The role of SmpB and the ribosomal decoding center in licensing tmRNA entry into stalled ribosomes

In bacteria, stalled ribosomes are recycled by a hybrid transfer-messenger RNA (tmRNA). Like tRNA, tmRNA is aminoacylated with alanine and is delivered to the ribosome by EF-Tu, where it reacts with the growing polypeptide chain. tmRNA entry into stalled ribosomes poses a challenge to our understanding of ribosome function because it occurs in the absence of a codon-anticodon interaction. Instead, tmRNA entry is licensed by the binding of its protein partner, SmpB, to the ribosomal decoding center. We analyzed a series of SmpB mutants and found that its C-terminal tail is essential for tmRNA accommodation but not for EF-Tu activation. We obtained evidence that the tail likely functions as a helix on the ribosome to promote accommodation and identified key residues in the tail essential for this step. In addition, our mutational analysis points to a role for the conserved K(131)GKK tail residues in trans-translation after peptidyl transfer to tmRNA, presumably EF-G-mediated translocation or translation of the tmRNA template. Surprisingly, analysis of A1492, A1493, and G530 mutants reveals that while these ribosomal nucleotides are essential for normal tRNA selection, they play little to no role in peptidyl transfer to tmRNA. These studies clarify how SmpB interacts with the ribosomal decoding center to license tmRNA entry into stalled ribosomes.     

Evolutionary Relationships among Actinophages and a Putative Adaptation for Growth in Streptomyces spp.

The genome sequences of eight Streptomyces phages are presented, four of which were isolated for this study. Phages R4, TG1, ϕHau3, and SV1 were isolated previously and have been exploited as tools for understanding and genetically manipulating Streptomyces spp. We also extracted five apparently intact prophages from recent Streptomyces spp. genome projects and, together with six phage genomes in the database, we analyzed all 19 Streptomyces phage genomes with a view to understanding their relationships to each other and to other actinophages, particularly the mycobacteriophages. Fifteen of the Streptomyces phages group into four clusters of related genomes. Although the R4-like phages do not share nucleotide sequence similarity with other phages, they clearly have common ancestry with cluster A mycobacteriophages, sharing many protein homologues, common gene syntenies, and similar repressor-stoperator regulatory systems. The R4-like phage ϕHau3 and the prophage StrepC.1 (from Streptomyces sp. strain C) appear to have hijacked a unique adaptation of the streptomycetes, i.e., use of the rare UUA codon, to control translation of the essential phage protein, the terminase. The Streptomyces venezuelae generalized transducing phage SV1 was used to predict the presence of other generalized transducing phages for different Streptomyces species.     

Effects of Route of Inoculation and Viral Genetic Variation on Antibody Responses to Polyomavirus SV40 in Syrian Golden Hamsters

Genetic variants of polyomavirus SV40 are powerful agents with which to define viral effects on cells and carcinogenesis pathways. We hypothesized that differences in biologic variation among viral strains affect the process of viral infection and are reflected in antibody responses to the viral nonstructural large T-antigen (TAg) protein but not in neutralizing antibody responses against the inoculated viral particles. We analyzed the production of TAg antibody and neutralizing antibody in Syrian golden hamsters that were inoculated with SV40 viral strains by intracardiac, intravenous, or intraperitoneal routes and remained tumor free. Compared with the intraperitoneal route, intravascular (that is, intravenous, intracardiac) inoculation resulted in increased frequency of responsiveness to TAg but not in higher TAg antibody titers. The intravascular route was superior both for eliciting neutralizing antibody responses and for higher titers of those responses. Viruses with complex regulatory regions induced TAg antibody more often than did viruses with simple regulatory regions after intraperitoneal but not intravascular injections, with no differences in antibody titers. This viral genetic variation had no effect on neutralizing antibody production after intraperitoneal or intravascular inoculations or on neutralizing antibody titers achieved. These findings confirm that SV40 variants differ in their biologic properties. Route of inoculation combined with viral genetic variation significantly influence the development of serum antibodies to SV40 TAg in tumor-free hamsters. Route of inoculation—but not viral genetic variation—is an important factor in production of neutralizing antibody to SV40.Abbreviations: TAg, large T antigenPolyomavirus SV40 was discovered as an unrecognized contaminant of early poliovaccines³⁸ and was shown promptly to be an oncogenic virus.^9,15,17,18 Syrian golden hamsters are the small animal model that is susceptible to SV40 tumorigenicity.^{7,9-13,18,27,35} Since its discovery, SV40 has proven to be an outstanding tool for the discovery of mechanisms underlying carcinogenesis and for viral influences on cellular processes.^{1,3,5,19,23,30}Genetic variants of SV40 exist.^{16,20,26,28,33,34,36,37,41} This variation typically occurs in the viral regulatory region, in which some strains have duplications or rearrangements (or both) in the enhancer region, and in the C-terminal region of the large T-antigen (TAg) gene, in which nucleic acid variations may result in amino acid changes in the protein. TAg is an essential viral replication protein and the major viral oncoprotein. An important question is whether SV40 viral variants differ in their biologic properties, including in host responses to infections, as this could affect the spectrum of viral disease pathogenesis. We previously have shown that the viral regulatory region influences SV40 tumor induction in hamsters^35,40 and vertical transmission of the virus in hamsters²⁹ and that the route of inoculation influences SV40-mediated carcinogenesis.²⁷ Because TAg is not a component of the virus particle but instead is synthesized in virus-infected cells, we hypothesized that differences in antibody responses to TAg reflect biologic variation among viral strains with respect to the process of viral infection. In contrast, we expected that neutralizing antibody responses arise primarily against the injected viral particles, which represent a single serotype, and therefore are less informative about viral variation. We report here that an analysis revealed that the route of inoculation—in combination with viral genetic variation—significantly influences the development of serum antibodies to SV40 large TAg but not the titer of those antibodies in virus-exposed, tumor-free hamsters. The route of inoculation—but not viral genetic variation—influenced both the frequency of development and the titers of virus-neutralizing antibodies in the same animals.     

