Anthracological evidence suggests naturalness of <Emphasis Type="Italic">Pinus pinaster</Emphasis> in inland southwestern Iberia

The study of well-preserved archaeological charcoals in the pre-Roman Iron Age settlement of Castillejos II (Badajoz, Spain) is used to reconstruct environmental conditions and land-use practices in vegetation landscapes in the southwest of the Iberian Peninsula before the arrival of Roman civilization. The results support that, while evergreen Quercus forests dominated during the Holocene, Pinus pinaster existed as a natural element of southwestern Iberian Peninsula vegetation. Although its presence could be linked to anthropogenic disturbance or fire history, it is suggested that P. pinaster populations survived during the Holocene in the region, mixed with oaks or in monospecific stands in mountain enclaves. This hypothesis contrasts with previous assumptions that P. pinaster was not autochthonous in the area.     

Molecular cloning and characterization of the polymorphic<Emphasis Type="Italic"> MHC</Emphasis> class II<Emphasis Type="Italic"> DBB</Emphasis> from the tammar wallaby (<Emphasis Type="Italic">Macropus eugenii</Emphasis>)

Genes of the major histocompatibility complex (MHC) have been characterized in all extant lineages of mammals. The tammar wallaby (Macropus eugenii) is well established as a model marsupial species; however, no classical MHC sequences have been described from this species. We have isolated two MHC class II -chain sequences from a tammar wallaby spleen cDNA library using a tammar MHC class II probe. These sequences belong to the marsupial MHC class II DBB gene family. Two additional DBB sequences were amplified from tammar wallaby genomic DNA. All four sequences were obtained from the same individual, indicating that there are at least two DBB loci in the tammar wallaby.Nucleotide sequence data reported are available in the GenBank database under the accession numbers AY438038–AY438042     

Conjugal transfer using the bacteriophage ϕC31 <Emphasis Type="Italic">att</Emphasis>/<Emphasis Type="Italic">int</Emphasis> system and properties of the <Emphasis Type="Italic">attB</Emphasis> site in <Emphasis Type="Italic">Streptomyces ambofaciens</Emphasis>

To facilitate molecular genetic studies of Streptomyces ambofaciens that produces spiramycin, a commercially important macrolide antibiotic used in human medicine against Gram-positive pathogenic bacteria, the conditions for the conjugal transfer of DNA from E. coli to S. ambofaciens were established using a bacteriophage ϕC31 att/int system. The transconjugation efficiency of S. ambofaciens varied with the medium used; the highest frequency was obtained on AS-1 medium containing 10 mM MgCl₂ without heat treatment of the spores. In addition, by cloning and sequencing the attB site, we identified that S. ambofaciens contains a single attB site within an ORF coding for a pirin homolog, and its attB site sequence shows 100% nt identity to the sequence of S. coelicolor and S. lividans, which have the highest efficiency in transconjugation using the ϕC31 att/int system.     

Expression and purification of <Emphasis Type="Italic">Zantedeschia aethiopica</Emphasis> agglutinin in <Emphasis Type="Italic">Escherichia </Emphasis><Emphasis Type="Italic">coli</Emphasis>

Recombinant Zantedeschia aethiopica agglutinin (ZAA) was expressed in Escherichia coli as N-terminal His-tagged fusion. After induction with isopropylthio-β-d-galactoside (IPTG), the recombinant ZAA was purified by metal-affinity chromatography. The purified ZAA protein was applied in anti-fungal assay and the result showed that recombinant ZAA had anti-fungal activity towards leaf mold (Fulvia fulva), one of the most serious phytopathogenic fungi causing significant yield loss of crops. This study suggests that ZAA could be an effective candidate in genetic engineering of plants for the control of leaf mold.     

Expression and characterization of <Emphasis Type="Italic">Trichoderma virens</Emphasis> UKM-1 endochitinase in <Emphasis Type="Italic">Escherichia</Emphasis> <Emphasis Type="Italic">coli</Emphasis>

A gene encoding endochitinase from Trichoderma virens UKM-1 was cloned and expressed in E. coli BL21 (DE3). Both the endochitinase gene and its cDNA sequences were obtained. The endochitinase gene encodes 430 amino acids from an open reading frame comprising of 1,690 bp nucleotide sequence with three introns. The endochitinase was expressed as soluble and active enzyme at 20°C when induced with 1 mM IPTG. Maximum activity was observed at 4 h of post-induction time. SDS-PAGE showed that the purified endochitinase exhibited a single band with molecular weight of 42 kDa. Biochemical characterization of the enzyme displayed a near neutral pH characteristic with an optimum pH at 6.0 and optimum temperature at 50°C. The enzyme is stable between pH 3.0–7.0 and is able to retain its activity from 30 to 60°C. The presence of Mg²⁺ and Ca²⁺ ions increased the enzyme activity up to 20%. The purified enzyme has a strong affinity towards colloidal chitin and low effect on ethyl cellulose and D-cellubiose which are non-chitin related substrates. HPLC analysis from the chitin hydrolysis showed the release of (GlcNAc)₃, (GlcNAc)₂ and GlcNAc, in which (GlcNAc)₂ was the main product.     

