Hydroxylations of substituted naphthalenes by Escherichia coli expressing aromatic dihydroxylating dioxygenase genes from polycyclic aromatic hydrocarbon-utilizing marine bacteria

Bioconversion experiments of various mono- or di-substituted naphthalenes such as dimethylnaphthalenes were carried out using the cells of Escherichia coli that expressed aromatic dihydroxylating dioxygenase genes (phnA1A2A3A4 and phdABCD) from polycyclic aromatic hydrocarbon-utilizing marine bacteria, Nocardioides sp. KP7 and Cycloclasticus sp. A5, respectively. We found that the former dioxygenase PhnA1A2A3A4 had broad substrate preference for these compounds and often was able to hydroxylate their methyl groups. Specifically, 1,4-dimethylnaphthalene was predominantly bioconverted into 1,4-dihydroxymethylnaphthalene.     

Trim41 is required to regulate chromosome axis protein dynamics and meiosis in male mice

Meiosis is a hallmark event in germ cell development that accompanies sequential events executed by numerous molecules. Therefore, characterization of these factors is one of the best strategies to clarify the mechanism of meiosis. Here, we report tripartite motif-containing 41 (TRIM41), a ubiquitin ligase E3, as an essential factor for proper meiotic progression and fertility in male mice. Trim41 knockout (KO) spermatocytes exhibited synaptonemal complex protein 3 (SYCP3) overloading, especially on the X chromosome. Furthermore, mutant mice lacking the RING domain of TRIM41, required for the ubiquitin ligase E3 activity, phenocopied Trim41 KO mice. We then examined the behavior of mutant TRIM41 (ΔRING-TRIM41) and found that ΔRING-TRIM41 accumulated on the chromosome axes with overloaded SYCP3. This result suggested that TRIM41 exerts its function on the chromosome axes. Our study revealed that Trim41 is essential for preventing SYCP3 overloading, suggesting a TRIM41-mediated mechanism for regulating chromosome axis protein dynamics during male meiotic progression.     

Systematic analysis of SNARE localization in the filamentous fungus Aspergillus oryzae

In spite of their great importance for both applied and basic biology, studies on vesicular trafficking in filamentous fungi have been so far very limited. Here, we identified 21 genes, which might be a total set, encoding putative SNARE proteins that are key factors for vesicular trafficking, taking advantage of available whole genome sequence in the filamentous fungus Aspergillus oryzae. The subsequent systematic analysis to determine the localization of putative SNAREs using EGFP-fused chimeras revealed that most putative SNAREs show similar subcellular distribution to their counterparts in the budding yeast. However, there existed some characteristic features of SNAREs in A. oryzae, such as SNARE localization at/near the septum and the presence of apparently non-redundant plasma membrane Qa-SNAREs. Overall, this analysis allowed us to provide an overview of vesicular trafficking and organelle distribution in A. oryzae.     

A genetically explicit model of speciation by sensory drive within a continuous population in aquatic environments

Background  

The sensory drive hypothesis predicts that divergent sensory adaptation in different habitats may lead to premating isolation upon secondary contact of populations. Speciation by sensory drive has traditionally been treated as a special case of speciation as a byproduct of adaptation to divergent environments in geographically isolated populations. However, if habitats are heterogeneous, local adaptation in the sensory systems may cause the emergence of reproductively isolated species from a single unstructured population. In polychromatic fishes, visual sensitivity might become adapted to local ambient light regimes and the sensitivity might influence female preferences for male nuptial color. In this paper, we investigate the possibility of speciation by sensory drive as a byproduct of divergent visual adaptation within a single initially unstructured population. We use models based on explicit genetic mechanisms for color vision and nuptial coloration.     

