Construction and characterization of a fosmid library for pathogenic bacterium Vibrio anguillarum

Vibrio anguillarum is a common bacterial pathogen in fish. However, little is known about its pathogenic mechanism, in part, because the entire genome has not been completely sequenced. We constructed a fosmid library for V. anguillarum containing 960 clones with an average insert size of 37.7 kb and 8.6-fold genome coverage. We characterized the library by end-sequencing 50 randomly selected clones. This generated 93 sequences with a total length of 57 485 by covering 1.4% of the whole genome. Of these sequences, 58 (62.4%) were homologous to known genes, 30 (32.3%) were genes with hypothetical functions, and the remaining 5 (5.3%) were unknown genes. We demonstrated the utility of this library by PCR screening of 10 genes. This resulted in an average of 6.2 fosmid clones per screening. This fosmid library offers a new tool for gene screening and cloning of V. anguillarum, and for comparative genomic studies among Vibrio species.

Construction and characterization of a bacterial artificial chromosome library of marine macroalga Porphyra yezoensis (Rhodophyta)

A large-DNA-fragment library is necessary for research into the Porphyra genome. In this study, a bacterial artificial chromosome (BAC) library of Porphyra yezoensis was constructed and characterized. The library contains 54,144 BAC clones with an average insert size of about 65 kb and fewer than 0.7% of clones without large inserts. Therefore, its capacity is more than 6.6 P. yezoensis genome equivalents, and the probability of recovering any nuclear DNA sequence from the library is higher than 99%. The library shows good fidelity and stability. A putative trehalose-6-phosphate synthase (TPS) gene was successfully screened out from the library. The above results show that the library is useful for gene cloning and genomic research in P. yezoensis.

Discovery of genes involved in defense/immunity functions in a haemocytes cDNA library from Fenneropenaeus chinensis by ESTs annotation

Crustacean haemocytes play important roles in the host immune response including recognition, phagocytosis, melanization, cytotoxicity and inter-cellular signal communication. Expressed sequence tags (ESTs) analysis is proved to be an efficient approach not only for gene discovery, but also for gene expression profiles performance. In order to further understand the innate immune system and defense mechanisms of Chinese shrimp at molecular level, complementary DNA library is constructed from the haemocyte tissue of Fenneropenaeus chinensis. A total of 2371 cDNA clones are successfully sequenced and the average sequence length is 460 bp. About 50% are identified as orthologs of known genes from other organisms by BLASTx and BLASTn program. By sequences comparability and analysis, 34 important genes including 177 ESTs are identified that may be involved in defense or immune functions in shrimp based on the known knowledge. These genes are categorized into five categories according to their putative functions in shrimp immune system: 13 genes are different types of antimicrobial peptides (AMP, penaeidin, antilipopolysaccharide factor, etc.), and their proportion is about 3 8%; 11 genes belong to prophenoloxidase system (prophenoloxidase, serine proteinase, serine proteinase inhibitor, etc.), and their proportion is about 32%; five genes have high homology with clotting protein (lectin, transglutaminase, etc), and their proportion is about 15%; three genes may be involved in inter-cell signal communication (peroxinectin, integrin), and their proportion is about 9%; two genes have been identified to be chaperone proteins (Hsc70, thioredoxin peroxidase), and their proportion is about 6%. These EST sequences enrich our understanding of the immune genes of F chinensis and will help farther experimental research into immune factors and improve our knowledge of the immune mechanisms of shrimp. (c) 2007 Elsevier B.V. All rights reserved.

Sequence analysis of Arthrospira maxima based on fosmid library

Arthrospira (Spirulina) (Setchell& Gardner) is an important cyanobacterium not only in its nutritional potential but in its special biological characteristics. An unbiased fosmid library of Arthrospira maxima FACHB438 that contains 4300 clones was constructed. The size distribution of insert fragments is from 15.5 to 48.9 kb and the average size is 37.6 kb. The recombination frequency is 100%. Therefore the library is 29.9 equivalents to the Arthrospira genome size of 5.4 Mb. A total of 719 sample clones were randomly chosen from the library and 602 available sequences, which consisted of 307,547 bases, covering 5.70% of the whole genome. The codon usage of A. maxima was not strongly biased. GC content at the first position of codons (46.9%) was higher than the second (39.8%) and the third (45.5%) positions. GC content of the genome was 43.6%. Of these sequences, 287 (47.7%) showed high similarities to known genes, 63 (10.5%) to hypothetical genes and the remaining 252 (41.8%) had no significant similarities. The assigned genes were classified into 22 categories with respect to different biological roles. Remarkably, the high presence of 25 sequences (4.2%) encoding reverse transcriptase indicates the RT gene may have multiple copies in the A. maxima genome and might play an important role in the evolutionary history and metabolic regulation. In addition, the sequences encoding the ATP-binding cassette transport system and the two-component signal transduction system were the second and third most frequent genes, respectively. These genomic features provide some clues as to the mechanisms by which this organism adapts to the high concentration of bicarbonate and to the high pH environment.

