Nucleosome compaction facilitates HP1γ binding to methylated H3K9

The α, β and γ isoforms of mammalian heterochromatin protein 1 (HP1) selectively bind to methylated lysine 9 of histone H3 via their chromodomains. Although the phenotypes of HP1-knockout mice are distinct for each isoform, the molecular mechanisms underlying HP1 isoform-specific function remain elusive. In the present study, we found that in contrast to HP1α, HP1γ could not bind tri-methylated H3 lysine 9 in a reconstituted tetra-nucleosomes when the nucleosomes were in an uncompacted state. The hinge region connecting HP1''s chromodomain and chromoshadow domain contributed to the distinct recognition of the nucleosomes by HP1α and HP1γ. HP1γ, but not HP1α, was strongly enhanced in selective binding to tri-methylated lysine 9 in histone H3 by the addition of Mg²⁺ or linker histone H1, which are known to induce compaction of nucleosomes. We propose that this novel property of HP1γ recognition of lysine 9 in the histone H3 tail in different nucleosome structures plays a role in reading the histone code.     

Enhanced chemiluminescence of 6-(4-methoxyphenyl)imidazo[1,2-a]pyrazin-3(7H)-one by attachment of a cyclomaltooligosaccharide (cyclodextrin). Attachment of cyclomaltononaose (delta-cyclodextrin)

2-Methyl-6-(4-methoxyphenyl)imidazo[1,2-a]pyrazin-3(7H)-one (MCLA) is an oxygen-induced chemiluminescent compound. It has been shown that the chemiluminescence can be enhanced by forming a cyclomaltooligosaccharide (cyclodextrin)-bound MCLA, and therefore, in continuation of the survey of the types of cyclodextrins, in this study, MCLA was attached to the secondary hydroxyl face of delta-cyclodextrin, which consists of nine D-glucose units. Although the oxygen-induced chemiluminescence efficiency of delta-cyclodextrin-bound MCLA in a pH 8.0 aqueous phosphate buffer was 12 times greater than that of MCLA, the efficiency was markedly lower than that of gamma-cyclodextrin-bound MCLA, which exhibited the highest chemiluminescence efficiency in the previous investigation. Although fluorescence efficiency and light-emitter formation efficiency for delta-cyclodextrin-bound MCLA were similar to those for gamma-cyclodextrin-bound MCLA, singlet-excited state formation efficiency for delta-cyclodextrin-bound MCLA was lower than that for gamma-cyclodextrin-bound MCLA. This study distinctly indicated the optimum cyclodextrin for construction of greatly luminescent cyclodextrin-bound MCLA is gamma-cyclodextrin.     

The MRX Complex Ensures NHEJ Fidelity through Multiple Pathways Including Xrs2-FHA–Dependent Tel1 Activation

Because DNA double-strand breaks (DSBs) are one of the most cytotoxic DNA lesions and often cause genomic instability, precise repair of DSBs is vital for the maintenance of genomic stability. Xrs2/Nbs1 is a multi-functional regulatory subunit of the Mre11-Rad50-Xrs2/Nbs1 (MRX/N) complex, and its function is critical for the primary step of DSB repair, whether by homologous recombination (HR) or non-homologous end joining. In human NBS1, mutations result truncation of the N-terminus region, which contains a forkhead-associated (FHA) domain, cause Nijmegen breakage syndrome. Here we show that the Xrs2 FHA domain of budding yeast is required both to suppress the imprecise repair of DSBs and to promote the robust activation of Tel1 in the DNA damage response pathway. The role of the Xrs2 FHA domain in Tel1 activation was independent of the Tel1-binding activity of the Xrs2 C terminus, which mediates Tel1 recruitment to DSB ends. Both the Xrs2 FHA domain and Tel1 were required for the timely removal of the Ku complex from DSB ends, which correlates with a reduced frequency of imprecise end-joining. Thus, the Xrs2 FHA domain and Tel1 kinase work in a coordinated manner to maintain DSB repair fidelity.     

Analysis of the NH2-Terminal 83rd Amino Acid of Escherichia coli GyrA in Quinolone-Resistance

Artificial mutations of Gyrase A protein (GyrA) in Escherichia coli by site-directed mutagenesis were generated to analyze quinolone-resistant mechanisms. By genetic analysis of gyrA genes in a gyrA temperature sensitive (Ts) background, exchange of Ser at the NH₂-terminal 83rd position of GyrA to Trp, Leu, Phe, Tyr, Ala, Val, and Ile caused bacterial resistance to the quinolones, while exchange to Gly, Asn, Lys, Arg and Asp did not confer resistance. These results indicate that it is the most important for the 83rd amino acid residue to be hydrophobic in expressing the phenotype of resistance to the quinolones. These findings also suggest that the hydroxyl group of Ser would not play a major role in the quinolone-gyrase interaction and Ser83 would not interact directly with other amino acid residues.     

