Extrachromosomal transposition of the transposable element Minos in embryos of the cricket Gryllus bimaculatus

Effective germline transformation of insects has been shown to depend on the right choice of transposon system and selection marker. In this study the promoter region of a Gryllus cytoplasmic actin (GbA3/4) gene was isolated and characterized, and was used to drive the expression of Minos transposase in embryos of the cricket Gryllus bimaculatus. Active Minos transposase was produced in these embryos as monitored through established transposon excision and interplasmid transposition assays. In contrast, Drosophila melanogaster hsp70 promoter, previously used to express Minos transposase in a number of insect species and insect cell lines, failed to produce any detectable Minos transposase activity, as recorded by using the very sensitive transposon excision assay. In addition, the GbA3/4 promoter was found to drive expression of enhanced green fluorescent protein (eGFP) predominantly in vitellophages of the developing Gryllus eggs when a plasmid carrying a GbA3/4 promoter-eGFP fusion gene was transiently injected into embryos. These results strongly support the use of Minos transposons marked with the GbA3/4 promoter-eGFP for the genetic transformation of this emerging model insect species.     

Involvement of Wingless/Armadillo signaling in the posterior sequential segmentation in the cricket, Gryllus bimaculatus (Orthoptera), as revealed by RNAi analysis

In insects, there are two different modes of segmentation. In the higher dipteran insects (like Drosophila), their segmentation takes place almost simultaneously in the syncytial blastoderm. By contrast, in the orthopteran insects (like Schistocerca (grasshopper)), the anterior segments form almost simultaneously in the cellular blastoderm and then the remaining posterior part elongates to form segments sequentially from the posterior proliferative zone. Although most of their orthologues of the Drosophila segmentation genes may be involved in their segmentation, little is known about their roles. We have investigated segmentation processes of Gryllus bimaculatus, focusing on its orthologues of the Drosophila segment-polarity genes, G. bimaculatus wingless (Gbwg), armadillo (Gbarm) and hedgehog (Gbhh). Gbhh and Gbwg were observed to be expressed in the each anterior segment and the posterior proliferative zone. In order to know their roles, we used RNA interference (RNAi). We could not observed any significant effects of RNAi for Gbwg and Gbhh on segmentation, probably due to functional replacement by another member of the corresponding gene families. Embryos obtained by RNAi for Gbarm exhibited abnormal anterior segments and lack of the abdomen. Our results suggest that GbWg/GbArm signaling is involved in the posterior sequential segmentation in the G. bimaculatus embryos, while Gbwg, Gbarm and Gbhh are likely to act as the segment-polarity genes in the anterior segmentation similarly as in Drosophila.     

Estimation of receptor-ligand interactions by the use of a two-marker system in affinity capillary electrophoresis

The study of receptor-ligand interactions by affinity capillary electrophoresis (ACE) requires an accurate form of analysis. Here, we examine the use of two noninteracting standards (markers) in the analysis of binding constant data in ACE studies. This concept is demonstrated using two model systems: carbonic anhydrase B (CAB, EC 4.2.1.1) and arylsulfonamides, and vancomycin (Van) from Streptomyces orientalis and the dipeptide N-acetyl-d-Ala-d-Ala. In this procedure a plug of receptor and noninteracting standards is injected, and analysis of the change in the relative migration time ratio of the receptor, relative to the noninteracting standards, as a function of the concentration of the ligand yields a value for the binding constant. The findings described here demonstrate that data from ACE studies can best be analyzed using two noninteracting standards, yielding values comparable to those estimated using other binding and ACE techniques.     

even-skipped has gap-like, pair-rule-like, and segmental functions in the cricket Gryllus bimaculatus, a basal, intermediate germ insect (Orthoptera)

Developmental mechanisms of segmentation appear to be varied among insects in spite of their conserved body plan. Although the expression patterns of the segment polarity genes in all insects examined imply well conserved function of this class of genes, expression patterns and function of the pair-rule genes tend to exhibit diversity. To gain further insights into the evolution of the segmentation process and the role of pair-rule genes, we have examined expression and function of an ortholog of the Drosophila pair-rule gene even-skipped (eve) in a phylogenetically basal insect, Gryllus bimaculatus (Orthoptera, intermediate germ cricket). We find that Gryllus eve (Gb'eve) is expressed as stripes in each of the prospective gnathal, thoracic, and abdominal segments and as a broad domain in the posterior growth zone. Dynamics of stripe formation vary among Gb'eve stripes, representing one of the three modes, the segmental, incomplete pair-rule, and complete pair-rule mode. Furthermore, we find that RNAi suppression of Gb'eve results in segmentation defects in both anterior and posterior regions of the embryo. Mild depletion of Gb'eve shows a pair-rule-like defect in anterior segments, while stronger depletion causes a gap-like defect showing deletion of anterior and posterior segments. These results suggest that Gb'eve acts as a pair-rule gene at least during anterior segmentation and also has segmental and gap-like functions. Additionally, Gb'eve may be involved in the regulation of hunchback and Krüppel expression. Comparisons with eve functions in other species suggest that the Gb'eve function may represent an intermediate state of the evolution of pair-rule patterning by eve in insects.     

