Increasing production in Korean shrimp farms with white-spot syndrome virus PCR-negative brood stock

White-spot syndrome virus (WSSV) is a devastating, infectious virus affecting shrimp. Although sensitive techniques involving PCR have been developed to assist farmers in screening shrimp (brood stock) for WSSV prior to stocking ponds, such practices have not yet been applied in Korea. Despite the rationality of implementing screening, there has been some doubt as to whether the stocking of WSSV-PCR-negative fry epidemiologically decreases white-spot disease outbreaks. Here, we report a retrospective analysis of data from shrimp farms in the western coast of Korea where WSSV-PCR-negative brood stocks were used to stock rearing ponds. A total of 366 shrimp from Heuksan Island were sampled for WSSV with PCR. Of the tested shrimp, 7.2% (28 brood stocks) were identified as WSSV positive; only WSSV-PCR-negative shrimp were used for brood stocks. Total unit production (final shrimp production/ the area of the ponds) was higher, at 1.96, in ponds where WSSV-PCR-negative shrimp were used, as compared with 1.02 in other ponds in Korea in 2004. This retrospective analysis of WSSV in Korea may be useful to the shrimp aquaculture industry, suggesting a testable hypothesis that may contribute to the eventual control of WSSV outbreaks.     

Optimal conditions for cryopreservation of primary chicken embryo kidney cells with dimethyl sulfoxide

Conditions were evaluated for optimum cryopreservation of primary chicken embryo kidney (CEK) cells. The recovery of viable CEK cells was best (50.8% viability) when the concentration of dimethyl sulfoxide (DMSO) in the freezing medium was 20% (v/v). The viability of primary CEK cells was not influenced by the concentration of calf serum in the freezing medium, the duration of storage at −70°C before storage in liquid nitrogen, cell concentration, or the method of addition or dilution of DMSO. Thawed cells recovered and grew in complete growth medium similarly to cells freshly isolated from kidney, and influenza viruses produced plaques in the monolayer. The cryopreservation procedures described here may facilitate maintenance of a standard stock of primary CEK cells for laboratories where preparation of primary CEK cells is not an option.     

Characterization of a family 45 glycosyl hydrolase from Fibrobacter succinogenes S85

Fibrobacter succinogenes is one of the most active cellulolytic bacteria ever isolated from the rumen, but enzymes from F. succinogenes capable of hydrolyzing native (insoluble) cellulose at a rapid rate have not been identified. However, the genome sequence of F. succinogenes is now available, and it was hoped that this information would yield new insights into the mechanism of cellulose digestion. The genome has a single family 45 beta-glucanase gene, and some of the enzymes in this family have good activity against native cellulose. The gene encoding the family 45 glycosyl hydrolase from F. succinogenes S85 was cloned into Escherichia coli JM109(DE3) using pMAL-c2 as a vector. Recombinant E. coli cells produced a soluble fusion protein (MAL-F45) that was purified on a maltose affinity column and characterized. MAL-F45 was most active on carboxymethylcellulose between pH 6 and 7 and it hydrolyzed cellopentaose and cellohexaose but not cellotetraose. It also cleaved p-nitrophenyl-cellopentose into cellotriose and p-nitrophenyl-cellobiose. MAL-F45 produced cellobiose, cellotriose and cellotetraose from acid swollen cellulose and bacterial cellulose, but the rate of this hydrolysis was much too low to explain the rate of cellulose digestion by growing cultures. Because the F. succinogenes S85 genome lacks dockerin and cohesin sequences, does not encode any known processive cellulases, and most of its endoglucanase genes do not encode carbohydrate binding modules, it appears that F. succinogenes has a novel mechanism of cellulose degradation.     

Isolation and characterization of two novel colon cancer cell lines from Chinese patients

Incidence of colon cancer has increased rapidly in China. Although many colon cancer cell lines have been established previously, most of them were derived from patients from western countries. Epidemiological, clinical, cytogenetic, and molecular biological studies showed that there are considerable differences between Chinese and western countries colon cancer patients. Therefore, establishment of novel colon cancer cell line from Chinese is useful for studying the racial difference of this disease and can be important for studying the pathogenesis of colon cancer in China. In our laboratory, two novel continuous human colon cancer cell lines, SHT-1 and SHH-1, have been established in vitro from Chinese patients, and both cell lines have been passaged for 4 yr, and they have been continuously subcultured with more than 800 population doubling and without signs of senescence. Both cell lines were obtained from primary tumor tissues during colon cancer surgery. Cells grew rapidly with a doubling time of 36–39 h and a plating efficiency of 26–28%. These cells exhibited an epithelial morphology and expressed cytokeratin. Tumor developed in severe combined immunodeficient (SCID) mice 4–6 wk after inoculated subcutaneously with the cultured cancer cells. Karyotypic analysis and comparative genomic hybridization (CGH) analysis in SHT-1 cells revealed a hypertriploid modal number of 76 with numerous numerical and structural abnormalities previously linked to colon cancer. In another cell line (SHH-1), CGH analysis revealed that −1p13 was the only cytogenetic anomaly.     

