Polyamine modulon in yeast-Stimulation of COX4 synthesis by spermidine at the level of translation.

We proposed that a group of genes whose expression is enhanced by polyamines at the level of translation in Escherichia coli and mammalian cells be referred to as a "polyamine modulon". In Saccharomyces cerevisiae, proteins whose synthesis is enhanced by polyamines at the level of translation were searched for using a polyamine-requiring mutant of S. cerevisiae deficient in ornithine decarboxylase (YPH499 Deltaspe1). Addition of spermidine to the medium recovered the spermidine content and enhanced cell growth of the YPH499 Deltaspe1 mutant by 3-5-fold. Under these conditions, synthesis of COX4, one of the subunits of cytochrome C oxidase (complex IV), was enhanced by polyamines about 2.5-fold at the level of translation. Accordingly, the COX4 gene is the first member of a polyamine modulon in yeast. Polyamines enhanced COX4 synthesis through stimulation of the ribosome shunting of the stem-loop structures (hairpin structures) during the scanning of the 5'-untranslated region (5'-UTR) of COX4 mRNA by 40S ribosomal subunit-Met-tRNA(i) complex.

Phylum Verrucomicrobia representatives share a compartmentalized cell plan with members of bacterial phylum Planctomycetes.

BACKGROUND: The phylum Verrucomicrobia is a divergent phylum within domain Bacteria including members of the microbial communities of soil and fresh and marine waters; recently extremely acidophilic members from hot springs have been found to oxidize methane. At least one genus, Prosthecobacter, includes species with genes homologous to those encoding eukaryotic tubulins. A significant superphylum relationship of Verrucomicrobia with members of phylum Planctomycetes possessing a unique compartmentalized cell plan, and members of the phylum Chlamydiae including human pathogens with a complex intracellular life cycle, has been proposed. Based on the postulated superphylum relationship, we hypothesized that members of the two separate phyla Planctomycetes and Verrucomicrobia might share a similar ultrastructure plan differing from classical prokaryote organization. RESULTS: The ultrastructure of cells of four members of phylum Verrucomicrobia - Verrucomicrobium spinosum, Prosthecobacter dejongeii, Chthoniobacter flavus, and strain Ellin514 - was examined using electron microscopy incorporating high-pressure freezing and cryosubstitution. These four members of phylum Verrucomicrobia, representing 3 class-level subdivisions within the phylum, were found to possess a compartmentalized cell plan analogous to that found in phylum Planctomycetes. Like all planctomycetes investigated, they possess a major pirellulosome compartment containing a condensed nucleoid and ribosomes surrounded by an intracytoplasmic membrane (ICM), as well as a ribosome-free paryphoplasm compartment between the ICM and cytoplasmic membrane. CONCLUSION: A unique compartmentalized cell plan so far found among Domain Bacteria only within phylum Planctomycetes, and challenging our concept of prokaryote cell plans, has now been found in a second phylum of the Domain Bacteria, in members of phylum Verrucomicrobia. The planctomycete cell plan thus occurs in at least two distinct phyla of the Bacteria, phyla which have been suggested from other evidence to be related phylogenetically in the proposed PVC (Planctomycetes-Verrucomicrobia-Chlamydiae) superphylum. This planctomycete cell plan is present in at least 3 of 6 subdivisions of Verrucomicrobia, suggesting that the common ancestor of the verrucomicrobial phylum was also compartmentalized and possessed such a plan. The presence of this compartmentalized cell plan in both phylum Planctomycetes and phylum Verrucomicrobia suggest that the last common ancestor of these phyla was also compartmentalized.

Ribosomally synthesized peptides with antimicrobial properties: biosynthesis, structure, function, and applications.

Ribosomally synthesized peptides with antimicrobial properties (antimicrobial peptides-AMPs) are produced by eukaryotes and prokaryotes and represent crucial components of their defense systems against microorganisms. Although they differ in structure, they are nearly all cationic and very often amphiphilic, which reflects the fact that many of them attack their target cells by permeabilizing the cell membrane. They can be roughly categorized into those that have a high content of a certain amino acid, most often proline, those that contain intramolecular disulfide bridges, and those with an amphiphilic region in their molecule if they assume an alpha-helical structure. Most of the known ribosomally synthesized peptides with antimicrobial functions have been identified and studied during the last 20 years. As a result of these studies, new knowledge has been acquired into biology and biochemistry. It has become evident that these peptides may be developed into useful antimicrobial additives and drugs. The use of two-peptide antimicrobial peptides as replacement for clinical antibiotics is promising, though their applications in preservation of foods (safe and effective for use in meat, vegetables, and dairy products), in veterinary medicine, and in dentistry are more immediate. This review focuses on the current status of some of the main types of ribosomally synthesized AMPs produced by eucaryotes and procaryotes and discusses the novel antimicrobial functions, new developments, e.g. heterologous production of bacteriocins by lactic acid bacteria, or construction of multibacteriocinogenic strains, novel applications related to these peptides, and future research paradigms.

Internal ribosome entry site of encephalomyocarditis virus RNA is unable to direct translation in Saccharomyces cerevisiae.

To evaluate the potential of the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) to promote efficient expression of foreign genes in the yeast, S. cerevisiae, we have constructed E. coli-yeast shuttle vectors in which the EMCV 5' non-coding region was fused to the reporter gene, human prothymosin alpha. Efficiency of translation of corresponding RNA transcripts in mammalian cell-free systems was highly dependent on the sequence context and/or position of the initiation codon. No translation of these IRES-dependent mRNAs occurred in S. cerevisiae.

