warranted to determine the frequency of migration of L. floridensis to aerial plant parts, when predation on P. latus could occur.     

Indel patterns of the plastid DNA<Emphasis Type="Italic"> trnL</Emphasis>–<Emphasis Type="Italic">trnF</Emphasis> region within the genus<Emphasis Type="Italic"> Poa</Emphasis> (Poaceae)

The use of insertion/deletion (indel) patterns from sequences of the trnL intron and trnL–F intergenic spacer (IGS) in finding plastid genome types of the genus Poa L. was studied. New sequences for 23 taxa (P. alpina, P. badensis, P. bulbosa, P. crassipes, P. molinerii, P. annua, P. chaixii, P. granitica, P. pratensis, P. sibirica, P. remota, P. botryoides, P. cenisia, P. compressa, P. laxa, P. margilicola, P. media, P. nemoralis, P. palustris, P. pannonica, P. pirinica, P. riphaea, and P. sejuncta) and 18 previously published sequences, which represent 11 of the 13 sections listed for Poa in Flora Europaea, were investigated. Collections were made primarily in central Europe. Indel patterns, despite sampling less than 0.7% of the plastid genome, produced four taxa groupings that were congruent with the major divisions obtained in intensive, previously published restriction-site studies. Insertion/deletion events in the trnL intron and trnL–trnF IGS were in nearly all cases unique to a single pattern group and thus provided almost no information about relationships among these groups. Indels did, however, provide a meaningful infrageneric classification criterion for Poa. They can serve as useful tools in studying relationships within this genus.     

Molecular characterization of <Emphasis Type="Italic">Vibrio cholerae</Emphasis> Δ<Emphasis Type="Italic">relA</Emphasis> Δ<Emphasis Type="Italic">spoT</Emphasis> double mutants

In Escherichia coli cellular levels of pppGpp and ppGpp, collectively called (p)ppGpp, are maintained by the products of two genes, relA and spoT. Like E. coli, Vibrio cholerae also possesses relA and spoT genes. Here we show that similar to E. coli, V. cholerae ΔrelA cells can accumulate (p)ppGpp upon carbon starvation but not under amino acid starved condition. Although like in E. coli, the spoT gene function was found to be essential in V. cholerae relA ⁺ background, but unlike E. coli, several V. cholerae ΔrelA ΔspoT mutants constructed in this study accumulated (p)ppGpp under glucose starvation. The results suggest a cryptic source of (p)ppGpp synthesis in V. cholerae, which is induced upon glucose starvation. Again, unlike E. coli ΔrelA ΔspoT mutant (ppGpp⁰ strain), the V. cholerae ΔrelA ΔspoT mutants showed certain unusual phenotypes, which are (a) resistance towards 3-amino-1,2,4-triazole (AT); (b) growth in nutrient poor M9 minimal medium; (c) ability to stringently regulate cellular rRNA accumulation under glucose starvation and (d) initial growth defect in nutrient rich medium. Since these phenotypes of ΔrelA ΔspoT mutants could be reverted back to ΔrelA phenotypes by providing SpoT in trans, it appears that the spoT gene function is crucial in V. cholerae. Part of this work was presented at the International Symposium on Chemical Biology, Kolkata, India, 7–9 March 2007.     

Cryopreservation of cell suspension cultures of <Emphasis Type="Italic">Taxus</Emphasis> × <Emphasis Type="Italic">media</Emphasis> and <Emphasis Type="Italic">Taxus floridana</Emphasis>

Different lines of cell suspension cultures of Taxus × media Rehd. and Taxus floridana Nutt. were cryopreserved with a two-step freezing method using a simple and inexpensive freezing container instead of a programmable freezer. Four to seven days old suspension cell cultures were precultured in growth medium supplemented with 0.5 M mannitol for 2 d. The medium was then replaced with cryoprotectant solution (1 M sucrose, 0.5 M glycerol and 0.5 M dimethylsulfoxide) and the cells incubated on ice for 1 h. Before being plunged into liquid nitrogen, cells were frozen with a cooling rate of approximately −1 °C per min to −80 °C. The highest post-thaw cell viability was 90 %. The recovery was line dependent. The cryopreservation procedure did not alter the nuclear DNA content of the cell lines. The results indicate that cryopreservation of Taxus cell suspension cultures using inexpensive freezing container is possible.     

Does sequence polymorphism of <Emphasis Type="Italic">FLC</Emphasis> paralogues underlie flowering time QTL in <Emphasis Type="Italic">Brassica </Emphasis><Emphasis Type="Italic">oleracea</Emphasis>?

Previous locations of flowering time (FT) QTL in several Brassica species, coupled with Arabidopsis synteny, suggest that orthologues of the genes FLC, FY or CONSTANS might be the candidates. We focused on FLC, and cloned paralogous copies in Brassica oleracea, obtained their genomic DNA sequences, and confirmed their locations relative to those of known FT-QTL by genetical mapping. They varied in total length mainly due to the variable size of the first and last introns. A high level of identity was observed among Brassica FLC genes at the amino acid level but non-synonymous differences were present. Comparative analysis of the promoter and intragenic regions of BoFLC paralogues with Arabidopsis FLC revealed extensive differences in overall structure and organisation but showed high conservation within those segments known to be essential in regulating FLC expression. Four B. oleracea FLC copies (BoFLC1, BoFLC3, BoFLC4 and BoFLC5) were located to their respective linkage groups based on allelic sequence variation in lines from a doubled haploid population. All except BoFLC4 were within the confidence intervals of known FT-QTL. Sequence data indicated that relevant non-synonymous polymorphisms were present between parents A12DHd and GDDH33 for BoFLC genes. However, BoFLC alleles segregated independently of FT in backcrosses while the study provided evidence that BoFLC4 and BoFLC5 contain premature stop codons and so could not contribute to flowering time variation. Therefore, there is strong evidence against any of the 4 BoFLC being FT-QTL candidates in this population.