In vitro selected peptides bind with thymidylate synthase mRNA and inhibit its translation

Thymidylate synthase (TS), an essential enzyme for catalyzing the biosynthesis of thymidylate, is a critical therapeutic target in cancer therapy. Recent studies have shown that TS functions as an RNA-binding protein by interacting with two different sequences on its own mRNA, thus, repressing translational efficiency. In this study, peptides binding TS RNA with high affinity were isolated using mRNA display from a large peptide library (>10(13) different sequences). The randomized library was subjected up to twelve rounds of in vitro selection and amplification. Comparing the amino acid composition of the selected peptides (12th round, R12) with those from the initial random library (round zero, R0), the basic and aromatic residues in the selected peptides were enriched significantly, suggesting that these peptide regions might be important in the peptide-TS mRNA interaction. Categorizing the amino acids at each random position based on their physicochemical properties and comparing the distributions with those of the initial random pool, an obvious basic charge characteristic was found at positions 1, 12, 17 and 18, suggesting that basic side chains participate in RNA binding. Secondary structure prediction showed that the selected peptides of R12 pool represented a helical propensity compared with R0 pool, and the regions were rich in basic residues. The electrophoretic gel mobility shift and in vitro translation assays showed that the peptides selected using mRNA display could bind TS RNA specifically and inhibit the translation of TS mRNA. Our results suggested that the identified peptides could be used as new TS inhibitors and developed to a novel class of anticancer agents.

Characterization and mapping of expressed sequence tags isolated from a subtracted cDNA library of Litopenaeus vannamei injected with white spot syndrome virus

A large number of polymorphic simple sequence repeats (SSRs) or microsatellites are needed to develop a genetic map for shrimp. However, developing an SSR map is very time-consuming, expensive, and most SSRs are not specifically linked to gene loci of immediate interest. We report here on our strategy to develop polymorphic markers using expressed sequence tags (ESTs) by designing primers flanking single or multiple SSRs with three or more repeats. A subtracted cDNA library was prepared using RNA from specific pathogen-free (SPF) Litopenaeus vannamei juveniles (similar to 1 g) collected before (0) and after (48 h) inoculation with the China isolate of white spot syndrome virus (WSSV). A total of 224 clones were sequenced, 194 of which were useful for homology comparisons against annotated genes in NCBI nonredundant (nr) and protein databases, providing 179 sequences encoded by nuclear DNA, 4 mitochondrial DNA, and 11 were similar to portions of WSSV genome. The nuclear sequences clustered in 43 groups, 11 of which were homologous to various ESTs of unknown function, 4 had no homology to any sequence, and 28 showed similarities to known genes of invertebrates and vertebrates, representatives of cellular metabolic processes such as calcium ion balance, cytoskeleton mRNAs, and protein synthesis. A few sequences were homologous to immune system-related (allergens) genes and two were similar to motifs of the sex-lethal gene of Drosophila. A large number of EST sequences were similar to domains of the EF-hand superfamily (Ca2+ binding motif and FRQ protein domain of myosin light chains). Single or multiple SSRs with three or more repeats were found in approximately 61 % of the 179 nuclear sequences. Primer sets were designed from 28 sequences representing 19 known or putative genes and tested for polymorphism (EST-SSR marker) in a small test panel containing 16 individuals. Ten (53%) of the 19 putative or unknown function genes were polymorphic, 4 monomorphic, and 3 either failed to satisfactorily amplify genomic DNA or the allele amplification conditions need to be further optimized. Five polymorphic ESTs were genotyped with the entire reference mapping family, two of them (actin, accession #CX535973 and shrimp allergen arginine kinase, accession #CX535999) did not amplify with all offspring of the IRMF panel suggesting presence of null alleles, and three of them amplified in most of the IRM F offspring and were used for linkage analysis. EF-hand motif of myosin light chain (accession #CX535935) was placed in ShrimpMap's linkage group 7, whereas ribosomal protein S5 (accession #CX535957) and troponin I (accession #CX535976) remained unassigned. Results indicate that (a) a large number of ESTs isolated from this cDNA library are similar to cytoskeleton mRNAs and may reflect a normal pathway of the cellular response after im infection with WSSV, and (b) primers flanking single or multiple SSRs with three or more repeats from shrimp ESTs could be an efficient approach to develop polymorphic markers useful for linkage mapping. Work is underway to map additional SSR-containing ESTs from this and other cDNA libraries as a plausible strategy to increase marker density in ShrimpMap.

