Polypyrimidine Tract Binding Protein Prevents Activity of an Intronic Regulatory Element That Promotes Usage of a Composite 3��-Terminal Exon

Alternative splicing of 3′-terminal exons plays a critical role in gene expression by producing mRNA with distinct 3′-untranslated regions that regulate their fate and their expression. The Xenopus α-tropomyosin pre-mRNA possesses a composite internal/3′-terminal exon (exon 9A9′) that is differentially processed depending on the embryonic tissue. Exon 9A9′ is repressed in non-muscle tissue by the polypyrimidine tract binding protein, whereas it is selected as a 3′-terminal or internal exon in myotomal cells and adult striated muscles, respectively. We report here the identification of an intronic regulatory element, designated the upstream terminal exon enhancer (UTE), that is required for the specific usage of exon 9A9′ as a 3′-terminal exon in the myotome. We demonstrate that polypyrimidine tract binding protein prevents the activity of UTE in non-muscle cells, whereas a subclass of serine/arginine rich (SR) proteins promotes the selection of exon 9A9′ in a UTE-dependent way. Morpholino-targeted blocking of UTE in the embryo strongly reduced the inclusion of exon 9A9′ as a 3′-terminal exon in the endogenous mRNA, demonstrating the function of UTE under physiological circumstances. This strategy allowed us to reveal a splicing pathway that generates a mRNA with no in frame stop codon and whose steady-state level is translation-dependent. This result suggests that a non-stop decay mechanism participates in the strict control of the 3′-end processing of the α-tropomyosin pre-mRNA.     

The CR3 motif of Rrp44p is important for interaction with the core exosome and exosome function

The 10-subunit RNA exosome is involved in a large number of diverse RNA processing and degradation events in eukaryotes. These reactions are carried out by the single catalytic subunit, Rrp44p/Dis3p, which is composed of three parts that are conserved throughout eukaryotes. The exosome is named for the 3′ to 5′ exoribonuclease activity provided by a large C-terminal region of the Rrp44p subunit that resembles other exoribonucleases. Rrp44p also contains an endoribonuclease domain. Finally, the very N-terminus of Rrp44p contains three Cys residues (CR3 motif) that are conserved in many eukaryotes but have no known function. These three conserved Cys residues cluster with a previously unrecognized conserved His residue in what resembles a metal-ion-binding site. Genetic and biochemical data show that this CR3 motif affects both endo- and exonuclease activity in vivo and both the nuclear and cytoplasmic exosome, as well as the ability of Rrp44p to associate with the other exosome subunits. These data provide the first direct evidence that the exosome-Rrp44p interaction is functionally important and also provides a molecular explanation for the functional defects when the conserved Cys residues are mutated.     

Cholinesterase activity in some species of theAllium genus

In partly purified protein complexes obtained from 22 species of theAllium genus and 6 cultivars ofAllium cepa the activity of cholinesterases was detected and measured using the method of Ellman et al. The degree of its inhibition with 10^-4 M neostigmine was also tested. It was found that the activity of cholinesterase differed in individual species up to two hundred times, while the differences in the inhibitory activity of 10^-4 M neostigmine occurred only in a few cases. Individual sections and cultivars could not be characterized on the basis of the differences in the activities of the cholinesterases. Of all the sections that ofPhyllodolon shows the highest average activity. In the case of the tested cultivars distinctly the lowest activity was observed in cv. Kastická. The values of the enzymatic activity measured by Ellman’s method in this plant material include the activity of specific and unspecific cholinesterases and the part uninhibitable by neostigmine.     

Effect of lowered incubation temperatures on nucleic acid and protein synthesis by a mesophilic and a psychrophilic bacterium

The authors studied changes in the synthesis of nucleic acids (RNA, DNA) and protein by a mesophilic strain ofEscherichia coli B and a psychrophilic strain ofPseudomonas fluorescens at a low incubation temperature giving tenfold prolongation of the generation time. It was found that lowering the incubation temperature was followed by an increase in the intracellular nucleic acid content during the lag phase and the phase of accelerated growth, in which maximum nucleic acid (NA) values were reached. As a result, the total NA level in the cell also remained relatively high during further proliferation, when the increase in NA (particularly RNA) slows down at low incubation temperatures. Proteosynthesis, however, fell in the mesophilic culture. The smaller effect of a lowered temperature on DNA biosynthesis was manifested specifically in the lag phase ofEscherichia coli, in which disproportion developed between the amount of DNA (which was synthesized at a relatively higher rate) and RNA; this was afterwards equalized by a temporary break in DNA production. Pronounced differences in the given types of biosynthesis were found only in the mesophilic culture, while at suboptimal temperatures the metabolism of the psychrophilic strain slowed down but no marked changes occurred.     

Ontogenetic change in effectiveness of chemical defence against different predators in Oxycarenus true bugs

Many prey species change their antipredator defence during ontogeny, which may be connected to different potential predators over the life cycle of the prey. To test this hypothesis, we compared reactions of two predator taxa – spiders and birds – to larvae and adults of two invasive true bug species, Oxycarenus hyalinipennis and Oxycarenus lavaterae (Heteroptera: Oxycarenidae) with life-stage-specific chemical defence mechanisms. The reactions to larvae and adults of both true bug species strikingly differed between the two predator taxa. The spiders were deterred by the defences of adult bugs, but the larval defences were ineffective against them. By contrast, birds attacked the larvae considerably less often than the adult bugs. The results indicate a predator-specific ontogenetic change in defence effectiveness of both Oxycarenus species. The change in defence is likely linked to the life-stage-specific composition of secretions in both species: whereas secretions of larvae are dominated by unsaturated aldehydes, secretions of adults are rich in terpenoids, which probably serve dual function of defensive chemicals and pheromones. Our results highlight the variation in defence between different life stages and the importance of testing responses of different types of predators.     

Thermal stabilisation of the short DNA duplexes by acridine-4-carboxamide derivatives

The short oligodeoxynucleotide (ODN) probes are suitable for good discrimination of point mutations. However, the probes suffer from low melting temperatures. In this work, the strategy of using acridine-4-carboxamide intercalators to improve thermal stabilisation is investigated. The study of large series of acridines revealed that optimal stabilisation is achieved upon decoration of acridine by secondary carboxamide carrying sterically not demanding basic function bound through a two-carbon linker. Two highly active intercalators were attached to short probes (13 or 18 bases; designed as a part of HFE gene) by click chemistry into positions 7 and/or 13 and proved to increase the melting temperate (T_m) of the duplex by almost 8°C for the best combination. The acridines interact with both single- and double-stranded DNAs with substantially preferred interaction for the latter. The study of interaction suggested higher affinity of the acridines toward the GC- than AT-rich sequences. Good discrimination of two point mutations was shown in practical application with HFE gene (wild type, H63D C > G and S65C A > C mutations). Acridine itself can also serve as a fluorophore and also allows discrimination of the fully matched sequences from those with point mutations in probes labelled only with acridine.