Diversity of<Emphasis Type="Italic"> Mhc</Emphasis> class I and IIB genes in house sparrows (<Emphasis Type="Italic">Passer domesticus</Emphasis>)

In order to understand the expression and evolution of host resistance to pathogens, we need to examine the links between genetic variability at the major histocompatibility complex (Mhc), phenotypic expression of the immune response and parasite resistance in natural populations. To do so, we characterized the Mhc class I and IIB genes of house sparrows with the goal of designing a PCR-based genotyping method for the Mhc genes using denaturing gradient gel electrophoresis (DGGE). The incredible success of house sparrows in colonizing habitats worldwide allows us to assess the importance of the variability of Mhc genes in the face of various pathogenic pressures. Isolation and sequencing of Mhc class I and IIB alleles revealed that house sparrows have fewer loci and fewer alleles than great reed warblers. In addition, the Mhc class I genes divided in two distinct lineages with different levels of polymorphism, possibly indicating different functional roles for each gene family. This organization is reminiscent of the chicken B complex and Rfp-Y system. The house sparrow Mhc hence appears to be intermediate between the great reed warbler and the chicken Mhc, both in terms of numbers of alleles and existence of within-class lineages. We specifically amplified one Mhc class I gene family and ran the PCR products on DGGE gels. The individuals screened displayed between one and ten DGGE bands, indicating that this method can be used in future studies to explore the ecological impacts of Mhc diversity.     

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.     

Expression of<Emphasis Type="Italic"> Sulfolobus solfataricus</Emphasis> α-glucosidase in<Emphasis Type="Italic"> Lactococcus lactis</Emphasis>

The industrial potential to use extreme thermophilic microorganisms and their enzymes lies in applications in which the temperature cannot be adjusted (cooled) at will. The production of enzymes from wild-type thermophiles is very low, therefore, for industrial applications, it is necessary to use recombinant microorganisms. In this paper, the cloning of a heat-stable -glucosidase from Sulfolobus solfataricus using lactic acid bacteria as expression system is reported. The extremophilic -glucosidase was cloned in Lactococcus lactis and correctly folded despite being expressed at a lower temperature. The recombinant cells were assayed for enzyme residual activity at 75 °C in order to analyze the direct use of whole cells as biocatalysts. Maximum activity corresponded to 40 U/l in static cultures. The protein yield was further improved by optimizing fermentation and reached 600 U/l in batch mode. Microfiltration led to an even higher enzyme production of 850 U/l as a result of increased biomass. The overall production of -glucosidase using the engineered L. lactis strain in microfiltration fermentation is 1,000-fold higher than obtained using the wild-type.     

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.     

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     

<Emphasis Type="Italic">Tracembler</Emphasis> – software for <Emphasis Type="Italic">in-silico</Emphasis> chromosome walking in unassembled genomes

Background  

Whole genome shotgun sequencing produces increasingly higher coverage of a genome with random sequence reads. Progressive whole genome assembly and eventual finishing sequencing is a process that typically takes several years for large eukaryotic genomes. In the interim, all sequence reads of public sequencing projects are made available in repositories such as the NCBI Trace Archive. For a particular locus, sequencing coverage may be high enough early on to produce a reliable local genome assembly. We have developed software, Tracembler, that facilitates in silico chromosome walking by recursively assembling reads of a selected species from the NCBI Trace Archive starting with reads that significantly match sequence seeds supplied by the user.     

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.     

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.     

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.     

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.     

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.     

Molecular population genetics of the<Emphasis Type="Italic">β-esterase</Emphasis> gene cluster of<Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

We have investigated nucleotide polymorphism at theβ-esterase gene cluster including theEst-6 gene andψEst-6 putative pseudogene in four samples ofDrosophila melanogaster derived from natural populations of southern Africa (Zimbabwe), Europe (Spain), North America (USA: California), and South America (Venezuela). A complex haplotype structure is revealed in bothEst-6 andψEst-6. Total nucleotide diversity is twice inψEst-6 as inEst-6; diversity is higher in the African sample than in the non-African ones. Strong linkage disequilibrium occurs within theβ-esterase gene cluster in non-African samples, but not in the African one. Intragenic gene conversion events are detected withinEst-6 and, to a much greater extent, withinyEst-6; intergenic gene conversion events are rare. Tests of neutrality with recombination are significant for theβ-esterase gene cluster in the non-African samples but not significant in the African one. We suggest that the demographic history (bottleneck and admixture of genetically differentiated populations) is the major factor shaping the pattern of nucleotide polymorphism in theb-esterase gene cluster. However there are some ’footprints’ of directional and balancing selection shaping specific distribution of nucleotide polymorphism within the cluster. Intergenic epistatic selection betweenEst-6 andψEst-6 may play an important role in the evolution of theβ-esterase gene cluster preserving the putative pseudogene from degenerative destruction and reflecting possible functional interaction between the functional gene and the putative pseudogene.Est-6 andyEst-6 may represent an indivisible intergenic complex (‘intergene’) in which each single component (Est-6 orψEst-6) cannot separately carry out the full functional role.     

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.     

Occurrence of<Emphasis Type="Italic">Fusarium</Emphasis> toxins in the 1999’s harvest

Continuing the monitoring ofFusarium toxins in cereals, we investigated 245 samples of wheat, barley, triticale and oats in 1999. 84 samples out of 100 analysed forFusarium could be found to be infected. The most prominentFusarium species detected whereF. avenaceum, F. poae, F. detected whereF. avenaceum, F. poae, F. graminearum, andF. sporotrichoides. The level of mycotoxin contamination of the samples varied depending on their origins and was in general very low in comparison with the result obtained in samples of the previous year. There where only some wheat samples with deoxynivalenol (DON) concentrations beyond existing advisory levels. The average DON concentration of all samples was 0.35 mg/kg with a median of 0.007 mg/kg. 3-Acetyldeoxynivalenol and zearalenone (ZEA) could only be detected at minor concentrations (below 0.1 and 0.05 mg/kg, respectively) in less than 10% of the samples. The analysis of commercial cereal flour reflects this situation. Flour bought in the first quarter of 1999, which was suspected to contain a high portion of the 1998 harvest, was contaminated by DON to a higher extent than those purchased in 2000. The average DON concentration in the flour samples of 1999 and 2000 was 0.35 mg/kg and 0.23 mg/kg, respectively. Although the general mycotoxin level in the 1999 harvest was lower as in 1998 there were some highly contaminated samples that had mainly been grown in fields with either maize or other cereals as previous crop and reduced tillage. The combination of maize as previous crop and non-tillage could be stated as most unsuitable, which promotes enhanced mycotoxin contamination, and should therefore be avoided.