Demonstration and Characterization of the Heterodimerization of ZnT5 and ZnT6 in the Early Secretory Pathway

The majority of CDF/ZnT zinc transporters form homo-oligomers. However, ZnT5, ZnT6, and their orthologues form hetero-oligomers in the early secretory pathway where they load zinc onto zinc-requiring enzymes and maintain secretory pathway functions. The details of this hetero-oligomerization remain to be elucidated, and much more is known about homo-oligomerization that occurs in other CDF/ZnT family proteins. Here, we addressed this issue using co-immunoprecipitation experiments, mutagenesis, and chimera studies of hZnT5 and hZnT6 in chicken DT40 cells deficient in ZnT5, ZnT6, and ZnT7 proteins. We found that hZnT5 and hZnT6 combine to form heterodimers but do not form complexes larger than heterodimers. Mutagenesis of hZnT6 indicated that the sites present in transmembrane domains II and V in which many CDF/ZnT proteins have conserved hydrophilic amino acid residues are not involved in zinc binding of hZnT6, although they are required for zinc transport in other CDF/ZnT family homo-oligomers. We also found that the long N-terminal half of hZnT5 is not necessary for its functional interaction with hZnT6, whereas the cytosolic C-terminal tail of hZnT5 is important in determining hZnT6 as a partner molecule for heterodimer formation. In DT40 cells, cZnT5 variant lacking the N-terminal half was endogenously induced during periods of endoplasmic reticulum stress and so seemed to function to supply zinc to zinc-requiring enzymes under these conditions. The results outlined here provide new information about the mechanism of action through heterodimerization of CDF/ZnT proteins that function in the early secretory pathway.     

Comparison of axonemal proteins from two kinds of Tetrahymena. I. Different characteristics of dyneins in heat stability

Tetrahymena thermophila could still swim after incubation of the cell body at 40°C for 30 min, whereas Tetrahymena pyriformis did not show any motility after the treatment. Turbidity measurements revealed that axonemes of T. pyriformis lost ATP-dependent sliding activity by the heat treatment, whereas those of T. thermophilia still had the activity under the same conditions. In connection with this difference in susceptibility to high temperature, the biochemical characteristics of dyneins were compared between the two species of Tetrahymena. Axonemal dyneins from the two species had significant vanadate-sensitive ATPase activity even after the heat treatment. Native gel electrophoresis and the following two-dimensional electrophoresis showed that the outer arm dynein of T. thermophilia is more stable in maintaining native configuration than that of T. pyriformis against the heat treatment, although both treated dyneins keep three (α, β and γ) subunits. Analysis by peptide mapping demonstrated that β- and γ-subunits of the outer arm dynein are considerably different in amino acid sequences between the two species. These results imply that dynein of T. thermophilia changed their amino acid sequences and biochemical characteristics to adapt to high temperature.     

One-step generation of multiple transgenic mouse lines using an improved Pronuclear Injection-based Targeted Transgenesis (i-PITT)

Background

The pronuclear injection (PI) is the simplest and widely used method to generate transgenic (Tg) mice. Unfortunately, PI-based Tg mice show uncertain transgene expression due to random transgene insertion in the genome, usually with multiple copies. Thus, typically at least three or more Tg lines are produced by injecting over 200 zygotes and the best line/s among them are selected through laborious screening steps. Recently, we developed technologies using Cre-loxP system that allow targeted insertion of single-copy transgene into a predetermined locus through PI. We termed the method as PI-based Targeted Transgenesis (PITT). A similar method using PhiC31-attP/B system was reported subsequently.

Results

Here, we developed an improved-PITT (i-PITT) method by combining Cre-loxP, PhiC31-attP/B and FLP-FRT systems directly under C57BL/6N inbred strain, unlike the mixed strain used in previous reports. The targeted Tg efficiency in the i-PITT typically ranged from 10 to 30%, with 47 and 62% in two of the sessions, which is by-far the best Tg rate reported. Furthermore, the system could generate multiple Tg mice simultaneously. We demonstrate that injection of up to three different Tg cassettes in a single injection session into as less as 181 zygotes resulted in production of all three separate Tg DNA containing targeted Tg mice.

Conclusions

The i-PITT system offers several advantages compared to previous methods: multiplexing capability (i-PITT is the only targeted-transgenic method that is proven to generate multiple different transgenic lines simultaneously), very high efficiency of targeted-transgenesis (up to 62%), significantly reduces animal numbers in mouse-transgenesis and the system is developed under C57BL/6N strain, the most commonly used pure genetic background. Further, the i-PITT system is freely accessible to scientific community.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1432-5) contains supplementary material, which is available to authorized users.