The herpes simplex virus type 1 US11 gene product is a phosphorylated protein found to be non-specifically associated with both ribosomal subunits.

Microsequencing of a cyanogen bromide peptide obtained from a basic phosphoprotein co-sedimenting with purified ribosomes extracted from herpes simplex virus type 1-infected human epidermoid carcinoma 2 cells identified this protein as a product of the true late US11 gene. An antibody was raised against a recombinant fusion protein expressed in Escherichia coli from a plasmid carrying 75% of the US11 coding sequence including the carboxy terminus. This antibody was used to probe Western blots carried out under various conditions of one- and two-dimensional electrophoresis. The electrophoretic behaviour of the immunoreactive proteins offered further proof that they were indeed products of the US11 gene. This US11 protein, which has phosphates on multiple serine residues, is brought into the cell by the virion and found to be present within ribosome fractions early after infection. This association with ribosomes is non-specific and due to probable aggregation or oligomerization of this proline-rich basic protein allowing its co-sedimentation with ribosomes during the different subcellular fractionation steps used for the purification of ribosomal subunits.

Construction of a bifunctional mRNA in the mouse by using the internal ribosomal entry site of the encephalomyocarditis virus.

Picornaviral mRNAs have been shown to possess special structures in their 5' nontranslated regions (5'NTRs) that provide sites for internal binding of ribosomes and thus direct cap-independent translation. The translational cis-acting elements for ribosomal internal entry into the 5'NTR of encephalomyocarditis virus (EMCV), a member of family Picornaviridae, have been named the internal ribosomal entry site (IRES). All of the published experiments regarding the IRES function of the picornavirus 5'NTR, however, were performed with cell extracts in vitro or with tissue culture cells in transient assay systems. In this study, we examined the IRES function of the EMCV 5'NTR in chimeric mouse embryos and demonstrated that this element does in fact work stably in mouse embryos as well as in embryonic stem (ES) cells. By using a dicistronic vector, pWH8, consisting of a promoter-driven neomycin resistance gene (neo) followed by the EMCV 5'NTR-lacZ sequence, we showed that more than half of the ES cells made G418 resistant by the vector stained positive for beta-galactosidase (beta-gal). On Northern (RNA) blots, all of the clones analyzed revealed a transcript of the expected size containing both the beta-gal and the neo cistrons. These results indicate that dicistronic mRNAs are produced from the stably integrated vector in those ES clones and that both of the cistrons are translated to produce functional proteins. The chimeric embryos derived from these ES clones also stained positive for beta-gal, suggesting that the bifunctional mRNAs are active in the embryos. This dicistronic vector system provides a novel tool by which to obtain temporally and spatially coordinated expression of two different genes driven by a single promoter in a single cell in mice.

Characterization of avirulent mutant Legionella pneumophila that survive but do not multiply within human monocytes.

Legionella pneumophila, the causative agent of Legionnaires' disease, is a Gram-negative bacterium and a facultative intracellular parasite that multiplies in human monocytes and alveolar macrophages. In this paper, mutants of L. pneumophila avirulent for human monocytes were obtained and extensively characterized. The mutants were obtained by serial passage of wild-type L. pneumophila on suboptimal artificial medium. None of 44 such mutant clones were capable of multiplying in monocytes or exerting a cytopathic effect on monocyte monolayers. Under the same conditions, wild-type L. pneumophila multiplied 2.5-4.5 logs, and destroyed the monocyte monolayers. The basis for the avirulent phenotype was an inability of the mutants to multiply intracellularly. Both mutant and wild-type bacteria bound to and were ingested by monocytes, and both entered by coiling phagocytosis. Thereafter, their intracellular destinies diverged. The wild-type formed a distinctive ribosome-lined replicative phagosome, inhibited phagosome-lysosome fusion, and multiplied intracellularly. The mutant did not form the distinctive phagosome nor inhibit phagosome-lysosome fusion. The mutant survived intracellularly but did not replicate in the phagolysosome. In all other respects studied, the mutant and wild-type bacteria were similar. They had similar ultrastructure and colony morphology; both formed colonies of compact and diffuse type. They had similar structural and secretory protein profiles and LPS profile by PAGE. Both the mutant and wild-type bacteria were completely resistant to human complement in the presence or absence of high titer anti-L. pneumophila antibody. The mutant L. pneumophila have tremendous potential for enhancing our understanding of the intracellular biology of L. pneumophila and other parasites that follow a similar pathway through the mononuclear phagocyte. Such mutants also show promise for enhancing our understanding of immunity to L. pneumophila, and they may serve as prototypes in the development of safe and effective vaccines against intracellular pathogens.

Isolation and characterization of cytoplasmic inclusions from influenza A virus-infected cells.

Influenza A viruses induce the accumulation of electron-dense inclusions in the cytoplasm of infected cells during the latter stages of the replication cycle. Cell fractionation studies showed that these inclusions could be recovered in subcellular fractions containing ribosomes and polysomes. Isolation of these inclusions was accomplished by procedures involving RNase treatment of these fractions followed by repurification, or by fluorocarbon extraction and gradient centrifugation. Electron microscopy indicated that the isolated inclusions exhibited a major periodicity of approximately 8 nm with minor periodicities of approximately 4 nm. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the influenza virus coded nonstructural protein was the only protein component present in isolated inclusions.

Association of ribosomes with intracellular vesicular stomatitis virus particles.

Ribosomes are observed intimately associated with nucleocapsids of vesicular, stomatitis virus, especially those that line structures that are either cytoplasmic vesicles or invaginations of the plasma membrane.