DP-Bind: a web server for sequence-based prediction of DNA-binding residues in DNA-binding proteins

This article describes DP-Bind, a web server for predicting DNA-binding sites in a DNA-binding protein from its amino acid sequence. The web server implements three machine learning methods: support vector machine, kernel logistic regression and penalized logistic regression. Prediction can be performed using either the input sequence alone or an automatically generated profile of evolutionary conservation of the input sequence in the form of PSI-BLAST position-specific scoring matrix (PSSM). PSSM-based kernel logistic regression achieves the accuracy of 77.2%, sensitivity of 76.4% and specificity of 76.6%. The outputs of all three individual methods are combined into a consensus prediction to help identify positions predicted with high level of confidence. AVAILABILITY: Freely available at http://lcg.rit.albany.edu/dp-bind. SUPPLEMENTARY INFORMATION: http://lcg.rit.albany.edu/dp-bind/dpbind_supplement.html.     

Identification of structural genes for Clostridium botulinum type C neurotoxin-converting phage particles

The structural genes for strain C-Stockholm (c-st) phage particles, a representative type C toxin-converting phage of Clostridium botulinum, have been determined. First, by determining the N-terminal amino acid sequences of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) bands of c-st phage particles, it became clear that four proteins, 14, 25, 32 and 42 kDa, are the products of the ORFs, cst166, cst165, cst160 and cst164, respectively, of the c-st phage genome. The Western blot analyses reacting these phage bands with an antiphage serum prepared previously indicated that the products of cst165 and cst160 are the main proteins of the phage particles. Then, six candidates for the phage structural proteins, including cst165 and cst160 gene products, were prepared as recombinant proteins. Also, the protein corresponding to the cst164 gene product was excised from SDS-PAGE gels. The antibodies against these seven proteins were prepared in rabbits, and finally, the reaction of these antibodies to the c-st phage particles was analyzed by electron microscopy. It was concluded that a sheath protein and a head protein of the c-st phage are the products of genes cst160 and cst165, respectively, and that these two proteins are conserved in the other three converting phages, but not in the nonconverting phage.     

Protein complexity, gene duplicability and gene dispensability in the yeast genome

Using functional genomic and protein structural data we studied the effects of protein complexity (here defined as the number of subunit types in a protein) on gene dispensability and gene duplicability. We found that in terms of gene duplicability the major distinction in protein complexity is between hetero-complexes, each of which includes at least two different types of subunits (polypeptides), and homo-complexes, which include monomers and complexes that consist of only subunits of one polypeptide type. However, gene dispensability decreases only gradually as the number of subunit types in a protein complex increases. These observations suggest that the dosage balance hypothesis can explain well gene duplicability of complex proteins, but cannot completely explain the difference in dispensabilities between hetero-complex subunits. It is likely that knocking out a gene coding for a hetero-complex subunit would disrupt the function of the whole complex, so that the deletion effect on fitness would increase with protein complexity. We also found that multi-domain polypeptide genes are less dispensable but more duplicable than single-domain polypeptide genes. Duplicate genes derived from the whole genome duplication event in yeast are more dispensable (except for ribosomal protein genes) than other duplicate genes. Further, we found that subunits of the same protein complex tend to have similar expression levels and similar effects of gene deletion on fitness. Finally, we estimated that in yeast the contribution of duplicate genes to genetic robustness against null mutation is approximately 9%, smaller than previously estimated. In yeast, protein complexity may serve as a better indicator of gene dispensability than do duplicate genes.     

The complete nucleotide sequence and gene organization of the mitochondrial genome of the bumblebee, Bombus ignitus (Hymenoptera: Apidae)

The complete 16,434-bp nucleotide sequence of the mitogenome of the bumble bee, Bombus ignitus (Hymenoptera: Apidae), was determined. The genome contains the base composition and codon usage typical of metazoan mitogenomes. An unusual feature of the B. ignitus mitogenome is the presence of five tRNA-like structures: two each of the tRNALeu(UUR)-like and tRNASer(AGN)-like sequences and one tRNAPhe-like sequence. These tRNA-like sequences have proper folding structures and anticodon sequences, but their functionality in their respective amino acid transfers remained uncertain. Among these sequences, the tRNALeu(UUR)-like sequence and the tRNASer(AGN)-like sequence are seemingly located within the A+T-rich region. This tRNASer(AGN)-like sequence is highly unusual in that its sequence homology is very high compared to the tRNAMet of other insects, including Apis mellifera, but it contains the anticodon ACT, which designates it as tRNASer(AGN). All PCG and rRNAs are conserved in positions observed most frequently in insect mitogenome structures, but the positions of the tRNAs are highly variable, presenting a new arrangement for an insect mitogenome. As a whole, the B. ignitus mitogenome contains the highest A+T content (86.9%) found in any of the complete insects mt sequences determined to date. All protein-coding sequences started with a typical ATN codon. Nine of the 13 PCGs have a complete termination codon (all TAA), but the remaining four genes terminate with the incomplete TA or T. All tRNAs have the typical clover-leaf structures of mt tRNAs, except for tRNASer(AGN), in which the DHU arm forms a simple loop. All anticodons of B. ignitus tRNAs are identical to those of A. mellifera. In the A+T-rich region, a highly conserved sequence block that was previously described in Orthoptera and Diptera was also present. The stem-and-loop structures that may play a role in the initiation of mtDNA replication were also found in this region. Phylogenetic analysis among three corbiculate tribes, represented by Melipona bicolor (Meliponini), A. mellifera (Apini), and B. ignitus (Bombini), showed the closest relationship between M. bicolor and B. ignitus.