Construction and characterization of a copy number-inducible fosmid library of Xanthomonas oryzae pathovar oryzae MAFF311018

A fosmid library of Xanthomonas oryzae pathovar oryzae MAFF311018 (T7174), the causative agent of bacterial blight on rice, was constructed and characterized. The average fosmid library insert size was > 34 kb, and 967 clones were uniquely positioned on its sequenced genome. The entire Xoo MAFF311018 genome was covered by end-sequenced clones with at least 5 kb of overlap. The fosmid vector contains both the single-copy Escherichia coli fertility factor origin, which enhances fosmid stability, and the multi-copy IncPα origin, allowing amplification of copy number upon induction with l-arabinose. Real-time quantitative PCR on 12 randomly picked fosmid library clones determined that fosmid copy number increased 8- to 58-fold after 5 hour induction. This library provides a new resource for complementation experiments and systematic functional studies in Xoo and related species.     

Expression of maize prolamins in Escherichia coli

We have constructed a cDNA expression library of developing corn (Zea mays L.) endosperm using plasmid pUC8 as vector and Escherichia coli strain DH1 as host. The expression library was screened with non-radioactive immunological probes to detect the expression of gamma-zein and alpha-zein. When anti-gamma-zein antibody was used as the probe, 23 colonies gave positive reactions. The lengths of cDNA inserts of the 23 colonies were found to be 250–900 base pairs. When anti-alpha zein antibody was used, however, fewer colonies gave positive reactions. The library was also screened by colony-hybridization with ³²P-labeled DNA probes. Based on immunological and hybridization screening of the library and other evidence, we conclude that alpha-zein was either toxic to E. coli cells or rapidly degraded whereas gamma-zein and its fragments were readily expressed.     

Babesia argentina, Plasmodium vivax and P. falciparum: antigenic cross-reactions

An indirect fluorescent antibody test was used to analyze the antigenic relationships between Babesia argentina, a parasite of cattle, and two human malaria parasites, Plasmodium falciparum and Plasmodium vivax. Elevated antibody titers to P. falciparum were found in cattle infected with B. argentina. Some persons infected with P. falciparum or P. vivax were found to produce antibodies to B. argentina. Explanations for the occurrence of these cross reactions are considered.     

The use of Tn5 transposable elements in a gene trapping strategy for the protozoan Leishmania

The use of transposable elements as a gene-trapping strategy is a powerful tool for gene discovery. Herein we describe the development of a transposable system, based on the bacterial Tn5 transposon, which has been used successfully in Leishmania braziliensis. The transposon carries the neomycin phosphotransferase gene, which is expressed only when inserted in-frame with a Leishmania gene present in the target DNA. Four cosmid clones from a L. braziliensis genomic library were used as targets in transposition reactions and four insertional libraries were constructed and transfected in L. braziliensis. Clones resistant to G418 were selected and analysed by immunofluorescence in order to identify the subcellular localisation of the protein coded by the trapped gene. A definitive subcellular localisation for neomycin phosphotransferase/targeted protein fusion was not obtained in any of the four Leishmania clones investigated. However, the constructed transposable element is highly efficient considering the frequency of insertion in large targets and is therefore a useful tool for functional genetic studies in Leishmania. Our data confirm the utility of the Tn5 transposon system for insertion of sequencing priming sites into target DNA. Furthermore, the high frequency of insertion and even distribution are important in studying genomic regions bearing long and polymorphic repetitive sequences.     

Differential gene expression of the honey bees Apis mellifera and A. cerana induced by Varroa destructor infection

Varroa destructor mite is currently the most serious threat to the world bee industry. Differences in mite tolerance are reported between two honey bee species Apis mellifera and Apis cerana. Differential gene expression of two honey bee species induced by V. destructor infection was investigated by constructing two suppression subtractive hybridization (SSH) libraries, as first steps toward elucidating molecular mechanisms of Varroa tolerance. From the SSH libraries, we obtained 289 high quality sequences which clustered into 132 unique sequences grouped in 26 contigs and 106 singlets where 49 consisted in A. cerana subtracted library and 83 in A. mellifera. Using BLAST, we found that 85% sequences had counterpart known genes whereas 15% were undescribed. A Gene Ontology analysis classified 51 unique sequences into different functional categories. Eight of these differentially expressed genes, representative of different regulation patterns, were confirmed by qRT-PCR. Upon the mite induction, the differentially expressed genes from both bee species were different, except hex 110 gene, which was up-regulated in A. cerana but down-regulated in A. mellifera, and Npy-r gene, which was down-regulated in both species. In general, most of the differential expression genes were involved in metabolic processes and nerve signaling. The results provide information on the molecular response of these two bee species to Varroa infection.     

