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.     

Unraveling the genetic basis of fast l-arabinose consumption on top of recombinant xylose-fermenting Saccharomyces cerevisiae

One major challenge in the bioconversion of lignocelluloses into ethanol is to develop Saccharomyces cerevisiae strains that can utilize all available sugars in biomass hydrolysates, especially the d -xylose and l -arabinose that cannot be fermented by the S. cerevisiae strain naturally. Here, we integrated an l -arabinose utilization cassette (AUC) into the genome of an efficient d -xylose fermenting industrial diploid S. cerevisiae strain CIBTS0735 to make strain CIBTS1972. After evolving on arabinose, CIBTS1974 with excellent fermentation capacity was obtained. A comparison between genome sequences of strains CIBTS1974 and CIBTS1972 revealed that the copy number of the AUC had increased from 1 to 12. We then constructed the AUC null-mutant CIBTS1975 and gradually rescued the l -arabinose utilization defect by integrating AUC iteratively. On the other hand, the parental strain CIBTS0735 was able to acquire the same performance as CIBTS1974 by the direct introduction of 12 copies of the AUC; the performance was further improved by adding two more copies. Besides, we found that not the two transporters present in the AUC were both needed during l -arabinose utilization, GAL2 was necessary and STP2 was not essential. We have described for the first time that a high copy number of AUC is sufficient for the strain to metabolize l -arabinose efficiently independent of evolution.     

Rapid prototyping of proteins: Mail order gene fragments to assayable proteins within 24 hours

In this study, we present a minimal template design and accompanying methods to produce assayable quantities of custom sequence proteins within 24 hr from receipt of inexpensive gene fragments from a DNA synthesis vendor. This is done without the conventional steps of plasmid cloning or cell-based amplification and expression. Instead the linear template is PCR amplified, circularized, and isothermally amplified using a rolling circle polymerase. The resulting template can be used directly with cost-optimized, scalably-manufactured Escherichia coli extract and minimal supplement reagents to perform cell-free protein synthesis (CFPS) of the template protein. We demonstrate the utility of this template design and 24 hr process with seven fluorescent proteins (sfGFP, mVenus, mCherry, and four GFP variants), three enzymes (chloramphenicol acetyltransferase, a chitinase catalytic domain, and native subtilisin), a capture protein (anti-GFP nanobody), and 2 antimicrobial peptides (BP100 and CA(1–7)M(2–9)). We detected each of these directly from the CFPS reaction using colorimetric, fluorogenic, and growth assays. Of especial note, the GFP variant sequences were found from genomic screening data and had not been expressed or characterized before, thus demonstrating the utility of this approach for phenotype characterization of sequenced libraries. We also demonstrate that the rolling circle amplified version of the linear template exhibits expression similar to that of a complete plasmid when expressing sfGFP in the CFPS reaction. We evaluate the cost of this approach to be $61/mg sfGFP for a 4 hr reaction. We also detail limitations of this approach and strategies to overcome these, namely proteins with posttranslational modifications.     

Rapid detection of Phytophthora nicotianae by simple DNA extraction and real‐time loop‐mediated isothermal amplification assay

Phytophthora nicotianae is a phytopathogenic oomycete with a wide host range and worldwide distribution. Rapid detection and diagnosis at the early stages of disease development are important for the effective control of P. nicotianae. In this study, we designed a simple and rapid loop‐mediated isothermal amplification (LAMP)‐based detection method for P. nicotianae. We tested three DNA extraction methods and selected the Kaneka Easy DNA Extraction Kit version 2, which is rapid and robust for LAMP‐based detection. The designed primers were tested using mycelial DNA from 35 species (81 isolates) of Phytophthora, 12 species (12 isolates) of Pythium, one isolate of Phytopythium and one isolate each from seven other soil‐borne pathogens. All of the 42 P. nicotianae isolates were detected by these primers, and no other isolates gave positive results. Three isolates were tested for the sensitivity of the reaction, and the lowest amounts of template DNA that could be detected were 10 fg for two isolates and 1 fg for the third. The target was detected within 25 min in all tested samples, including DNA extracted from both inoculated and naturally infected plants. In contrast, PCR assays with P. nicotianae‐specific primers failed or showed weakened detection in several samples. Thus, we found that the rapid DNA extraction and LAMP assay methods developed in this study can be used to detect P. nicotianae with high sensitivity, specificity and stability.     

Flow cytometry: an improved method for the selection of highly productive gene-amplified CHO cells using flow cytometry.

In previous work, we clarified the relationship between the productivity and stability of gene-amplified cells and the location of the amplified gene. The location of the amplified gene enabled us to classify resistant cells into two types. One type of resistant cell group, in which the amplified genes were observed near the telomeric region, was named the "telomere type." The other type of cell group, in which the amplified genes were observed in other chromosomal regions, was named the "other type." The phenotypes of these two types of cells are very different. In this experiment, using a fluorescein isothiocyanate-labeled methotrexate (F-MTX) reagent with flow cytometry, we were easily able to distinguish between highly productive cells and the other types of cells. The level of fluorescence differed according to the difference in resistance to MTX. Based on this new finding, highly productive gene-amplified cells could be isolated from heterogeneous gene-amplified cell pools more easily than by the method of limiting-dilution assay. The limiting-dilution method requires several months to obtain highly productive gene-amplified cells, while our flow-cytometry-based method of selection requires only a few weeks.     

Microsatellites reveal clonal structure of populations of the thelytokous ant platythyrea punctata (F. Smith) (Hymenoptera; formicidae)

Parthenogenesis is often thought to constitute an evolutionary dead end as compared with sexual reproduction because genetic recombination is limited or nonexistent in parthenogenetic populations. Yet there are many species to demonstrate that parthenogenesis can initially be extremely successful under certain environmental conditions. In this study we used microsatellite markers to investigate the genetic structure of four natural populations of the neotropical thelytokous parthenogenetic ant Platythyrea punctata. Ten dinucleotide microsatellites were isolated from a partial genomic library of P. punctata. Five of these were found to be polymorphic. In a subsequent analysis of 314 workers taken from 51 colonies, we detected low intraspecific levels of variation at all loci, expressed both in the number of alleles detected and heterozygosities observed. Surprisingly, we found almost no differentiation within populations. Populations rather had a clonal structure, with all individuals from all colonies usually sharing the same genotype. Only in one colony from Puerto Rico did some workers have an additional genotype. This low level of genotypic diversity probably reflects the predominance of thelytoky in P. punctata, together with genetic bottlenecks and founder effects. Cross-species amplification of all 10 loci in 29 ant species comprising four different subfamilies yielded positive amplification products in only a limited number of species.