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.     

Characteristics of β-lactamases and their genes (<Emphasis Type="Italic">blaA</Emphasis> and <Emphasis Type="Italic">blaB</Emphasis>) in <Emphasis Type="Italic">Yersinia intermedia</Emphasis> and <Emphasis Type="Italic">Y. frederiksenii</Emphasis>

Background  

The presence of β-lactamases in Y. enterocolitica has been reported to vary with serovars, biovars and geographical origin of the isolates. An understanding of the β-lactamases in other related species is important for an overall perception of antibiotic resistance in yersiniae. The objective of this work was to study the characteristics of β-lactamases and their genes in strains of Y. intermedia and Y. frederiksenii, isolated from clinical and non-clinical sources in India.     

Retiolitid graptolites from the collection of Hermann Jaeger II: <Emphasis Type="Italic">Cometograptus</Emphasis>, <Emphasis Type="Italic">Spinograptus</Emphasis> and <Emphasis Type="Italic">Plectograptus</Emphasis>

Graptolites from the Jaeger collection at the Museum für Naturkunde (Berlin, Germany) provide important information on structural details of Silurian (Wenlock–Ludlow) retiolitids as well as for the biostratigraphic and biogeographic distribution of these magnificent graptolites. Species of the genera Cometograptus, Spinograptus and Plectograptus are described from isolated glacial boulder material, collected in northern Germany and from shale specimens found in the Lower Graptolite Shale of Thuringia. The biostratigraphic placement of material derived from glacial erratic boulders, however, is far from being precise. The fauna associated with the neotype of Plectograptus macilentus in the ‘Unterer Graptolithenschiefer’ of Thuringia is discussed and illustrated. Cometograptus alfeisenacki from the Cyrtograptus lundgreni Biozone is recognized as a new species. The genus is discovered for the first time in North German glacial erratic boulders.     

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.     

Fertility and precocity of <Emphasis Type="Italic">Osmunda</Emphasis> × <Emphasis Type="Italic">intermedia</Emphasis> offspring in culture

The feasibility of later-generation hybrid production in ferns has not been previously studied, although it is a significant factor in relation to reproductive isolation. Osmunda × intermedia, a hybrid between O. japonica and O. lancea, is semifertile and has moderate spore germination rates. Under the artificial conditions of this study, F2 and F3 offspring were formed. Some of the F2 offspring showed precocity, and some of the F3 offspring also showed precocity. This fertility suggests that introgressive hybridization might be ongoing in nature. This also indicates a currently unknown genetic control over the timing of fertile frond production in Osmunda.     

Cryopreservation of hairy root cultures of <Emphasis Type="Italic">Maesa lanceolata</Emphasis> and <Emphasis Type="Italic">Medicago truncatula</Emphasis>

To study the production of secondary metabolites of Maesa lanceolata and Medicago truncatula, hairy root cultures of both plant species were established. Because maintenance of large numbers of cultures is laborious and costly, we developed a cryopreservation protocol and stored different isolated lines over time. Using encapsulation-dehydration, high survival rates were observed for both Maesa and Medicago hairy roots. Root tips were isolated and encapsulated in calcium-alginate beads, containing 0.1 M sucrose. The encapsulated hairy roots were precultured for 3 days using basal medium containing high sucrose concentrations. Medicago root tip growth during the preculturing time lead to unwanted outgrowth which could be tempered by addition of plant growth inhibitors. After preculturing, the beads were dehydrated in the air flow of a laminar flow until 35–40% of the initial bead weight was reached. Dehydrated beads were plunged into liquid nitrogen and after different storage times thawed in a water bath at 40°C. The survival rates were 90% for Maesa and 53% for Medicago, which are sufficient to allow implementation in large storage experimental set-ups.     