Evolution of carotene desaturation: the complication of a simple pathway

In a series of desaturation reactions, the trienoic structures of phytoene and diapophytoene are extended to a maximum of 15 or 11 conjugated double bonds, respectively. After the cloning of several genes from bacteria and eukaryotes, the desaturation reactions were first analyzed in a heterologous host by functional genetic complementation. In addition, different desaturases were heterologously expressed and the reactions studied in vitro. This revealed that in archaea, non-photosynthetic prokaryotes and fungi the desaturases differ significantly from convergently evolved desaturases in cyanobacteria, Chlorobaculum (old name Chlorobium) species and eukaryotic photosynthetic organisms including plants. Detailed analysis of the desaturation reactions including the determination of the substrates converted by the enzymes, the intermediates and the products formed in the reactions revealed the bacterial all-trans desaturation pathway catalyzed by a single enzyme and the cyanobacterial/plant type poly-cis desaturation pathway which involves two closely related desaturases. This indicates that in the course of evolution of carotenogenesis from bacteria via cyanobacteria to plants, the simple situation of one enzyme for the entire reaction sequence from phytoene to all-trans lycopene changed to a more complex process. Three individual enzymes, newly acquired phytoene and ζ-carotene desaturases, as well as a carotene isomerase which is phylogenetically related to CrtI are involved. Only the CrtI-type enzymes seem to have the property to catalyze cis to trans conversion of carotenes.     

Begin at the beginning: A BAC-end view of the passion fruit (Passiflora) genome

Background

The passion fruit (Passiflora edulis) is a tropical crop of economic importance both for juice production and consumption as fresh fruit. The juice is also used in concentrate blends that are consumed worldwide. However, very little is known about the genome of the species. Therefore, improving our understanding of passion fruit genomics is essential and to some degree a pre-requisite if its genetic resources are to be used more efficiently. In this study, we have constructed a large-insert BAC library and provided the first view on the structure and content of the passion fruit genome, using BAC-end sequence (BES) data as a major resource.

Results

The library consisted of 82,944 clones and its levels of organellar DNA were very low. The library represents six haploid genome equivalents, and the average insert size was 108 kb. To check its utility for gene isolation, successful macroarray screening experiments were carried out with probes complementary to eight Passiflora gene sequences available in public databases. BACs harbouring those genes were used in fluorescent in situ hybridizations and unique signals were detected for four BACs in three chromosomes (n = 9). Then, we explored 10,000 BES and we identified reads likely to contain repetitive mobile elements (19.6% of all BES), simple sequence repeats and putative proteins, and to estimate the GC content (~42%) of the reads. Around 9.6% of all BES were found to have high levels of similarity to plant genes and ontological terms were assigned to more than half of the sequences analysed (940). The vast majority of the top-hits made by our sequences were to Populus trichocarpa (24.8% of the total occurrences), Theobroma cacao (21.6%), Ricinus communis (14.3%), Vitis vinifera (6.5%) and Prunus persica (3.8%).

Conclusions

We generated the first large-insert library for a member of Passifloraceae. This BAC library provides a new resource for genetic and genomic studies, as well as it represents a valuable tool for future whole genome study. Remarkably, a number of BAC-end pair sequences could be mapped to intervals of the sequenced Arabidopsis thaliana, V. vinifera and P. trichocarpa chromosomes, and putative collinear microsyntenic regions were identified.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-816) contains supplementary material, which is available to authorized users.     

Development of Full-Length cDNAs from Chinese Cabbage (Brassica rapa Subsp. pekinensis) and Identification of Marker Genes for Defence Response

Arabidopsis belongs to the Brassicaceae family and plays an important role as a model plant for which researchers have developed fine-tuned genome resources. Genome sequencing projects have been initiated for other members of the Brassicaceae family. Among these projects, research on Chinese cabbage (Brassica rapa subsp. pekinensis) started early because of strong interest in this species. Here, we report the development of a library of Chinese cabbage full-length cDNA clones, the RIKEN BRC B. rapa full-length cDNA (BBRAF) resource, to accelerate research on Brassica species. We sequenced 10 000 BBRAF clones and confirmed 5476 independent clones. Most of these cDNAs showed high homology to Arabidopsis genes, but we also obtained more than 200 cDNA clones that lacked any sequence homology to Arabidopsis genes. We also successfully identified several possible candidate marker genes for plant defence responses from our analysis of the expression of the Brassica counterparts of Arabidopsis marker genes in response to salicylic acid and jasmonic acid. We compared gene expression of these markers in several Chinese cabbage cultivars. Our BBRAF cDNA resource will be publicly available from the RIKEN Bioresource Center and will help researchers to transfer Arabidopsis-related knowledge to Brassica crops.     

