Synthetic inhibitors of the processing of pretransfer RNA by the ribonuclease P ribozyme: enzyme inhibitors which act by binding to substrate

2,2'-p-Phenylene bis[6-(4-methyl-1-piperazinyl)]benzimidazole, 2,2'-bis(3,5-dihydroxyphenyl)-6,6'-bis benzimidazole, and 2,2'-bis(4-hydroxyphenyl)-6,6'-bis benzimidazole are shown by UV-visible and fluorescence spectrophotometry to be strong ligands for tRNA, giving simple, hyperbolic binding isotherms with apparent dissociation constants in the micromolar range. Hydroxyl radical footprinting indicates that they may bind in the D and T loops. On the basis of this tRNA recognition as a rationale, they were tested as inhibitors of the processing of precursor tRNAs by the RNA subunit of Escherichia coli RNase P (M1 RNA). Preliminary studies show that inhibition of the processing of Drosophila tRNA precursor molecules by phosphodiester bond cleavage, releasing the extraneous 5'-portion of RNA and the mature tRNA molecule, was dependent on both the structure of the inhibitor and the structure of the particular tRNA precursor substrate for tRNA(Ala), tRNA(Val), and tRNA(His). In more detailed followup using the tRNA(His) precursor as the substrate, experiments to determine the concentration dependence of the reaction showed that inhibition took time to reach its maximum extent. I(50) values (concentrations for 50% inhibition) were between 5.3 and 20.8 microM, making these compounds among the strongest known inhibitors of this ribozyme, and the first inhibitors of it not based on natural products. These compounds effect their inhibition by binding to the substrate of the enzyme reaction, making them examples of an unusual class of enzyme inhibitors. They provide novel, small-molecule, inhibitor frameworks for this endoribonuclease ribozyme.     

Effects of sugar on vegetative development and floral transition in Arabidopsis

Although sugar has been suggested to promote floral transition in many plant species, growth on high concentrations (5% [w/v]) of sucrose (Suc) significantly delayed flowering time, causing an increase in the number of leaves at the time of flowering in Arabidopsis. The effect of high concentrations of Suc seemed to be metabolic rather than osmotic. The delay of floral transition was due to extension of the late vegetative phase, which resulted in a delayed activation of LFY expression. In addition, growth on low concentrations (1% [w/v]) of Suc slightly inhibited flowering in wild-type plants. This delay resulted from effects on the early vegetative phase. This inhibition was more pronounced in tfl1, an early flowering mutant, than in the wild type. Although 1% (w/v) Suc was reported to promote floral transition of late-flowering mutants such as co, fca, and gi, floral transition in these mutants was delayed by a further increase in Suc concentration. These results suggest that sugar may affect floral transition by activating or inhibiting genes that act to control floral transition, depending on the concentration of sugars, the genetic background of the plants, and when the sugar is introduced. Growth on 1% (w/v) Suc did not restore the reduced expression levels of FT and SOC1/AGL20 in co or fca mutants. Rather, expression of FT and SOC1/AGL20 was repressed by 1% (w/v) Suc in wild-type background. The possible effects of sugar on gene expression to promote floral transition are discussed.     

Comparison of ES cell fate in sandwiched aggregates and co-cultured aggregates during blastocyst formation by monitored GFP expression

Markers and the means to detect them are required to monitor the fate of living cells. However, few suitable markers for living cells were known until a green fluorescent protein (GFP) was discovered. We have established mouse embryonic stem (ES) cell lines that express mutant GFP under the chicken beta-actin (CAG) promoter. Using these cell lines, we were able to follow the migration of ES cells during blastocyst formation both in sandwiching and coculture methods, even if only a single ES cell was used. Furthermore, the contribution of ES cells to the inner cell mass (ICM) was easily estimated at the blastocyst stage. We compared sandwiching with coculture aggregation relative to the contribution of the ES cell in the ICM, and the results indicated that there was no difference in the ratios of chimeric embryos having ICM contributed from cultured ES cells. Furthermore, an aggregated single ES cell was able to contribute three or four cells to the ICM at the blastocyst stage. Thus we conclude that one, instead of two, embryos is enough to make aggregation with ES cells, and a single ES cell attached to an embryo is enough to produce germline chimeras. Moreover, we could clearly observe single cell fate during blastocyst formation. This suggests that our established cell line can be used for monitoring single cell fate in vivo. In addition, we have shown that up to five doses of 30 sec of UV irradiation using GFP filters have no effect on the embryonic development.     

Accumulation of anthranilic acid and N-glucosylanthranilic acid by a Corynebacterium glutamicum mutant resistant to DL-serine hydroxamate

During a study on the effect of DL-serine hydroxamate on Corynebacterium glutamicum (JCM1318, a wild strain), a mutant resistant to the drug, strain TO3002, was isolated. This mutant accumulated five Ehrlich's reagent positive fluorescent substances in the culture medium. Two major and one minor fluorescent products were isolated by preparative high-performance liquid chromatography following charcoal column chromatography from the culture supernatant. One major product was identified as anthranilic acid whose molecular ion was confirmed to be 137 by a measurement of liquid chromatography-mass spectrometry (LC-MS), and NMR spectrum coincided with that of anthranilic acid. LC-MS spectra of another major and the minor product showed that they had the same molecular weight of 299. This major product was supported to be N-glucosylanthranilic acid (N-o-carboxyphenyl-1-beta-glucosylamine) by two-dimensional (1)H and (13)C NMR analyses. The minor product was speculated to be an Amadori compound derived from N-glucosylanthranilic acid. N-Glucosylanthranilic acid accumulated in the early phase, then decreased in the late phase of the culture. In contrast, the accumulation of anthranilic acid increased remarkably in the late phase of the fermentation. Based on this phenomenon, it was assumed that N-glucosylanthranilic acid once accumulated was decomposed to form anthranilic acid, at least in large part, with the progress of fermentation. The strain TO3002 showed a leaky requirement for L-tryptophan or indole (but did not for anthranilic acid) and resistance to DL-serine hydroxamate.     

