High-affinity hnRNP A1 binding sites and duplex-forming inverted repeats have similar effects on 5' splice site selection in support of a common looping out and repression mechanism

High-affinity binding sites for the hnRNP A1 protein stimulate the use of a distal 5' splice site in mammalian pre-mRNAs. Notably, strong A1-mediated shifts in splice site selection are not accompanied by equivalent changes in the assembly of U1 snRNP-containing complexes on competing 5' splice sites. To explain the above results, we have proposed that an interaction between hnRNP A1 molecules bound to high-affinity sites loops out the internal 5' splice site. Here, we present additional evidence in support of the looping out model. First, replacing A1 binding sites with sequences that can generate a loop through RNA duplex formation activates distal 5' splice site usage in an equivalent manner. Second, increasing the distance between the internal 5' splice site and flanking A1 binding sites does not compromise activation of the distal 5' splice site. Similar results were obtained with pre-mRNAs carrying inverted repeats. Using a pre-mRNA containing only one 5' splice site, we show that splicing is repressed when flanked by two high-affinity A1 binding sites or by inverted repeats, and that inactivation of the internal 5' splice site is sufficient to elicit a strong increase in the use of the distal donor site. Our results are consistent with the view that the binding of A1 to high-affinity sites promotes loop formation, an event that would repress the internal 5' splice site and lead to distal 5' splice site activation.     

Ordered Cloned DNA Map of the Genome of Vibrio cholerae 569B and Localization of Genetic Markers

By using a low-resolution macrorestriction map as the foundation (R. Majumder et al., J. Bacteriol. 176:1105–1112, 1996), an ordered cloned DNA map of the 3.2-Mb chromosome of the hypertoxinogenic strain 569B of Vibrio cholerae has been constructed. A cosmid library the size of about 4,000 clones containing more than 120 Mb of V. cholerae genomic DNA (40-genome equivalent) was generated. By combining landmark analysis and chromosome walking, the cosmid clones were assembled into 13 contigs covering about 90% of the V. cholerae genome. A total of 92 cosmid clones were assigned to the genome and to regions defined by NotI, SfiI, and CeuI macrorestriction maps. Twenty-seven cloned genes, 9 rrn operons, and 10 copies of a repetitive DNA sequence (IS1004) have been positioned on the ordered cloned DNA map.     

Nitrate Reductase is Needed for Methyl Jasmonate-Mediated Arsenic Toxicity Tolerance of Rice by Modulating the Antioxidant Defense System,Glyoxalase System and Arsenic Sequestration Mechanism

Journal of Plant Growth Regulation - The study was conducted to consider the role of nitrate reductase (NR)-synthesized nitric oxide (NO) in the methyl jasmonate (MJ)-induced tolerance of arsenic...     

Genetic effects of acridine compounds.

Acridines and a very large number of acridine derivatives are used in enormous quantities both in medicine and industry. The mutagenic action of these compounds has been demonstrated in a wide variety of organisms and is known to occur both in the dark as well as in the presence of light (photodynamic action). At the molecular level, acridines have been shown to cause frameshift mutations of both the addition and deletion types, a characteristic which has been of tremendous help in elucidating the nature of the genetic code. These and various other biological effects of acridines, such as inhibition of DNA repair, curing of plasmids and cell-growth inhibition, are examined in this review.     

Contribution of a caffeine-sensitive recombinational repair pathway to survival and mutagenesis in UV-irradiated Schizosaccharomyces pombe

Summary Cells of wild-type Schizosacharomyces pombe exposed to UV radiation in either G1 or G2 phase show enhanced inactivation of colony-forming ability if plated in the presence of caffeine. This UV-sensitization by caffeine is abolished in both G1 and G2 phase cells by the rad1 mutation; since both caffeine and the rad1 mutation markedly reduce recombinational events, this suggests that a recombinational repair process is active in cells irradiated either in G1 or G2 phase. A prereplicative or sister chromatid exchange recombinational process appears to account for caffeine-sensitive repair of UV-damage in G2 cells (which possess at the time of radiation exposure the duplicated genome necessary for recombination), since caffeine-sensitive repair begins immediately and is completed before resumption of DNA synthesis. In contrast, since caffeine-sensitive repair of UV-damage in G1 cells displays a considerable lag and then occurs concomitantly with DNA synthesis, it appears that G1 cells must acquire a second genome in order to accomplish a caffeine-sensitive recovery process. Since a duplicated genome is required for caffeinesensitive repair, all such repair would seem to involve a recombinational mechanism. In G1 cells the process may be a post-replication recombinational mechanism. Since G2 phase cells are considerably more UV-resistant than G1 phase cells, the prereplicative recombinational process appears to be a much more efficient process for dealing with UV-induced damage than the post-replication mechanism.UV-induced mutagenesis was examined in wildtype and rad mutants using a forward mutation system. Rad mutants which show higher UV-induced mutation rates than wild-type retain UV-sensitization by caffeine (and thus presumably retain the recombinational mechanism). In contrast, rad strains which are relatively UV-immutable compared to wild-type do not possess the caffeine-sensitive UV-repair process. The recombinational process therefore may be the major pathway responsible for UV-induced mutation.AECL Reference No. 6251; NRC Publication No. 16999     