Expression profiles of <Emphasis Type="Italic">amh</Emphasis> and <Emphasis Type="Italic">foxl2</Emphasis> in <Emphasis Type="Italic">Schizothorax kozlovi</Emphasis>, and their response to temperature during the early developmental stage

To elucidate the role of amh and foxl2 in sex differentiation of the teleost fish Schizothorax kozlovi, the full-length cDNAs were cloned from the mature testis and ovary by rapid amplification of cDNA ends (RACE), and their relative mRNA expression levels were determined by quantitative real-time polymerase chain reaction among tissues and temperature groups. The complete amh and foxl2 cDNAs of S. kozlovi were 2060 bp and 1750 bp, which encoded 568 and 306 amino acids, respectively. The amh were expressed only in gonads, while foxl2 was expressed in the gills, brain and gonads, both exhibiting relatively high tissue specificity. The amh exhibited sex-specific expression pattern in the gonads. No sex differences in the foxl2 expression were observed in the brain and gonads, but significant sex differences were found in the gills. No significant differences were found in the foxl2 expression, from the larval to the juvenile stage, and also between different temperature groups. However, significant differences were found in the expression levels of amh from the larval (12–63 days posthatching (dph)) to the juvenile stage (190 dph), and also among the \(18{^{\circ }}\hbox {C}\) and \(10{^{\circ }}\hbox {C}\) groups at 31 dph. This result suggested that amh plays an important role in male sex differentiation of S. kozlovi during the early developmental stage, but no similar effect was observed in foxl2.     

<Emphasis Type="Italic">Apis mellifera</Emphasis> and <Emphasis Type="Italic">Osmia cornuta</Emphasis> as carriers for the secondary spread of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> on apple flowers

The efficiency of two pollinators, Apis mellifera L. (Hymenoptera: Apidae) and the mason bee Osmia cornuta (Latreille) (Hymenoptera: Megachilidae), as carriers of biocontrol agents (BCA) from flower to flower (secondary colonisation) was investigated on apple cv ‘Golden Delicious’. The BCA tested was Bacillus subtilis, strain BD170 (Biopro®) developed for the control of the ‘fire blight’ caused by Erwinia amylovora (Burril) Winslow et al. The two insect species were studied as secondary BCA carriers on apple plants in pots under net screened tunnels. Their behaviour and capacity to deposit the BCA in the most receptive flower parts were compared both by washing, diluting and plating the flower organs on a recovery medium and by means of PCR analyses based on a molecular marker. O. cornuta showed better performances with respect to A. mellifera. For the field trials, pollinators were introduced in four apple orchards. During apple’s flowering, the BD170 (100 g hl^?l) was sprayed once in two fields, and twice in the others. The pollinators’ efficacy in carrying the BCA from sprayed flowers to the stigmas of newly opened ones at different times after the spray treatment was evaluated. The detection of the BCA was performed by PCR analysis. The percentages of positive PCR flower samples were higher in the internal treated areas of the fields with respect to the external untreated ones, but the high colonisation level found in the latter and in the flowers opened in both areas several days after the treatment(s) demonstrated that pollinators can play an important role as secondary carriers.     

Metabolic engineering of <Emphasis Type="Italic">Lilium</Emphasis> × <Emphasis Type="Italic">formolongi</Emphasis> using multiple genes of the carotenoid biosynthesis pathway

Lilium × formolongi was genetically engineered by Agrobacterium-mediated transformation with the plasmid pCrtZW-N8idi-crtEBIY, which contains seven enzyme genes under the regulation of the CaMV 35S promoter. In the transformants, ketocarotenoids were detected in both calli and leaves, which showed a strong orange color. In transgenic calli, the total amount of carotenoids [133.3 μg/g fresh weight (FW)] was 26.1-fold higher than in wild-type calli. The chlorophyll content and photosynthetic efficiency in transgenic orange plantlets were significantly lowered; however, after several months of subculture, they had turned into plantlets with green leaves that showed significant increases in chlorophyll and photosynthetic efficiency. The total carotenoid contents in leaves of transgenic orange and green plantlets were quantified at 102.9 and 135.2 μg/g FW, respectively, corresponding to 5.6- and 7.4-fold increases over the levels in the wild-type. Ketocarotenoids such as echinenone, canthaxanthin, 3′-hydroxyechinenone, 3-hydroxyechinenone, and astaxanthin were detected in both transgenic calli and orange leaves. A significant change in the type and composition of ketocarotenoids was observed during the transition from orange transgenic plantlets to green plantlets. Although 3′-hydroxyechinenone, 3-hydroxyechinenone, astaxanthin, and adonirubin were absent, and echinenone and canthaxanthin were present at lower levels, interestingly, the upregulation of carotenoid biosynthesis led to an increase in the total carotenoid concentration (+31.4%) in leaves of the transgenic green plantlets.     

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.     

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.     

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.