An eight-nucleotide sequence in the potato virus X 3' untranslated region is required for both host protein binding and viral multiplication.

Gel retardation and UV-cross-linking techniques were used to demonstrate that two tobacco proteins, with approximate molecular masses of 28 and 32 kDa, bind to a site within the 3' region of potato virus X (PVX) genomic RNA. The protein binding is specific, in that a 50-fold excess of unlabeled probe prevents formation of the complexes but no reduction is observed with a 2,000-fold molar excess of yeast tRNA. Complex formation is inhibited by poly(U) but is relatively unaffected by poly(A), poly(G), or poly(C-I). PVX RNA-host protein complex formation occurs in vitro at salt concentrations up to 400 mM. Deletion mapping indicates that the proteins bind within the 3' untranslated region (UTR) of PVX genomic RNA and that an 8-nucleotide U-rich sequence (5'-UAUUUUCU) is required for the binding. Deletion of the 8-nucleotide U-rich region from the 3' UTR of a sensitive PVX reporter virus that carries the luciferase gene in place of the PVX coat protein gene results in a more than 70,000-fold reduction in luciferase expression in tobacco protoplasts. RNA probes carrying the sequence GCGC in place of the central four contiguous uridines of the 8-nucleotide U-rich motif fail to bind host protein at detectable levels, and the same mutation, when introduced into the PVX reporter virus, eliminates viral multiplication. Mutations of 1 or 2 nucleotides within the same four uridines reduced both binding of host proteins and replication of reporter virus. These results indicate that the 8-nucleotide U-rich motif within the PVX 3' UTR is important for some aspect of viral multiplication and suggest that host protein binding plays a role in the process.     

A novel method for the evaluation of proximal tubule epithelial cellular necrosis in the intact rat kidney using ethidium homodimer

Background  

Ethidium homodimer is a cell-membrane impermeant nuclear fluorochrome that has been widely used to identify necrotic cells in culture. Here, we describe a novel technique for evaluating necrosis of epithelial cells in the proximal tubule that involves perfusing ethidium homodimer through the intact rat kidney. As a positive control for inducing necrosis, rats were treated with 3.5, 1.75, 0.87 and 0.43 mg/kg mercuric chloride (Hg²⁺, intraperitoneal), treatments which have previously been shown to rapidly cause dose-dependent necrosis of the proximal tubule. Twenty-four h after the administration of Hg²⁺, ethidium homodimer (5 μM) was perfused through the intact left kidney while the animal was anesthetized. The kidney was then removed, placed in embedding medium, frozen and cryosectioned at a thickness of 5 μm. Sections were permeabilized with -20°C methanol and then stained with 4',6-diamidino-2-phenylindole (DAPI) to label total nuclei. Total cell number was determined from the DAPI staining in random microscopic fields and the number of necrotic cells in the same field was determined by ethidium homodimer labeling.     

Legionella pneumophila in Cooling Towers: Fluctuations in Counts, Determination of Genetic Variability by Pulsed-Field Gel Electrophoresis (PFGE), and Persistence of PFGE Patterns

The concentrations of Legionella pneumophila in cooling towers may vary considerably over short periods of time, producing significant fluctuations throughout the year. Despite genetic variability, in small geographical areas the same indistinguishable pulsed-field gel electrophoresis patterns may be shared among different cooling towers and persist over time.     

HPLC photofingerprinting of conformational peculiarities and transitions in oligonucleotide duplexes.

Two self-complementary sequence-isomeric decadeoxyribonucleotides were exposed to UV light under conditions in which they assume duplex structures. After that they were analyzed in the denatured state by reversed-phase high-performance liquid chromatography (HPLC). Characterization of the separated photoproducts allowed localization of cyclobutane pyrimidine dimers in the sequences of the modified oligonucleotides. For [d(GGAAATTTCC)]2, which is known to contain in its central part a stretch of rigid B'-conformation with decreased mobility of constituent bases, lower yields of thymine dimers, as compared with that for ordinary B-form [d(CCTTTAAAGG)]2, were found. On the contrary, mixed thymine-cytosine heterodimers generated in the former oligonucleotide demonstrate the increase in photoreactivity of these residues at the B'-B junction. This is probably due to the peculiar conformation adopted by this decanucleotide. Stimulation of B'-B transition, by increasing the temperature before melting, reduced an inhibition of thymine photodimer formation. During the melting of both oligonucleotides yields of all identified photoinduced cyclobutadipyrimidines were reduced. Possible influences of some metal cations on the stability of the B'-form were also studied by this photoprobing technique. The present study demonstrates the feasibility of HPLC photofingerprinting as a new approach for structural analysis of nucleic acids.     

The costs of egg production and incubation in great tits (Parus major).

The costs of egg production and incubation may have a crucial effect on avian reproductive decisions, such as clutch size and the timing of reproduction. We carried out a brood-size enlargement experiment on the great tit (Parus major), in which the birds had to lay and incubate extra eggs (full costs), only incubate extra eggs (free eggs) or did not pay any extra cost (free chicks) in obtaining a larger brood. We used female fitness (half the recruits produced plus female survival) as a fitness measure because it is the female which pays the costs of egg production and incubation, and because clutch size is under female control. Female fitness decreased with increasing costs (fitness of free chicks females is higher than that of free eggs females which is higher than that of full costs females). These fitness differences were due to differences in female survival rather than in the number of recruits produced. This is the first time that the costs of egg production and incubation have been estimated using such a complete fitness measure, including, as our measure does, the local survival to the following year of both the female and her offspring. Our results emphasize that reproductive decisions cannot be understood without taking egg production and incubation costs into account.     

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.