The effects of spacer sequences on silencing efficiency of plant RNAi vectors

RNA interference (RNAi) has been used to suppress gene expression in various eukaryotic organisms. In plants, RNAi can be induced by introduction of an RNAi vector that transcribes a self-complementary hairpin RNA. Most basic RNAi constructs have an inverted repeat interrupted with a spacer sequence. To test silencing capability of RNAi constructs, we developed an in vivo assay that is based on the RNAi-mediated changes of the α-linolenic acid content in hairy roots. A tobacco endoplasmic reticulum ω-3 fatty acid desaturase (NtFAD3) is the main enzyme for production of α-linolenic acid of root membrane lipids. Tobacco hairy roots transformed with the RNAi vectors against the NtFAD3 gene showed a decrease in α-linolenic acid content. The frequency of RNA silencing was more affected by spacer sequence than by spacer length, at least between 100 and 1800 bp. Since significant amounts of hairpin RNA against the NtFAD3 gene remained in the transgenic plants displaying a weak silencing phenotype, low degree of silencing was attributed to low efficiency of hairpin RNA processing mediated by Dicer-like proteins. Our results show the possibility of producing a broad range of the RNAi-induced silencing phenotypes by replacing the spacer sequence of RNAi construct.     

<Emphasis Type="Italic">Slc11a1</Emphasis> (<Emphasis Type="Italic">Nramp1</Emphasis>) alleles interact with acute inflammation loci to modulate wound-healing traits in mice

Lines of mice were obtained by selective breeding for maximum (AIRmax) or minimum (AIRmin) acute inflammation. They present distinct neutrophil influx and show frequency disequilibrium of the solute carrier family 11a member 1 (Slc11a1) alleles. This gene is involved in ion transport at the endosomes within macrophages and neutrophils, interfering in their activation. Homozygous AIRmax and AIRmin sublines for the Slc11a1 gene were produced to examine the interaction of this gene with the acute inflammatory loci. The present work investigated wound-healing traits in AIRmax and AIRmin mice, in F₁ and F₂ intercrosses, and in Slc11a1 sublines. Two-millimeter ear punches were made in the mice and hole closure was measured during 40 days. AIRmax mice demonstrated significant tissue repair while AIRmin mice did not. Significant differences between the responses of male and female mice were also observed. Wound-healing traits demonstrated a correlation with neutrophil influx in F₂ populations. AIRmax ^SS showed higher ear-wound closure than AIRmax ^RR mice, suggesting that the Slc11a1 S allele favored ear tissue repair. QTL analysis has detected two inflammatory loci modulating ear wound healing on chromosomes 1 and 14. These results suggest the involvement of the acute inflammation modifier QTL in the wound-healing phenotype.     

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.     

Selective fluorescence detection of 8-oxoguanosine with 8-oxoG-clamp

8-oxoguanosine, which is derived from the oxidation of guanosine (dG), is known to induce transversion mutations (G:C-->T:A) in DNA. The compounds with a small molecular weight for recognizing 8-oxoG were designed on the basis of the structure of the G-clamp, which is reported to have selective affinity toward guanosine. The G-clamp derivatives with the additional binding units toward 8-oxoG were effectively synthesized and named "8-oxoG-clamps." The 8-oxoG-clamp completely discriminated 8-oxoG from other nucleosides by fluorescence quenching.     

Hybridization and polyploidization effects on LTR-retrotransposon activation in potato genome

Hybridization and polyploidization are major forces in plant evolution and potatoes are not an exception. It is proposed that the proliferation of Long Terminal Repeat-retrotransposons (LTR-RT) is related to genome reorganization caused by hybridization and/or polyploidization. The main purpose of the present work was to evaluate the effect of interspecific hybridization and polyploidization on the activation of LTR-RT. We evaluated the proliferation of putative active LTR-RT in a diploid hybrid between the cultivated potato Solanum tuberosum and the wild diploid potato species S. kurtzianum, allotetraploid lines derived from this interspecific hybrid and S. kurtzianum autotetraploid lines (ktz-autotetraploid) using the S-SAP (sequence-specific amplified polymorphism) technique and normalized copy number determination by qPCR. Twenty-nine LTR-RT copies were activated in the hybrid and present in the allotetraploid lines. Major LTR-RT activity was detected in Copia-27, Copia-12, Copia-14 and, Gypsy-22. According to our results, LTR-RT copies were activated principally in the hybrid, there was no activation in allotetraploid lines and only one copy was activated in the autotetraploid.

