The gene transformer-2 of Anastrepha fruit flies (Diptera, Tephritidae) and its evolution in insects

Background: In the tephritids Ceratitis, Bactrocera and Anastrepha, the gene transformer provides the memory device for sex determination via its auto-regulation; only in females is functional Tra protein produced. To date, the isolation and characterisation of the gene transformer-2 in the tephritids has only been undertaken in Ceratitis, and it has been shown that its function is required for the female-specific splicing of doublesex and transformer pre-mRNA. It therefore participates in transformer auto-regulatory function. In this work, the characterisation of this gene in eleven tephritid species belonging to the less extensively analysed genus Anastrepha was undertaken in order to throw light on the evolution of transformer-2. Results: The gene transformer-2 produces a protein of 249 amino acids in both sexes, which shows the features of the SR protein family. No significant partially spliced mRNA isoform specific to the male germ line was detected, unlike in Drosophila. It is transcribed in both sexes during development and in adult life, in both the soma and germ line. The injection of Anastrepha transformer-2 dsRNA into Anastrepha embryos caused a change in the splicing pattern of the endogenous transformer and doublesex pre-mRNA of XX females from the female to the male mode. Consequently, these XX females were transformed into pseudomales. The comparison of the eleven Anastrepha Transformer-2 proteins among themselves, and with the Transformer-2 proteins of other insects, suggests the existence of negative selection acting at the protein level to maintain Transformer-2 structural features. Conclusions: These results indicate that transformer-2 is required for sex determination in Anastrepha through its participation in the female-specific splicing of transformer and doublesex pre-mRNAs. It is therefore needed for the auto-regulation of the gene transformer. Thus, the transformer/transfomer-2 > doublesex elements at the bottom of the cascade, and their relationships, probably represent the ancestral state ( which still exists in the Tephritidae, Calliphoridae and Muscidae lineages) of the extant cascade found in the Drosophilidae lineage ( in which tra is just another component of the sex determination gene cascade regulated by Sex-lethal). In the phylogenetic lineage that gave rise to the drosophilids, evolution co-opted for Sex-lethal, modified it, and converted it into the key gene controlling sex determination.

Mycovirus in Pseudocercospora griseola, the causal agent of angular leaf spot in common bean

Pseudocercospora griseola (Sacc.) Crous &. Braun is a widespread fungal phytopathogen that is responsible for angular leaf spot in the common bean (Phaseolus vulgaris L.). A number of fungal phytopathogens have been shown to harbour mycoviruses, and this possibility was investigated in populations of Pseudocercospora griseola. The total nucleic acid extracts of 61 fungal isolates were subjected to agarose gel electrophoresis. Small fragments (800-4800 bp) could be identified in 42 of the samples. The presence of dsRNA in isolate Ig838 was confirmed by treatment of total nucleic acid with DNase, RNase A, and nuclease S I. Transmission electron microscopy revealed the presence of viral-like particles 40 nm in diameter in the mycelia of 2 fungal isolates, namely 29-3 and Ig838. The transmission of dsRNA by means of conidia was 100% for isolate 29-3, but there was loss of 1-6 fragments of dsRNA in monosporic colonies of isolate Ig848. Cycloheximide treatment failed to inhibit the mycovirus in isolate 29-3, but proved efficient in the elimination of the 2.2, 2.0, 1.8, 1.2 and 1.0 kb fragments in 2 colonies of isolate Ig848. The occurrence of a mycovirus in Pseudocercospora griseola was demonstrated for the first time in the present study.

A non-invasive method for silencing gene transcription in honeybees maintained under natural conditions

in the Apis mellifera post-genomic era, RNAi protocols have been used in functional approaches. However, sample manipulation and invasive methods such as injection of double-stranded RNA (dsRNA) can compromise physiology and survival. To circumvent these problems, we developed a non-invasive method for honeybee gene knockdown, using a well-established vitellogenin RNAi system as a model. Second instar larvae received dsRNA for vitellogenin (dsVg-RNA) in their natural diet. For exogenous control, larvae received dsRNA for GFP (dsGFP-RNA). Untreated larvae formed another control group. Around 60% of the treated larvae naturally developed until adult emergence when 0.5 mu g of dsVg-RNA or dsGFP-RNA was offered while no larvae that received 3.0 mu g of dsRNA reached pupal stages. Diet dilution did not affect the removal rates. Viability depends not only on the delivered doses but also on the internal conditions of colonies. The weight of treated and untreated groups showed no statistical differences. This showed that RNAi ingestion did not elicit drastic collateral effects. Approximately 90% of vitellogenin transcripts from 7-day-old workers were silenced compared to controls. A large number of samples are handled in a relatively short time and smaller quantities of RNAi molecules are used compared to invasive methods. These advantages culminate in a versatile and a cost-effective approach. (c) 2008 Elsevier Ltd. All rights reserved.

Human endogenous RNAs: Implications for the immunomodulation of Toll-like receptor 3

Toll-like receptors (TLRs), a family of mammalian receptors, are able to recognize nucleic acids. TLR3 recognizes double-stranded (ds)RNA, a product of the replication of certain viruses. Polyinosinic-polycytidylic acid, referred to as poly(I:C), an analog of viral dsRNA, interacts with TLR3 thereby eliciting immunoinflammatory responses characteristic of viral infection or down-regulating the expression of chemokine receptor CXCR4. It is known that dsRNA also directly activates interferon (IFN)-induced enzymes, such as the RNA-dependent protein kinase (PKR). In the present study, the mRNA expression of TLR3, CXCR4, IFN gamma and PKR was investigated in a culture of peripheral blood mononuclear cells (PBMCs) stimulated with poly(I:C) and endogenous RNA from human PBMCs. No cytotoxic effect on the cells or on the proliferation of CD3(+), CD4(+) and CD8(+) cells was observed. TLR3 expression in the PBMCs in the presence of poly(I:C) was up-regulated 9.5-fold, and TLR3 expression in the PBMCs treated with endogenous RNA was down-regulated 1.8-fold (p=0.002). The same trend was observed for IFN gamma where in the presence of poly(I:C) an 8.7-fold increase was noted and in the presence of endogenous RNA a 3.1-fold decrease was observed. In the culture activated with poly(1:C), mRNA expression of CXCR4 increased 8.0-fold and expression of PKR increased 33.0-fold. Expression of these genes decreased in the culture treated with endogenous RNA when compared to the culture without stimulus. Thus, high expression of mRNA for TLR3, IFN gamma, CXCR4 and PKR was observed in the presence of poly(I:C) and low expression was observed in the cells cultured with endogenous RNA. In conclusion, TLR3 may play major physiological roles that are not in the context of viral infection. It is possible that RNA released from cells could contain enough double-stranded structures to regulate cell activation. The involvement of endogenous RNA in endogenous gene expression and its implications in the regulation thereof, are still being studied, and will have significant implications in the future.