Suppression of telomere-binding protein gene expression represses seed and fruit development in tomato.

Tomato (Solanum lycopersicum L.) plants were transformed with an antisense construct of a cDNA encoding tomato telomere-binding protein (LeTBP1) to describe the role of a telomere-binding protein at the whole plant level. Fruit size decreased corresponding to the degree of suppression of LeTBP1 expression. This inhibition of fruit development was likely due to a decrease in the number of seeds in the LeTBP1 antisense plants. Pollen fertility and pollen germination rate decreased in accordance with the degree of suppression of LeTBP1 expression. Ovule viability was also reduced in the LeTBP1 antisense plants. Although plant height was somewhat reduced in the antisense plants compared to the control plants, the number and weight of leaves were unaffected by LeTBP1 suppression. The number and morphology of flowers were also normal in the antisense plants. These indicate that reduced fertility in the antisense plants is not an indirect effect of altered vegetative growth. LeTBP1 expression was sensitive to temperature stress in wild-type plants. We conclude that LeTBP1 plays a critical role in seed and fruit development rather than vegetative growth and flower formation.

Suppression of dioxin mediated aryl hydrocarbon receptor transformation by ethanolic extracts of propolis.

Present study demonstrated that the ethanolic extracts of propolis containing higher concentrations of flavonoids suppressed 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced aryl hydrocarbon receptor transformation in a dose-dependent manner. The IC(50) values of propolis group 3 and group 12 were 1.2 and 3.6 microg/ml, respectively, indicating that propolis showed stronger antagonistic effects as compared with vegetable extracts.

The tRNA-dependent activation of arginine by arginyl-tRNA synthetase requires inter-domain communication.

The tRNA-dependent amino acid activation catalyzed by mammalian arginyl-tRNA synthetase has been characterized. A conditional lethal mutant of Chinese hamster ovary cells that exhibits reduced arginyl-tRNA synthetase activity (Arg-1), and two of its derived revertants (Arg-1R4 and Arg-1R5) were analyzed at the structural and functional levels. A single nucleotide change, resulting in a Cys to Tyr substitution at position 599 of arginyl-tRNA synthetase, is responsible for the defective phenotype of the thermosensitive and arginine hyper-auxotroph Arg-1 cell line. The two revertants have a single additional mutation resulting in a Met222 to Ile change for Arg-1R4 or a Tyr506 to Ser change for Arg-1R5. The corresponding mutant enzymes were expressed in yeast and purified. The Cys599 to Tyr mutation affects both the thermal stability of arginyl-tRNA synthetase and the kinetic parameters for arginine in the ATP-PP(i) exchange and tRNA aminoacylation reactions. This mutation is located underneath the floor of the Rossmann fold catalytic domain characteristic of class 1 aminoacyl-tRNA synthetases, near the end of a long helix belonging to the alpha-helix bundle C-terminal domain distinctive of class 1a synthetases. For the Met222 to Ile revertant, there is very little effect of the mutation on the interaction of arginyl-tRNA synthetase with either of its substrates. However, this mutation increases the thermal stability of arginyl-tRNA synthetase, thereby leading to reversion of the thermosensitive phenotype by increasing the steady-state level of the enzyme in vivo. In contrast, for the Arg-1R5 cell line, reversion of the phenotype is due to an increased catalytic efficiency of the C599Y/Y506S double mutant as compared to the initial C599Y enzyme. In light of the location of the mutations in the 3D structure of the enzyme modeled using the crystal structure of the closely related yeast arginyl-tRNA synthetase, the kinetic analysis of these mutants suggests that the obligatory tRNA-induced activation of the catalytic site of arginyl-tRNA synthetase involves interdomain signal transduction via the long helices that build the tRNA-binding domain of the enzyme and link the site of interaction of the anticodon domain of tRNA to the floor of the active site.

The efficiency of Escherichia coli selenocysteine insertion is influenced by the immediate downstream nucleotide.

Selenocysteine (Sec) incorporation requires the TGA opal codon and a downstream Sec insertion sequence (SECIS), which can be partially randomized and cloned into M13 pIII fusion constructs for phage display. This combinatorial approach provides a convenient non-radioactive assay that couples phage production to opal suppression. Two SECIS libraries were prepared, with the immediate downstream nucleotide either randomized (TGAN) or fixed as thymidine (TGAT). The TGAN library resulted in a majority of clones with a downstream purine and selenium-independent phage production, implicating the endo-genous tryptophan-inserting opal suppression pathway. Although the addition of sodium selenite to the growth medium did not affect phage production, it did increase the level of Sec insertion, as shown by the chemical reactivity of the resulting phage. The TGAT phage library yielded clones with strictly selenium-dependent phage production and reactivity consistent with the presence of Sec. These clones were prone to spontaneous mutation upon further propagation, however, resulting in loss of the selenium-dependent phenotype. We conclude that the immediate downstream nucleotide determines whether the endogenous opal suppression pathway competes with co-translational Sec insertion.

Pollen fertility restoration by nuclear gene Fr in CMS bean: nuclear-directed alteration of a mitochondrial population.

Two nuclear genes, Fr and Fr2, have been identified that restore pollen fertility to cytoplasmic male sterile (CMS) common bean (Phaseolus vulgaris L.) by apparently distinct mechanisms. Whereas Fr2 appears to suppress the expression of a male sterility associated mitochondrial sequence (designated pvs), Fr restores pollen fertility by causing the elimination of this unusual mitochondrial DNA-segment. To further investigate the mechanism of Fr action, Fr and Fr2 were cointroduced into the nucleus of a bean line containing the sterility inducing cytoplasm. When the effect of pvs was suppressed by Fr2, the presence of Fr no longer directed the elimination of the mitochondrial pvs sequence. This result suggests that the Fr function is dependent on proper expression of the pvs sequence. To evaluate the temporal and spatial patterns of Fr action, we undertook a polymerase chain reaction-based approach to trace the fate of the pvs sequence in different tissues of F2 and F3 fertile-restored plants derived from a genetic cross between a cytoplasmic male sterile line of common bean, CMS-Sprite (frfr), and fertility restorer line R351 (FrFr). We demonstrate that the Fr-directed disappearance of pvs sequence occurs during flower development. Elimination of the pvs sequence from developing megaspores results in permanent fertility restoration in the following generations. Genetic analysis demonstrated that permanent fertility restoration, that is, the complete elimination of pvs from reproductive tissues requires two doses of the Fr allele or the absence of fr in F2 individuals.(ABSTRACT TRUNCATED AT 250 WORDS)