UV-induced lethal sectoring and pure mutant clones in yeast

The induction of lethal sectoring and pure mutant clones by ultraviolet light has been studied in a homogeneous G₁ population of Saccharomyces cerevisiae grown in a normal growth medium. At the lowest UV dose of 250 ergs, which corresponds to a shoulder in the survival curve, all mutants appeared as pure clones. At higher doses the frequency of mosaic mutants progressively increased. These results indicate a relationship between the highest frequency of complete mutants and the maximum repair activity. In addition, the frequency of lethal sectoring at all doses tested was too low to account for the origin of pure mutant clones.     

A novel procedure for the recovery of hybrid products from protoplast fusion

Summary A protoplast mixture of two -mating type strains of Saccharomyces cerevisiae was encapsulated in a beaded form of calcium alginate gel. Part of the same protoplast mixture was embedded in a molten (50–55°C) agar overlay, a routine procedure for the recovery of regenerated colonies and hybrid products. The number of hybrid products isolated from the encapsulated protoplast mixtures was greater than that recovered from the agar overlay. The increase ranged from 40 to 1000 fold.     

Role of Ni-tolerant Bacillus spp. and Althea rosea L. in the phytoremediation of Ni-contaminated soils

In our current study, four nickel-tolerant (Ni-tolerant) bacterial species viz, Bacillus thuringiensis 002, Bacillus fortis 162, Bacillus subtilis 174, and Bacillus farraginis 354, were screened using Ni-contaminated media. The screened microbes exhibited positive results for synthesis of indole acetic acid (IAA), siderophore production, and phosphate solubilization. The effects of these screened microbes on Ni mobility in the soil, root elongation, plant biomass, and Ni uptake in Althea rosea plants grown in Ni-contaminated soil (200 mg Ni kg^?1) were evaluated. Significantly higher value for water-extractable Ni (38 mg kg^?1) was observed in case of Ni-amended soils inoculated with B. subtilis 174. Similarly, B. thuringiensis 002, B. fortis 162, and B. subtilis 174 significantly enhanced growth and Ni uptake in A. rosea. The Ni uptake in the shoots and roots of B. subtilis 174-inoculated plants enhanced up to 1.7 and 1.6-fold, respectively, as compared to that in the un-inoculated control. Bacterial inoculation also significantly improved the root and shoot biomass of treated plants. The current study presents a novel approach for bacteria-assisted phytoremediation of Ni-contaminated areas.     

Enhancement of the antiangiogenic activity of interleukin-12 by peptide targeted delivery of the cytokine to alphavbeta3 integrin

We engineered a fusion protein, mrIL-12vp [mouse recombinant interleukin (IL)-12 linked to vascular peptide], linking the vascular homing peptide CDCRGDCFC (RGD-4C), a ligand for alphavbeta3 integrin, to mrIL-12 to target IL-12 directly to tumor neovasculature. The fusion protein stimulated IFN-gamma production in vitro and in vivo, indicating its biological activity was consistent with mrIL-12. Immunofluorescence techniques showed mrIL-12vp specifically bound to alphavbeta3 integrin-positive cells but not to alphavbeta3 integrin-negative cells. In corneal angiogenesis assays using BALB/c mice treated with either 0.5 microg/mouse/d of mrIL-12vp or mrIL-12 delivered by subcutaneous continuous infusion, mrIL-12vp inhibited corneal neovascularization by 67% compared with only a slight reduction (13%) in angiogenesis in the mrIL-12-treated animals (P = 0.008). IL-12 receptor knockout mice given mrIL-12vp showed a marked decrease in the area of corneal neovascularization compared with mice treated with mrIL-12. These results indicate that mrIL-12vp inhibits angiogenesis through IL-12-dependent and IL-12-independent mechanisms, and its augmented antiangiogenic activity may be due to suppression of endothelial cell signaling pathways by the RGD-4C portion of the fusion protein. Mice injected with NXS2 neuroblastoma cells and treated with mrIL-12vp showed significant suppression of tumor growth compared with mice treated with mrIL-12 (P = 0.03). Mice did not show signs of IL-12 toxicity when treated with mrIL-12vp, although hepatic necrosis was present in mrIL-12-treated mice. Localization of IL-12 to neovasculature significantly enhances the antiangiogenic effect, augments antitumor activity, and decreases toxicity of IL-12, offering a promising strategy for expanding development of IL-12 for treatment of cancer patients.