Probing electron transfer reactions between two azurins from Alcaligenes xylosoxidans GIFU 1051 with optically active Ru complexes as molecular recognition probes: Importance of the 43rd residue

Electron transfer reactions between optically-active Ru^II/III complexes incorporating (S)-/(R)-amino acids, and the two azurins, azurin-1 (az-1Cu) and azurin-2 (az-2Cu) isolated from Alcaligenes xylosoxidans GIFU 1051, have been studied to probe molecular recognition sites on the two azurins. The Ru^II/III complexes are K[Ru^II(L)(bpy)] and [Ru^III(L)(bpy)], and have a tripodal ligand (L) derived from the (S)-/(R)-amino acids, which are in turn exchanged for other functional substituent groups, such as (S)-/(R)-phenylalanine, -leucine, -valine, -alanine, and -glutamic acid (L = (S)-/(R)-BCMPA, -BCMLE, -BCMVA, -BCMAL, and -BCMGA). In the oxidation reaction of az-1Cu^I promoted by the Ru^III complexes, the kinetic parameters exhibited enantio- and stereo-selectivities, while the same reaction of az-2Cu^I was less enantio- and stereo-selective. These differences suggest that the processes of formation of the activated states are different for the two azurins. On the other hand, such a difference has not been observed for az-1 and az-2 with respect to the reduction reactions promoted by both azurins Cu^II by the Ru^II complexes within the experimental error. This suggests that the neutrality of the Ru complexes is important for precise molecular recognition of azurins. His117 has been proposed as the electron transfer site. The local structures in the vicinity of the His117 side chain in the two azurins, are essentially identical with the exception of the 43rd residue, Val43 and Ala43 for az-1 and az-2, respectively. Electron transfer reactions between Ru^III complexes and a mutant azurin, V43A-az-1, were also carried out. Interestingly, the activation parameters estimated were very similar to those of az-2, indicating that the 43rd residue acts as the electron transfer site in azurins and provides rationalization for the different mechanisms of az-1 and az-2 in redox reactions.     

Using FAME Analysis to Compare, Differentiate, and Identify Multiple Nematode Species

We have adapted the Sherlock^® Microbial Identification system for identification of plant parasitic nematodes based on their fatty acid profiles. Fatty acid profiles of 12 separate plant parasitic nematode species have been determined using this system. Additionally, separate profiles have been developed for Rotylenchulus reniformis and Meloidogyne incognita based on their host plant, four species and three races within the Meloidogyne genus, and three life stages of Heterodera glycines. Statistically, 85% of these profiles can be delimited from one another; the specific comparisons between the cyst and vermiform stages of H. glycines, M. hapla and M. arenaria, and M. arenaria and M. javanica cannot be segregated using canonical analysis. By incorporating each of these fatty acid profiles into the Sherlock^® Analysis Software, 20 library entries were created. While there was some similarity among profiles, all entries correctly identified the proper organism to genus, species, race, life stage, and host at greater than 86% accuracy. The remaining 14% were correctly identified to genus, although species and race may not be correct due to the underlying variables of host or life stage. These results are promising and indicate that this library could be used for diagnostics labs to increase response time.     

Construction and evaluation of pMycoFos, a fosmid shuttle vector for Mycobacterium spp. with inducible gene expression and copy number control

Molecular tools for Gram-positive bacteria such as Mycobacterium are less well-developed than those for Gram-negatives such as Escherichiacoli. This has slowed the molecular-genetic characterisation of Mycobacterium spp, which is unfortunate, since this genus has high medical, environmental and industrial significance. Here, we developed a new Mycobacterium shuttle vector (pMycoFos, 12.5 kb, Km^R) which combines desirable features of several previous vectors (controllable copy number in E. coli, inducible gene expression in Mycobacterium) and provides a new multiple cloning site compatible with large inserts of high-GC content DNA. Copy number control in E. coli was confirmed by the increased Km^R of cultures after arabinose induction and the greater DNA yield of vector from arabinose-induced cultures. Measurement of beta-galactosidase activity in pMycoFos clones carrying the lacZ gene showed that in Mycobacterium smegmatis mc²-155, expression was inducible by acetamide, but in E. coli EPI300, the expression level was primarily determined by the vector copy number. Examination of protein profiles on SDS-PAGE gels confirmed the beta-galactosidase assay results. Construction of a fosmid library with the new vector confirmed that it could carry large DNA inserts. The new vector enabled the stable cloning and expression of an ethene monooxygenase gene cluster, which had eluded previous attempts at heterologous expression.     