The Translation Inhibitor Rocaglamide Targets a Bimolecular Cavity between eIF4A and Polypurine RNA

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Akt mediates self-renewal division of mouse spermatogonial stem cells

Spermatogonial stem cells have unique properties to self-renew and support spermatogenesis throughout their lifespan. Although glial cell line-derived neurotrophic factor (GDNF) has recently been identified as a self-renewal factor for spermatogonial stem cells, the molecular mechanism of spermatogonial stem cell self-renewal remains unclear. In the present study, we assessed the role of the phosphoinositide-3 kinase (PI3K)-Akt pathway using a germline stem (GS) cell culture system that allows in vitro expansion of spermatogonial stem cells. Akt was rapidly phosphorylated when GDNF was added to the GS cell culture, and the addition of a chemical inhibitor of PI3K prevented GS cell self-renewal. Furthermore, conditional activation of the myristoylated form of Akt-Mer (myr-Akt-Mer) by 4-hydroxy-tamoxifen induced logarithmic proliferation of GS cells in the absence of GDNF for at least 5 months. The myr-Akt-Mer GS cells expressed spermatogonial markers and retained androgenetic imprinting patterns. In addition, they supported spermatogenesis and generated offspring following spermatogonial transplantation into the testes of infertile recipient mice, indicating that they are functionally normal. These results demonstrate that activation of the PI3K-Akt pathway plays a central role in the self-renewal division of spermatogonial stem cells.     

CD9 is a surface marker on mouse and rat male germline stem cells

Spermatogenesis is dependent on a small population of stem cells. Despite the biological significance of spermatogonial stem cells, their analysis has been hampered by their scarcity. However, spermatogonial stem cells can be enriched by selection with an antibody against cell-surface molecules. In this investigation, we searched for new antigens expressed on spermatogonial stem cells. Using the spermatogonial transplantation technique, we examined expression of the CD9 molecule, which is commonly expressed on stem cells of other tissues. Selection of both mouse and rat testis cells with anti-CD9 antibody resulted in 5- to 7-fold enrichment of spermatogonial stem cells from intact testis cells, indicating that CD9 is commonly expressed on spermatogonial stem cells of both species. Therefore, CD9 may be involved in the common machinery in stem cells of many self-renewing tissues, and the identification of a common surface antigen on spermatogonial stem cells of different species has important implications for the development of a technique to enrich stem cells from other mammalian species.     

Evolutionary and environmental effects on the geographical adaptation of herbivory resistance in native and introduced <Emphasis Type="Italic">Solidago altissima</Emphasis> populations

To understand rapid evolution in plant resistance to herbivory, it is critical to determine how the genetic correlation among resistances varies genetically and/or environmentally. We conducted a reciprocal transplant experiment of tall goldenrod, Solidago altissima with multiple replicates within the native range (USA) and the introduced range (Japan) to explore the differences in phenotypic traits of resistance to multiple herbivorous insects and their relationships between and within the countries. The Japanese plants were more resistant to the lace bug, Corythucha marmorata, which had recently invaded Japan, but were more susceptible to other herbivorous insects compared to the USA plants. An antagonistic relationship was found between plant resistances to lace bugs and other herbivorous insects in both USA and Japanese plants. In addition, this relationship was more obvious in gardens with a high level of foliage damage than in gardens with a low level of foliage damage by other herbivorous insects. An antagonistic relationship between resistances to aphids and lace bugs was also observed in USA gardens, but not in Japanese garden. These results suggest that the strength of constraints on the evolution of plant resistance due to genetic trade-offs may differ among biotic environments, including community structure of herbivorous insects. Therefore, differences in herbivorous insect communities between the native and introduced ranges can result in the rapid evolution of greater resistance in plants in the introduced range than in the native range.     

Production of knockout mice by gene targeting in multipotent germline stem cells

Spermatogonial stem cells can convert into embryonic stem (ES) cell-like multipotent germline stem (mGS) cells in vitro and produce germline chimeras by blastocyst microinjection. Although homologous recombination was previously demonstrated in mGS cells, spermatogenesis was not found in chimeras, suggesting that they are not competent for germline modification. Here we conducted detailed analysis of chimeric animals to determine whether mGS cells retain germline potential after genetic manipulation. Spermatozoa that were deficient in the occludin gene could be recovered from animals that were chimeric with mGS cells that underwent homologous recombination. The phenotypes of the occludin knockout (KO) mice were similar to those reported for KO mice produced using ES cells, and the animals showed growth retardation, gastritis and male infertility. Furthermore, we found that heterozygous mGS cells acquire two copies of the G418-resistant genes and become homozygous for the targeted allele by culturing at high concentrations of G418. Cytogenetic analysis showed that the aneuploid mGS cells observed during genetic manipulation were trisomic for chromosome 8 or 11, which is a common chromosomal abnormality in ES cells. Thus, mGS cells can be used to produce KO animals, and this novel method of germline manipulation may prove useful in diverse mammalian species.     

High Prevalence of Anemia in Children and Adult Women in an Urban Population in Southern Brazil

This population-based study was designed to detect the prevalence of anemia in a healthy population of children (18 months to 7 years) and women (14 to 30 years) tested in 2006–2007 in the state of Rio Grande do Sul, Brazil as part of an effort to tackle this massive problem that still affects so many people in the XXI century. Anemia was defined according to the WHO. Capillary blood was measured and socioeconomic status was determined according to the Brazilian Association of Market Research Agencies. The median prevalence of anemia in 2198 children was 45.4% and in 1999 women 36.4%. Anemia decreased with age during childhood; although significantly more prevalent in lower classes individuals, it was also high in the upper classes. There are indirect evidences that the lack of iron supplementation and/or iron fortified food may play a role in it. Professionals and society wise measures of education have to be implemented in order to address possible biologic factors involved in childhood psychosocial development in southern Brazil.