Establishment of a cell-free translation system from rice callus extracts

ABSTRACT

Eukaryotic in vitro translation systems require large numbers of protein and RNA components and thereby rely on the use of cell extracts. Here we established a new in vitro translation system based on rice callus extract (RCE). We confirmed that RCE maintains its initial activity even after five freeze-thaw cycles and that the optimum temperature for translation is around 20°C. We demonstrated that the RCE system allows the synthesis of hERG, a large membrane protein, in the presence of liposomes. We also showed that the introduction of a bicistronic mRNA based on 2A peptide to RCE allowed the production of two distinct proteins from a single mRNA. Our new method thus facilitates laboratory-scale production of cell extracts, making it a useful tool for the in vitro synthesis of proteins for biochemical studies.     

Submergence tolerance in immature stages of the stalk-eyed fly Sphyracephala detrahens (Diptera: Diopsidae)

Sphyracephala detrahens (Walker, 1860) (Diptera: Diopsidae) inhabits the riparian zones of streams and rivers. Because of the limited dispersal ability of S. detrahens during egg, larval, and pupal stages, immature individuals are at risk of being submerged by floodwater after heavy rain. In this study, I evaluated the submergence tolerances of immatures of S. detrahens by comparing them to immatures of Drosophila melanogaster, which also feed on rotten fruits but are not restricted to the riparian zone. The results showed that S. detrahens eggs were susceptible to desiccation, but more than 80% of eggs hatched after full submergence. Later instar larvae were more resistant to full submergence than earlier instar larvae. The duration of submergence causing 50% pupation (PD₅₀) in the first, second, and third-instar larvae of S. detrahens were 15.88, 58.46, and 91.74 h, respectively. The PD₅₀ of the third-instar larvae of D. melanogaster was 20.01 h. Third-instar S. detrahens larvae continued to develop in water for a longer duration than D. melanogaster larvae of the same instar. In the pupal stages, late pupae tended to remain afloat longer than early pupae. The duration of submergence causing 50% emergence (ED₅₀) of adults from early and late pupae were 40.70 and 104.74 h, respectively. In the larval and pupal stages, individuals in the later developmental phases tended to be more tolerant to full submergence. The submergence tolerance of the immature stages of S. detrahens may reflect adaptation to an environment with fluctuating water levels.     

Fgf4 is required for left-right patterning of visceral organs in zebrafish

Fgf signaling plays essential roles in many developmental events. To investigate the roles of Fgf4 signaling in zebrafish development, we generated Fgf4 knockdown embryos by injection with Fgf4 antisense morpholino oligonucleotides. Randomized LR patterning of visceral organs including the liver, pancreas, and heart was observed in the knockdown embryos. Prominent expression of Fgf4 was observed in the posterior notochord and Kupffer's vesicle region in the early stages of segmentation. Lefty1, lefty2, southpaw, and pitx2 are known to play crucial roles in LR patterning of visceral organs. Fgf4 was essential for the expression of lefty1, which is necessary for the asymmetric expression of southpaw and pitx2 in the lateral plate mesoderm, in the posterior notochord, and the expression of lefty2 and lefty1 in the left cardiac field. Fgf8 is also known to be crucial for the formation of Kupffer's vesicle, which is needed for the LR patterning of visceral organs. In contrast, Fgf4 was required for the formation of cilia in Kupffer's vesicle, indicating that the role of Fgf4 in the LR patterning is quite distinct from that of Fgf8. The present findings indicate that Fgf4 plays a unique role in the LR patterning of visceral organs in zebrafish.     

Correction: Lack of discrimination of sex and maturity of conspecifics in the copulation attempts of the male stalk-eyed fly,Sphyracephala detrahens (Diptera: Diopsidae)

Journal of Ethology -