     

Establishment of hepatitis E virus infection‐permissive and ‐non‐permissive human hepatoma PLC/PRF/5 subclones

PLC/PRF/5 cells show limited permissiveness, meaning that almost all subclones are permissive; however, some subclones do not exhibit permissiveness for hepatitis E virus (HEV) infection. In this study, the single‐cell cloning of PLC/PRF/5 was performed and heterogeneous subclones characterized. Notably, the efficiency of intracellular virus replication did not correlate with the permissiveness for HEV infection. However, as well as binding permissive subclones, virus‐like particles bound non‐permissive subclones on various levels, suggesting that these subclones have some deficiencies in the attachment and entry steps of infection. Our data would be useful for investigating the HEV life cycle.     

Isolation of B subunit‐specific monoclonal antibody clones that strongly neutralize the toxicity of Shiga toxin 2

Shiga toxin 2 (Stx2)‐specific mAb‐producing hybridoma clones were generated from mice. Because mice tend to produce small amounts of B subunit (Stx2B)‐specific antibodies at the polyclonal antibody level after immunization via the parenteral route, mice were immunized intranasally with Stx2 toxoids with a mutant heat‐labile enterotoxin as a mucosal adjuvant; 11 different hybridoma clones were obtained in two trials. Six of them were A subunit (Stx2A)‐specific whereas five were Stx2B‐specific antibody‐producing clones. The in vitro neutralization activity of Stx2B‐specific mAbs against Stx2 was greater than that of Stx2A‐specific mAbs on HeLa229 cells. Furthermore, even at low concentrations two of the Stx2B‐specific mAbs (45 and 75D9) completely inhibited receptor binding and showed in vivo neutralization activity against a fivefold median lethal dose of Stx2 in mice. In western blot analysis, these Stx2B‐specific neutralization antibodies did not react to three different mutant forms of Stx2, each amino acid residue of which was associated with receptor binding. Additionally, the nucleotide sequences of the V_H and V_L regions of clones 45 and 75D9 were determined. Our Stx2B‐specific mAbs may be new candidates for the development of mouse‐human chimeric Stx2‐neutralizing antibodies which have fewer adverse effects than animal antibodies for enterohemorrhagic Escherichia coli infection.     

Pattern Selection by Dynamical Biochemical Signals

The development of multicellular organisms involves cells to decide their fate upon the action of biochemical signals. This decision is often spatiotemporally coordinated such that a spatial pattern arises. The dynamics that drive pattern formation usually involve genetic nonlinear interactions and positive feedback loops. These complex dynamics may enable multiple stable patterns for the same conditions. Under these circumstances, pattern formation in a developing tissue involves a selection process: why is a certain pattern formed and not another stable one? Herein we computationally address this issue in the context of the Notch signaling pathway. We characterize a dynamical mechanism for developmental selection of a specific pattern through spatiotemporal changes of the control parameters of the dynamics, in contrast to commonly studied situations in which initial conditions and noise determine which pattern is selected among multiple stable ones. This mechanism can be understood as a path along the parameter space driven by a sequence of biochemical signals. We characterize the selection process for three different scenarios of this dynamical mechanism that can take place during development: the signal either 1) acts in all the cells at the same time, 2) acts only within a cluster of cells, or 3) propagates along the tissue. We found that key elements for pattern selection are the destabilization of the initial pattern, the subsequent exploration of other patterns determined by the spatiotemporal symmetry of the parameter changes, and the speeds of the path compared to the timescales of the pattern formation process itself. Each scenario enables the selection of different types of patterns and creates these elements in distinct ways, resulting in different features. Our approach extends the concept of selection involved in cellular decision-making, usually applied to cell-autonomous decisions, to systems that collectively make decisions through cell-to-cell interactions.