Controlled Cre/loxP Site-Specific Recombination in the Developing Brain in Medaka Fish,Oryzias latipes

Background

Genetic mosaic techniques have been used to visualize and/or genetically modify a neuronal subpopulation within complex neural circuits in various animals. Neural populations available for mosaic analysis, however, are limited in the vertebrate brain.

Methodology/Principal Findings

To establish methodology to genetically manipulate neural circuits in medaka, we first created two transgenic (Tg) medaka lines, Tg (HSP:Cre) and Tg (HuC:loxP-DsRed-loxP-GFP). We confirmed medaka HuC promoter-derived expression of the reporter gene in juvenile medaka whole brain, and in neuronal precursor cells in the adult brain. We then demonstrated that stochastic recombination can be induced by micro-injection of Cre mRNA into Tg (HuC:loxP-DsRed-loxP-GFP) embryos at the 1-cell stage, which allowed us to visualize some subpopulations of GFP-positive cells in compartmentalized regions of the telencephalon in the adult medaka brain. This finding suggested that the distribution of clonally-related cells derived from single or a few progenitor cells was restricted to a compartmentalized region. Heat treatment of Tg(HSP:Cre x HuC:loxP-DsRed-loxP-GFP) embryos (0–1 day post fertilization [dpf]) in a thermalcycler (39°C) led to Cre/loxP recombination in the whole brain. The recombination efficiency was notably low when using 2–3 dpf embyos compared with 0–1 dpf embryos, indicating the possibility of stage-dependent sensitivity of heat-inducible recombination. Finally, using an infrared laser-evoked gene operator (IR-LEGO) system, heat shock induced in a micro area in the developing brains led to visualization of clonally-related cells in both juvenile and adult medaka fish.

Conclusions/Significance

We established a noninvasive method to control Cre/loxP site-specific recombination in the developing nervous system in medaka fish. This method will broaden the neural population available for mosaic analyses and allow for lineage tracing of the vertebrate nervous system in both juvenile and adult stages.     

Defect in the Formation of 70S Ribosomes Caused by Lack of Ribosomal Protein L34 Can Be Suppressed by Magnesium

To elucidate the biological functions of the ribosomal protein L34, which is encoded by the rpmH gene, the rpmH deletion mutant of Bacillus subtilis and two suppressor mutants were characterized. Although the ΔrpmH mutant exhibited a severe slow-growth phenotype, additional mutations in the yhdP or mgtE gene restored the growth rate of the ΔrpmH strain. Either the disruption of yhdP, which is thought to be involved in the efflux of Mg²⁺, or overexpression of mgtE, which plays a major role in the import of Mg²⁺, could suppress defects in both the formation of the 70S ribosome and growth caused by the absence of L34. Interestingly, the Mg²⁺ content was lower in the ΔrpmH cells than in the wild type, and the Mg²⁺ content in the ΔrpmH cells was restored by either the disruption of yhdP or overexpression of mgtE. In vitro experiments on subunit association demonstrated that 50S subunits that lacked L34 could form 70S ribosomes only at a high concentration of Mg²⁺. These results showed that L34 is required for efficient 70S ribosome formation and that L34 function can be restored partially by Mg²⁺. In addition, the Mg²⁺ content was consistently lower in mutants that contained significantly reduced amounts of the 70S ribosome, such as the ΔrplA (L1) and ΔrplW (L23) strains and mutant strains with a reduced number of copies of the rrn operon. Thus, the results indicated that the cellular Mg²⁺ content is influenced by the amount of 70S ribosomes.     

CAB-Align: A Flexible Protein Structure Alignment Method Based on the Residue-Residue Contact Area

Proteins are flexible, and this flexibility has an essential functional role. Flexibility can be observed in loop regions, rearrangements between secondary structure elements, and conformational changes between entire domains. However, most protein structure alignment methods treat protein structures as rigid bodies. Thus, these methods fail to identify the equivalences of residue pairs in regions with flexibility. In this study, we considered that the evolutionary relationship between proteins corresponds directly to the residue–residue physical contacts rather than the three-dimensional (3D) coordinates of proteins. Thus, we developed a new protein structure alignment method, contact area-based alignment (CAB-align), which uses the residue–residue contact area to identify regions of similarity. The main purpose of CAB-align is to identify homologous relationships at the residue level between related protein structures. The CAB-align procedure comprises two main steps: First, a rigid-body alignment method based on local and global 3D structure superposition is employed to generate a sufficient number of initial alignments. Then, iterative dynamic programming is executed to find the optimal alignment. We evaluated the performance and advantages of CAB-align based on four main points: (1) agreement with the gold standard alignment, (2) alignment quality based on an evolutionary relationship without 3D coordinate superposition, (3) consistency of the multiple alignments, and (4) classification agreement with the gold standard classification. Comparisons of CAB-align with other state-of-the-art protein structure alignment methods (TM-align, FATCAT, and DaliLite) using our benchmark dataset showed that CAB-align performed robustly in obtaining high-quality alignments and generating consistent multiple alignments with high coverage and accuracy rates, and it performed extremely well when discriminating between homologous and nonhomologous pairs of proteins in both single and multi-domain comparisons. The CAB-align software is freely available to academic users as stand-alone software at http://www.pharm.kitasato-u.ac.jp/bmd/bmd/Publications.html.     

Pollen morphology of andruris japonica (triuridaceae)

Pollen morphology of Andruris japonica (Triuridaceae) was investigated by light and electron microscopy. The pollen is monosulcate and has a size of 22–25 μm × 25–28 μm in polar view. In the non-apertural region the exine has gemmate to nearly verrucate protrusions of 0.4–0.5 μm in diameter and 0.3–0.5 μm in height, and a foot layer of 0.4–0.5 μm in thickness. The sporoderm of the apertural region has large gemmae of 0.7–0.8 μm in diameter and 0.6–0.7 μm in height, with a thin foot layer of 0.1 μm thickness.     

Calcineurin-Dependent Mitochondrial Disturbances in Calcium-Induced Apoptosis of Human Immunodeficiency Virus gp160-Expressing CD4+ Cells

In CD4+ UE160 cells with inducible expression of gp160, mechanisms of apoptosis induced by human immunodeficiency virus (HIV) Env protein were analyzed. Induction of gp160 caused intracellular calcium increase followed by the release of cytochrome c from mitochondria, which was inhibited by calcineurin inhibitors. Association of BAD with Bcl-xL was observed, and a portion of BAD was dephosphorylated after induction of gp160. These data suggested that calcineurin plays a role in the HIV Env-induced apoptosis in a mitochondrion-dependent way.