Noncompetitive counteractions of DNA polymerase epsilon and ISW2/yCHRAC for epigenetic inheritance of telomere position effect in Saccharomyces cerevisiae

Relocation of euchromatic genes near the heterochromatin region often results in mosaic gene silencing. In Saccharomyces cerevisiae, cells with the genes inserted at telomeric heterochromatin-like regions show a phenotypic variegation known as the telomere-position effect, and the epigenetic states are stably passed on to following generations. Here we show that the epigenetic states of the telomere gene are not stably inherited in cells either bearing a mutation in a catalytic subunit (Pol2) of replicative DNA polymerase epsilon (Pol epsilon) or lacking one of the nonessential and histone fold motif-containing subunits of Pol epsilon, Dpb3 and Dpb4. We also report a novel and putative chromatin-remodeling complex, ISW2/yCHRAC, that contains Isw2, Itc1, Dpb3-like subunit (Dls1), and Dpb4. Using the single-cell method developed in this study, we demonstrate that without Pol epsilon and ISW2/yCHRAC, the epigenetic states of the telomere are frequently switched. Furthermore, we reveal that Pol epsilon and ISW2/yCHRAC function independently: Pol epsilon operates for the stable inheritance of a silent state, while ISW2/yCHRAC works for that of an expressed state. We therefore propose that inheritance of specific epigenetic states of a telomere requires at least two counteracting regulators.     

Loss of PGC-specific expression of the orphan nuclear receptor ERR-beta results in reduction of germ cell number in mouse embryos

Estrogen related receptor beta (ERR-beta) is an orphan nuclear receptor specifically expressed in a subset of extra-embryonic ectoderm of post-implantation embryos. ERR-beta is essential for placental development since the ERR-beta null mutants die at 10.5dpc due to the placenta abnormality. Here, we show that the ERR-beta is specifically expressed in primordial germ cells (PGC), obviously another important cell type for reproduction. Expression of the ERR-beta mRNA in embryonic germ cells started at E11.5 as soon as PGC reached genital ridges, and persisted until E15-E16 in both sexes. Immunostaining with anti-ERR-beta antibody revealed that the ERR-beta protein is exclusively expressed in germ cells in both male and female gonads from E11.5 to E16. 5. To study function of the ERR-beta in PGC, we complemented placental defects of the ERR-beta null mutants with wild-type tetraploid embryos, and analyzed germ cell development in the rescued embryos. It was found that development of gonad and PGC was not apparently affected, but number of germ cells was significantly reduced in male and female gonads, suggesting that the ERR-beta appears to be involved in proliferation of gonadal germ cells. The rescued embryos could develop to term and grow up to adulthood. The rescued ERR-beta null male were found to be fertile, but both male and female null mutants exhibited behavioural abnormalities, implying that the ERR-beta plays important roles in wider biological processes than previously thought.     

Shp2 regulates SRC family kinase activity and Ras/Erk activation by controlling Csk recruitment

The protein-tyrosine phosphatase Shp2 plays an essential role in growth factor and integrin signaling, and Shp2 mutations cause developmental defects and/or malignancy. Previous work has placed Shp2 upstream of Ras. However, the mechanism of Shp2 action and its substrate(s) are poorly defined. Additional Shp2 functions downstream of, or parallel to, Ras/Erk activation also are proposed. Here, we show that Shp2 promotes Src family kinase (SFK) activation by regulating the phosphorylation of the Csk regulator PAG/Cbp, thereby controlling Csk access to SFKs. In Shp2-deficient cells, SFK inhibitory C-terminal tyrosines are hyperphosphorylated, and the tyrosyl phosphorylation of multiple SFK substrates, including Plcgamma1, is decreased. Decreased Plcgamma1 phosphorylation leads to defective Ras activation on endomembranes, and may help account for impaired Erk activation in Shp2-deficient cells. Decreased phosphorylation/activation of other SFK substrates may explain additional consequences of Shp2 deficiency, including altered cell spreading, stress fibers, focal adhesions, and motility.     

Genome-wide identification of NBS genes in<Emphasis Type="Italic"> japonica</Emphasis> rice reveals significant expansion of divergent non-TIR NBS-LRR genes

A complete set of candidate disease resistance ( R) genes encoding nucleotide-binding sites (NBSs) was identified in the genome sequence of japonica rice ( Oryza sativa L. var. Nipponbare). These putative R genes were characterized with respect to structural diversity, phylogenetic relationships and chromosomal distribution, and compared with those in Arabidopsis thaliana. We found 535 NBS-coding sequences, including 480 non-TIR (Toll/IL-1 receptor) NBS-LRR (Leucine Rich Repeat) genes. TIR NBS-LRR genes, which are common in A. thaliana, have not been identified in the rice genome. The number of non-TIR NBS-LRR genes in rice is 8.7 times higher than that in A. thaliana, and they account for about 1% of all of predicted ORFs in the rice genome. Some 76% of the NBS genes were located in 44 gene clusters or in 57 tandem arrays, and 16 apparent gene duplications were detected in these regions. Phylogenetic analyses based both NBS and N-terminal regions classified the genes into about 200 groups, but no deep clades were detected, in contrast to the two distinct clusters found in A. thaliana. The structural and genetic diversity that exists among NBS-LRR proteins in rice is remarkable, and suggests that diversifying selection has played an important role in the evolution of R genes in this agronomically important species. (Supplemental material is available online at .)Communicated by R. HagemannThe first three authors contributed equally to this work