Fine-scale vertical distribution of bacteria in the East Pacific deep-sea sediments determined via 16S rRNA gene T-RFLP and clone library analyses

Although the deep-sea sediments harbor diverse and novel bacteria with important ecological and environmental functions, a comprehensive view of their community characteristics is still lacking, considering the vast area and volume of the deep-sea sedimentary environments. Sediment bacteria vertical distribution and community structure were studied of the E272 site in the East Pacific Ocean with the molecular methods of 16S rRNA gene T-RFLP (terminal restriction fragment length polymorphism) and clone library analyses. Layered distribution of the bacterial assemblages was detected by both methods, indicating that the shallow sediments (40 cm in depth) harbored a diverse and distinct bacterial composition with fine-scale spatial heterogeneity. Substantial bacterial diversity was detected and nine major bacterial lineages were obtained, including Acidobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, Nitrospirae, Planctomycetes, Proteobacteria, and the candidate divisions OP8 and TM6. Three subdivisions of the Proteobacteria presented in our libraries, including the alpha-, gamma- and delta-Proteobacteria. Most of our sequences have low similarity with known bacterial 16S rRNA genes, indicating that these sequences may represent as-yet-uncultivated novel bacteria. Most of our sequences were related to the GenBank nearest neighboring sequences retrieved from marine sediments, especially from deep-sea methane seep, gas hydrate or mud volcano environments. Several sequences were related to the sequences recovered from the deep-sea hydrothermal vent or basalt glasses-bearing sediments, indicating that our deep-sea sampling site might be influenced to certain degree by the nearby hydrothermal field of the East Pacific Rise at 13A degrees N.

A bioaugmentation failure caused by phage infection and weak biofilm formation ability

Two biological aerated filters (BAF) were setup for ammonia removal treatment of the circulation water in a marine aquaculture. One of the BAFs was bioaugmented with a heterotrophic nitrifying bacterium, Lutimonas sp. H10, where the ammonia removal was not improved and the massive inoculation was even followed by a nitrification breakdown from day 9 to 18. The nitrification was remained stable in control BAF operated under the same conditions. Fluorescent in situ hybridization (FISH) with rRNA-targeted probes and cultivable method revealed that Lutimonas sp. H10 almost disappeared from the bioaugomented BAF within 3 d, and this was mainly due to the infection of a specific phage as revealed by flask experiment, plaque assay and transmission electron observation. Analyses of 16S rRNA gene libraries showed that bacterial groups from two reactors evolved differently and an overgrowth of protozoa was observed in the bioaugmented BAR Therefore, phage infection and poor biofilm forming ability of the inoculated strain are the main reasons for bioaugmentation failure. In addition, gazing by protozoa of the bacteria might be the reason for the nitrification breakdown in bioaugmented BAF during day 9-18.

Tactual Display of Consonant Voicing to Supplement Lipreading

This research is concerned with the development of tactual displays to supplement the information available through lipreading. Because voicing carries a high informational load in speech and is not well transmitted through lipreading, the efforts are focused on providing tactual displays of voicing to supplement the information available on the lips of the talker. This research includes exploration of 1) signal-processing schemes to extract information about voicing from the acoustic speech signal, 2) methods of displaying this information through a multi-finger tactual display, and 3) perceptual evaluations of voicing reception through the tactual display alone (T), lipreading alone (L), and the combined condition (L+T). Signal processing for the extraction of voicing information used amplitude-envelope signals derived from filtered bands of speech (i.e., envelopes derived from a lowpass-filtered band at 350 Hz and from a highpass-filtered band at 3000 Hz). Acoustic measurements made on the envelope signals of a set of 16 initial consonants represented through multiple tokens of C1VC2 syllables indicate that the onset-timing difference between the low- and high-frequency envelopes (EOA: envelope-onset asynchrony) provides a reliable and robust cue for distinguishing voiced from voiceless consonants. This acoustic cue was presented through a two-finger tactual display such that the envelope of the high-frequency band was used to modulate a 250-Hz carrier signal delivered to the index finger (250-I) and the envelope of the low-frequency band was used to modulate a 50-Hz carrier delivered to the thumb (50T). The temporal-onset order threshold for these two signals, measured with roving signal amplitude and duration, averaged 34 msec, sufficiently small for use of the EOA cue. Perceptual evaluations of the tactual display of EOA with speech signal indicated: 1) that the cue was highly effective for discrimination of pairs of voicing contrasts; 2) that the identification of 16 consonants was improved by roughly 15 percentage points with the addition of the tactual cue over L alone; and 3) that no improvements in L+T over L were observed for reception of words in sentences, indicating the need for further training on this task

Program: a record of the first 40 years of electronic library and information systems

Tedd, L.(2006). Program: a record of the first 40 years of electronic library and information systems. Program: electronic library and information systems,40(1), 11-26.

40 years of library and information studies education in Wales

Tedd, L.A. (2005). 40 years of library and information studies education in Wales. Education for Information, 23(1/2), 1-8.

Library co-operation and ICT in the UK: an overview

Tedd, L.A. (2003). Library co-operation and ICT in the UK: an overview. Information Services and Use, 23(4), 199-210

Systematic assessment of the training needs of health library staff

Urquhart, C., Spink, S., Thomas, R. & Durbin, J. (2005). Systematic assessment of the training needs of health library staff. Library and Information Research, 29(93), 35-42. Sponsorship: National Library for Health (NLH)

NHS Wales e-library portal evaluation (For Informing Healthcare Strategy implementation programme)

Thomas, R. & Urquhart, C. NHS Wales e-library portal evaluation. (For Informing Healthcare Strategy implementation programme). Aberystwyth: Department of Information Studies, University of Wales Aberystwyth Follow-on to NHS Wales User Needs study Sponsorship: Informing Healthcare, NHS Wales