Expression and functional analysis of flotillins in Dugesia japonica

FLOTILLIN-1 and FLOTILLIN-2 are membrane rafts associated proteins that have been implicated in insulin and growth factor signaling, endocytosis, cell migration, proliferation, differentiation, cytoskeleton remodeling and membrane trafficking. Furthermore, FLOTILLINs also play important roles in the progression of cancer and neurodegenerative diseases. In this study, the roles of flotillins are investigated in planarian Dugesia japonica. The results show that Djflotillin-1 and Djflotillin-2 play a key role in homeostasis maintenance and regeneration process by regulating the proliferation of the neoblast cells, they are not involved in the maintenance and regeneration of the central nervous system in planarians.     

Genome-wide expression analysis of roxarsone-stimulated growth of broiler chickens (Gallus gallus)

Roxarsone is a commonly used additive in chicken (Gallus gallus) industry. However, little is known on the intrinsic molecular mechanism via which the growth performance of birds improves. This study was therefore performed to investigate the expression profiles of genes induced by roxarsone. Fifty-six broiler chickens were divided into two groups, namely treated and untreated with roxarsone. The treated group was provided a diet of 45.4 mg/kg roxarsone medication and the other group acted as control. Data analysis showed that roxarsone consistently and significantly (P < 0.05) increased chicken growth performance. In addition to this a significant (P < 0.05) increase of arsenic residue in liver has been seen. Microarray expression analysis of 8935 genes in liver showed that 22 genes (10 up- and 12 down-regulated) had altered expression throughout the experimental periods. Two novel genes (GenBank accession no. GU724343 and GU724344) were cloned through rapid amplification of cDNA ends (RACE). Gene GU724343 was predicted to encode an unidentified protein and the second gene GU724344 was presumed to encode a new member of immunoglobulin-like receptor (CHIR) family. Our results suggested for the first time that the role of roxarsone could be mainly to modify the expression levels of cell growth, immunity/defense and energy metabolism associated genes, as a result promoting animal growth. Further research on these genes should help to increase the knowledge of improving animal productivity safely and effectively.     

Gene characterization, analysis of expression and in vitro synthesis of dihydroflavonol 4-reductase from [Citrus sinensis (L.) Osbeck

Dihydroflavonol 4-reductase (DFR, EC 1.1.1.219) catalyzes the reduction of dihydroflavonols to leucoanthocyanins, a key "late" step in the biosynthesis of anthocyanins. In this study we showed that a strong reduction in DFR expression occurs in the non-red orange cultivar (Navel and Ovale) compared to that of the red orange (Tarocco) suggesting that the enzyme could be involved in the lack of production of anthocyanins. Therefore, we isolated and compared the cDNAs, the genomic clones, as well as the promoter regions of blood and blond orange dfrs. Our data revealed that the cDNA sequences of pigmented and non-pigmented orange DFRs were 100% homologous and contained a 1017 bp open reading frame which encodes a protein of 338 amino acid residues, corresponding to a molecular mass of 38010.76 Da, with a theoretical pI of 5.96. Moreover, we found that there were no significant differences in non-coding regions (introns and 5' upstream region) of dfr sequences. Southern blot analysis of genomic DNA indicated that dfr was present as a single copy gene in both cultivars. From these findings the low expression level of blond orange dfr, which might play a role in the phenotypic change from blood to blond orange, is thought to be the result of a likely mutation in a regulatory gene controlling the expression of dfr. In addition, here we reported the successful expression of orange DFR cDNAs leading to an active DFR enzyme which converts dihydroquercetin to leucoanthocyanidin, thus confirming the involvement of the isolated genes in the biosynthesis of anthocyanins. Moreover, as far as we know, this is the first report concerning the in vitro expression of DFR from fruit flesh whose biochemical properties might be very different from those of other plant organ DFRs.     

Alkaline transfer of DNA to plastic membrane

DNA forms a stable complex with Gene Screen Plus membrane at alkaline pH. Based on this, a method of alkaline transfer of DNA from agarose gel to Gene Screen Plus membrane was elaborated. The procedure entails the use of 0.4 M NaOH for both, the DNA denaturation and DNA transfer steps. The alkaline transfer offers a higher hybridization efficiency and simplifies the transfer procedure as compared with the standard method of DNA transfer at neutral pH. In addition, it can be used to remove RNA contamination from the transferred DNA.     

1H, 13C, and 15N resonance assignments of a general stress protein GSP13 from Bacillus subtilis

GSP13 encoded by gene yugI is a general stress protein in Bacillus subtilis. The NMR assignments of the protein are essential for its structure determination.     

Calculating phenotypic similarity between genes using hierarchical structure data based on semantic similarity

Phenotypic similarity is correlated with a number of measures of gene function, such as relatedness at the level of direct protein-protein interaction. The phenotypic effect of a deleted or mutated gene, which is one part of gene annotation, has caught broad attention. However, there have been few measures to study phenotypic similarity with the data from Human Phenotype Ontology (HPO) database, therefore more analogous measures should be developed and investigated. We used five semantic similarity-based measures (Jiang and Conrath, Lin, Schlicker, Yu and Wu) to calculate the human phenotypic similarity between genes (PSG) with data from HPO database, and evaluated their accuracy with information of protein-protein interaction, protein complex, protein family, gene function or DNA sequence. Compared with the gene pairs that were random selected, the results of these methods were statistically significant (all P<0.001). Furthermore, we assessed the performance of these five measures by receiver operating characteristic (ROC) curve analysis, and found that most of them performed better than the previous methods. This work had proved that these measures based on semantic similarity for calculation of PSG were effective for hierarchical structure data. Our study contributes to the development and optimization of novel algorithms of PSG calculation and provides more alternative methods to researchers as well as tools and directions for PSG study.     

Variable intron/exon structure in the oligochaete lombricine kinase gene

Lombricine kinase is an annelid enzyme that belongs to the phosphagen kinase family of which creatine kinase and arginine kinase are the typical representatives. The enzymes play important roles in the cellular energy metabolism of animals. Biochemical, physiological and molecular information with respect to lombricine kinase is limited compared to other phosphagen kinases. This study presents data on the cDNA sequences of lombricine kinase from two smaller oligochaetes, Enchytraeus sp. and Stylaria sp. The deduced amino acid sequences are analyzed and compared with other selected phosphagen kinases. The intron/exon structure of the lombricine kinase gene was determined for these two species as well as two additional oligochaetes, Lumbriculus variegatus and Tubifex tubifex, and compared with available data for annelid phosphagen kinases. The data indicate the existence of a variable organization of the proposed 8-intron/9-exon gene structure. The results provide further insights in the evolution and position of these enzymes within the phosphagen kinase family.