Comparative molecular cytogenetics of major repetitive sequence families of three Dendrobium species (Orchidaceae) from Bangladesh

Background and Aims

Dendrobium species show tremendous morphological diversity and have broad geographical distribution. As repetitive sequence analysis is a useful tool to investigate the evolution of chromosomes and genomes, the aim of the present study was the characterization of repetitive sequences from Dendrobium moschatum for comparative molecular and cytogenetic studies in the related species Dendrobium aphyllum, Dendrobium aggregatum and representatives from other orchid genera.

Methods

In order to isolate highly repetitive sequences, a c₀t-1 DNA plasmid library was established. Repeats were sequenced and used as probes for Southern hybridization. Sequence divergence was analysed using bioinformatic tools. Repetitive sequences were localized along orchid chromosomes by fluorescence in situ hybridization (FISH).

Key Results

Characterization of the c₀t-1 library resulted in the detection of repetitive sequences including the (GA)_n dinucleotide DmoO11, numerous Arabidopsis-like telomeric repeats and the highly amplified dispersed repeat DmoF14. The DmoF14 repeat is conserved in six Dendrobium species but diversified in representative species of three other orchid genera. FISH analyses showed the genome-wide distribution of DmoF14 in D. moschatum, D. aphyllum and D. aggregatum. Hybridization with the telomeric repeats demonstrated Arabidopsis-like telomeres at the chromosome ends of Dendrobium species. However, FISH using the telomeric probe revealed two pairs of chromosomes with strong intercalary signals in D. aphyllum. FISH showed the terminal position of 5S and 18S–5·8S–25S rRNA genes and a characteristic number of rDNA sites in the three Dendrobium species.

Conclusions

The repeated sequences isolated from D. moschatum c₀t-1 DNA constitute major DNA families of the D. moschatum, D. aphyllum and D. aggregatum genomes with DmoF14 representing an ancient component of orchid genomes. Large intercalary telomere-like arrays suggest chromosomal rearrangements in D. aphyllum while the number and localization of rRNA genes as well as the species-specific distribution pattern of an abundant microsatellite reflect the genomic diversity of the three Dendrobium species.     

Peptides panned from a phage-displayed random peptide library are useful for the detection of Bacillus anthracis surrogates B. cereus 4342 and B. anthracis Sterne

Recent use of Bacillus anthracis as a bioweapon has highlighted the need for a sensitive monitoring system. Current bacterial detection tests use antibodies as bio-molecular recognition elements which have limitations with regard to time, specificity and sensitivity, creating the need for new and improved cost-effective high-affinity detection probes. In this study, we screened a commercially available bacteriophage-displayed random peptide library using Bacillus cereus 4342 cells as bait to identify peptides that could be used for detection of Bacillus. The method enabled us to identify two 12-amino acid consensus peptide sequences that specifically bind to B. cereus 4342 and B. anthracis Sterne, the nonpathogenic surrogates of B. anthracis strain. The two Bacillus-binding peptides (named BBP-1 and BBP-2) were synthesized with biotin tag to confirm their binding by four independent detection assays. Dot-blot analysis revealed that the peptides bind specifically to B. cereus 4342 and B. anthracis Sterne. Quantitative analysis of this interaction by ELISA and fluorometry demonstrated a detection sensitivity of 10² colony forming U/ml (CFU/ml) by both assays. When the peptides were used in combination with Qdots, the sensitivity was enhanced further by enabling detection of even a single bacterium by fluorescence microscopy. Immunoblot analysis and protein sequencing showed that BBP-1 and BBP-2 bound to the S-layer protein of B. anthracis Sterne. Overall, our findings validate the usefulness of synthetic versions of phage-derived peptides in combination with Qdot-liquid nanocrystals as high sensitivity bioprobes for various microbial detection platforms.     

Stepwise oxygenations of toluene and 4-nitrotoluene by a fungal peroxygenase

Fungal peroxygenases have recently been shown to catalyze remarkable oxidation reactions. The present study addresses the mechanism of benzylic oxygenations catalyzed by the extracellular peroxygenase of the agaric basidiomycete Agrocybe aegerita. The peroxygenase oxidized toluene and 4-nitrotoluene via the corresponding alcohols and aldehydes to give benzoic acids. The reactions proceeded stepwise with total conversions of 93% for toluene and 12% for 4-nitrotoluene. Using H₂¹⁸O₂ as the co-substrate, we show here that H₂O₂ is the source of the oxygen introduced at each reaction step. A. aegerita peroxygenase resembles cytochromes P450 and heme chloroperoxidase in catalyzing benzylic hydroxylations.     