The genome of <Emphasis Type="Italic">Streptomyces rimosus</Emphasis> subsp. <Emphasis Type="Italic">rimosus</Emphasis> shows a novel structure compared to other <Emphasis Type="Italic">Streptomyces</Emphasis> using DNA/DNA microarray analysis

DNA/DNA genome microarray analysis together with genome sequencing suggests that the genome of members of the genus Streptomyces would seem to have a common structure including a linear genomic structure, a core of common syntenous Actinomycete genes, the presence of species specific terminal regions and two intermediate group of syntenous genes that seem to be genus specific. We analyzed Streptomyces species using DNA/DNA microarray comparative genome analysis. Only Streptomyces rimosus failed to give a congruent genome pattern for the genes found in Streptomyces coelicolor. We expanded the analysis to include a number of strains related to the type strain of S. rimosus and obtained a similar divergence from the main body of Streptomyces species. These strains showed very close identity to the original strain with no gene deletion or duplication detected. The 16S rRNA sequences of these S. rimosus strains were confirmed as very similar to the S. rimosus sequences available from the Ribosomal Database Project. When the SSU ribosomal RNA phylogeny of S. rimosus is analyzed, the species is positioned at the edge of the Streptomyces clade. We conclude that S. rimosus represents a distinct evolutionary lineage making the species a worthy possibility for genome sequencing.     

Pyrethroid resistance in Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae) and implications for its management in Australia

Abstract  The study was conducted to characterise the underlying resistance mechanisms responsible for high levels of pyrethroid resistance in Frankliniella occidentalis (Pergande) in Australia. Seven commercially available pyrethroids (acrinathrin, alpha-cypermethrin, bifenthrin, deltamethrin, esfenvalerate, permethrin and tau-fluvalinate) were evaluated against seven F. occidentalis strains collected from ornamentals, fruit and vegetables in three states of Australia. A Potter spray tower was used to test for pyrethroid resistance and all field strains were found to be resistant, with resistance ratios ranging from 15-fold deltamethrin to 1300-fold tau-fluvalinate. The two most resistant strains were further tested for detoxification enzymes that could be involved in resistance. Three synergists, piperonyl butoxide (PBO), diethyl maleate (DEM) and profenofos, which, respectively, inhibit the enzymes cytochrome P-450 monooxygenases, glutathione S -transferases and esterases, were used. The synergism data indicate that multiple mechanisms may be involved in pyrethroid resistance in Australian populations of F. occidentalis . Among the three synergists, PBO considerably reduced pyrethroid resistance in the selected strains compared with DEM and profenofos. The practical implication for PBO use to suppress pyrethroid resistance in F. occidentalis is elaborated.     

A modified technique for semen collection by electroejaculation in the domestic cat

A method was developed for collecting feline semen by electroejaculation combined with the use of a urethral catheter. The catheter facilitated handling the small volumes of semen for laboratory analysis. In 14 cats, semen volumes ranged from 0.019 to 0.284 ml (mean 0.076) and spermatozoa counts of ejaculates collected in the catheter ranged from 0.32 to 49.60 x 10(6) (mean 11.64 x 10(6)). Nine individuals were evaluated for retrograde ejaculation by quantitation of spermatozoa in pre-ejaculation and post-ejaculation urine samples. No spermatozoa were detected in pre-ejaculation samples but post-ejaculation urine samples contained from 0 to 11.88 x 10(6) (mean 4.55 x 10(6)) spermatozoa. The antegrade portion of the ejaculate contained from 6.3 to 100% (mean 59.1%) of the total number of spermatozoa ejaculated.     

Initial verification of the resistance management strategy for Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae) in Australia

Shortly after the initial detection of western flower thrips (WFT), Frankiniella occidentalis (Pergande), in Australia during 1993 a resistance management strategy based on the alternation of chemical groups was implemented. This study aimed to verify this strategy by field testing α-cypermethrin against WFT with and without chemical alternation. Up to 114 times α-cypermethrin resistance (at LC50) was detected and resistance increased with and without chemical alternation; however, chemical alternation did significantly reduce numbers of thrips compared with a nonalternation strategy. Resistance has the potential to undermine the sustainable use of those chemicals to which there is no current detectable resistance. Consequently, chemicals with a high frequency and level of resistance against WFT need to be identified through monitoring and quickly eliminated from WFT chemical control recommendations.     

Stereotaxic Device for Optical Imaging of Mice Hind Feet

Imaging of in vivo model systems, especially mouse models, has revolutionized our understanding of normal and pathological developments. However, mice present several challenges for imaging. They are living and therefore breathing organisms with a fast heart rate (>500 beat/min), which necessitates the need for restraints and positioning controls that do not compromise their normal physiology. We present here a device that immobilizes the rear legs of a mouse while retaining the ability to position both the hind feet and legs for reproducible imaging deep below the skin''s surface. The device is highly adjustable to accommodate mice, 5 weeks of age and older. The function of this device is demonstrated by imaging the vasculature ∼250 μm beneath the skin in the hind leg. Whereas the overall dimensions are for a motorized stage (Märzhäuser Wetzlar GmbH, Wetzlar, Germany), minor modifications would allow it to be customized for use with most commercially available stages that accept an insert.