Comparative leaf development ofOsmunda lancea andO. japonica (osmundaceae): Heterochronic origin of rheophytic stenophylly

Comparative development of the narrow pinnules of rheophyticOsmunda lancea and of the broad pinnules of a related dryland species,O. japonica, was examined and the origin of rheophytic stenophylly was discussed. The mature leaves and their various parts ofO. lancea are smaller and narrower than those ofO. japonica. The young pinnules ofO. lancea at the initiation of cell expansion are smaller than those ofO. japonica. The growth pattern of the pinnules is fundamentally the same in the two species, but pinnule growth period is shorter inO. lancea than inO. japonica. While the largest growth rate in pinnule length is quite similar, inO. lancea the pinnules are less elongated and much less broadened during ontogeny. Cell expansion in the mesophyll and epidermis proceeds acropetally and toward the margin along the axes of costules and veins. Although the numbers of mesophyll and epidermal cells between two adjacent veinlets are almost the same inO. lancea andO. japonica, during the subsequent growth period inO. lancea, the cells expand to a smaller extent and the veinlets become more narrowly oblique to the costule. This oblique distortion of laminar segments framed by veins causes stenophylly, an allometric modification. The stenophylly ofO. lancea is believed to have arisen by heterochronic evolution, in particular, progenesis.     

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.     

A Naturally Occurring Canine Model of Autosomal Recessive Congenital Stationary Night Blindness

Congenital stationary night blindness (CSNB) is a non-progressive, clinically and genetically heterogeneous disease of impaired night vision. We report a naturally-occurring, stationary, autosomal recessive phenotype in beagle dogs with normal daylight vision but absent night vision. Affected dogs had normal retinas on clinical examination, but showed no detectable rod responses. They had “negative-type” mixed rod and cone responses in full-field ERGs. Their photopic long-flash ERGs had normal OFF-responses associated with severely reduced ON-responses. The phenotype is similar to the Schubert-Bornschein form of complete CSNB in humans. Homozygosity mapping ruled out most known CSNB candidates as well as CACNA2D4 and GNB3. Three remaining genes were excluded based on sequencing the open reading frame and intron-exon boundaries (RHO, NYX), causal to a different form of CSNB (RHO) or X-chromosome (NYX, CACNA1F) location. Among the genes expressed in the photoreceptors and their synaptic terminals, and mGluR6 cascade and modulators, reduced expression of GNAT1, CACNA2D4 and NYX was observed by qRT-PCR in both carrier (n = 2) and affected (n = 2) retinas whereas CACNA1F was down-regulated only in the affecteds. Retinal morphology revealed normal cellular layers and structure, and electron microscopy showed normal rod spherules and synaptic ribbons. No difference from normal was observed by immunohistochemistry (IHC) for antibodies labeling rods, cones and their presynaptic terminals. None of the retinas showed any sign of stress. Selected proteins of mGluR6 cascade and its modulators were examined by IHC and showed that PKCα weakly labeled the rod bipolar somata in the affected, but intensely labeled axonal terminals that appeared thickened and irregular. Dendritic terminals of ON-bipolar cells showed increased Goα labeling. Both PKCα and Goα labeled the more prominent bipolar dendrites that extended into the OPL in affected but not normal retinas. Interestingly, RGS11 showed no labeling in the affected retina. Our results indicate involvement of a yet unknown gene in this canine model of complete CSNB.     

Trace elements influenced by environmental changes in Lake Biwa: (I) Seasonal variations under suboxic hypolimnion conditions during 2007 and 2009

Dissolved oxygen (DO) in the Lake Biwa hypolimnion reached its lowest level of <1 mg kg^?1 in 2007. In this paper, we report the variations in the total dissolvable (TD), dissolved (D), and labile particulate (LP) fractions of Al, Si, P, V, Cr, Mn, Fe, Ni, Cu, Zn, As, Mo, W, and U in Lake Biwa 2007 and 2009. Al and Fe species were predominantly in the form of LP-Al and LP-Fe and were strongly correlated with one another (r = 0.99), suggesting that the weathering of aluminous minerals and the supply of clay mineral particles are the main factors that influence the distributions of Al and Fe. Although D-Al increased in the summer epilimnion, D-Fe was relatively low, probably as a result of uptake by plants. Reductive release of Fe from the bottom was not seen. Mn was also dominated by LP-Mn, but this fraction showed a different distribution to those of LP-Al and LP-Fe. The D-Mn and LP-Mn concentrations varied by factors of 700–1000 and showed marked increases in the bottom water during stratification in 2007. We believe that Mn²⁺ was released from the sediments and oxidized by DO in the bottom water. Ni, Cu, Zn, and Cr, which exist as cationic species, had LP/TD ratios of 0.1–0.7 and relatively uniform distributions. Si, P, V, As, Mo, W, and U, which form oxoacid species, had LP/TD ratios of 0–0.8. Si, P, and As were characterized by nutrient-like profiles, V, W, and U showed summer maxima in the epilimnion, and Mo had a uniform distribution. TD-Mo increased in the bottom water along with TD-Mn, while TD-V and TD-W showed significant decreases. These results are likely attributable to differences in the adsorption of these elements onto manganese oxides and iron hydroxides.