Wing morphometrics as a possible tool for the diagnosis of the Ceratitis fasciventris,C. anonae,C. rosa complex (Diptera,Tephritidae)

Previous attempts to resolve the Ceratitis FAR complex (Ceratitis fasciventris, Ceratitis anonae, Ceratitis rosa, Diptera, Tephritidae) showed contrasting results and revealed the occurrence of five microsatellite genotypic clusters (A, F1, F2, R1, R2). In this paper we explore the potential of wing morphometrics for the diagnosis of FAR morphospecies and genotypic clusters. We considered a set of 227 specimens previously morphologically identified and genotyped at 16 microsatellite loci. Seventeen wing landmarks and 6 wing band areas were used for morphometric analyses. Permutational multivariate analysis of variance detected significant differences both across morphospecies and genotypic clusters (for both males and females). Unconstrained and constrained ordinations did not properly resolve groups corresponding to morphospecies or genotypic clusters. However, posterior group membership probabilities (PGMPs) of the Discriminant Analysis of Principal Components (DAPC) allowed the consistent identification of a relevant proportion of specimens (but with performances differing across morphospecies and genotypic clusters). This study suggests that wing morphometrics and PGMPs might represent a possible tool for the diagnosis of species within the FAR complex. Here, we propose a tentative diagnostic method and provide a first reference library of morphometric measures that might be used for the identification of additional and unidentified FAR specimens.     

Synthesis of cis-TMCDA: Optimization and characterization of a key intermediate

We report optimized synthetic procedures to cis-TMCDA and cis-cyclohexane-1,2-diamine. Our new two-step approach is an efficient and low-cost method for synthesizing the ligand cis-TMCDA. In the preparation of cis-cyclohexane-1,2-diamine, the crystallization of the unexpected intermediate cis-cyclohexane-1,2-diammonium hydrogensulfate ethylsulfate made the subsequent reactions easier in their workup, as nickel(II) chloride impurities were eliminated.     

Larval settlement can explain the adult distribution of Mytilus californianus Conrad but not of M. galloprovincialis Lamarck or M. trossulus Gould in Moss Landing, central California: Evidence from genetic identification of spat

We investigated the spatial distribution of adult and newly settled mussels (Mytilus galloprovincialis Lamarck, Mytilus trossulus Gould and Mytilus californianus Conrad) on the shore at Moss Landing, California to test the hypothesis that adult distributions are a result of settlement patterns. Adult M. californianus were most abundant on a wave-exposed rocky jetty and adults of Blue mussels (M. trossulus and M. galloprovincialis) were more abundant inside the protected Moss Landing harbor. Using taxon-specific polymerase chain reactions, we monitored recruitment during continuous 1-2 week intervals on fibrous scrubbing pads for 12 months in 2002-2003. All mussel species settled in greatest numbers on the exposed jetty, and Blue mussels settled in greater numbers there than did M. californianus. Because Blue mussels settled abundantly where their adults were rare, post-settlement mortality appeared to be the strongest influence on adult distribution. In contrast, M. californianus settled mostly in their adult habitat.     

A randomized library approach to identifying functional lox site domains for the Cre recombinase

The bacteriophage P1 Cre/loxP site-specific recombination system is a useful tool in a number of genetic engineering processes. The Cre recombinase has been shown to act on DNA sequences that vary considerably from that of its bacteriophage recognition sequence, loxP. However, little is known about the sequence requirements for functional lox-like sequences. In this study, we have implemented a randomized library approach to identify the sequence characteristics of functional lox site domains. We created a randomized spacer library and a randomized arm library, and then tested them for recombination in vivo and in vitro. Results from the spacer library show that, while there is great plasticity, identity between spacer pairs is the most important factor influencing function, especially in in vitro reactions. The presence of one completely randomized arm in a functional loxP recombination reaction revealed that only three wild-type loxP arms are necessary for successful recombination in Cre-expressing bacteria, and that there are nucleotide preferences at the first three and last three positions of the randomized arm for the most efficiently recombined sequences. Finally, we found that in vitro Cre recombination reactions are much more stringent for evaluating which sequences can support efficient recombination compared to the 294-CRE system.