Highly specific gene silencing by artificial microRNAs in the unicellular alga Chlamydomonas reinhardtii

MicroRNAs (miRNAs) are small RNAs, 21 to 22 nucleotides long, with important regulatory roles. They are processed from longer RNA molecules with imperfectly matched foldback regions and they function in modulating the stability and translation of mRNA. Recently, we and others have demonstrated that the unicellular alga Chlamydomonas reinhardtii , like diverse multicellular organisms, contains miRNAs. These RNAs resemble the miRNAs of land plants in that they direct site-specific cleavage of target mRNA with miRNA-complementary motifs and, presumably, act as regulatory molecules in growth and development. Utilizing these findings we have developed a novel artificial miRNA system based on ligation of DNA oligonucleotides that can be used for specific high-throughput gene silencing in green algae.     

Lifetime selection on adult body size and components of body size in a waterstrider: opposing selection and maintenance of sexual size dimorphism

Abstract.— Sexual size dimorphism (SSD), the difference in body size between males and females, is common in almost all taxa of animals and is generally assumed to be adaptive. Although sexual selection and fecundity selection alone have often been invoked to explain the evolution of SSD, more recent views indicate that the sexes must experience different lifetime selection pressures for SSD to evolve and be maintained. We estimated selection acting on male and female adult body size (total length) and components of body size in the waterstrider Aquarius remigis during three phases of life history. Opposing selection pressures for overall body size occurred in separate episodes of fitness for females in both years and for males in one year. Specific components of body size were often the targets of the selection on overall body size. When net adult fitness was estimated by combining each individual's fitnesses from all episodes, we found stabilizing selection in both sexes. In addition, the net optimum overall body size of males was smaller than that of females. However, even when components of body size had experienced opposing selection pressures in individual episodes, no components appeared to be under lifetime stabilizing selection. This is the first evidence that contemporary selection in a natural population acts to maintain female size larger than male size, the most common pattern of SSD in nature.     

Calcium transients accompany ooplasmic segregation in zebrafish embryos

Through the injection of f -aequorin (a calcium-specific luminescent reporter), and the use of an imaging photon detector, transient localized elevations of free cytosolic calcium in the forming blastodisc (BD) and animal hemisphere cortex were visualized that correlated with ooplasmic segregation. The introduction of an appropriate concentration of the weak (K_D= 1.5 μmol/L) calcium buffer 5,5'-dibromo-BAPTA results in the dissipation of these calcium domains, and inhibits cytoplasmic streaming and the subsequent formation of a BD at the animal pole. These inhibitory actions are dependent on the final cytosolic concentration of buffer within the egg: ≥ 1.3 mmol/L blocks ooplasmic streaming; < 1.3 mmol/L eggs segregate normally. Injection of 5,5'-dimethyl-BAPTA (K_D= 0.15 μmol/L) to a final concentration of 1.5 mmol/L as a control has no effect on ooplasmic streaming. These results suggest that localized domains of elevated free cytosolic calcium are essential for ooplasmic segregation in zebrafish. Furthermore, a hypothetical model is presented linking these calcium transients to the contraction of a cortically located actin microfilament network as a possible mechanism providing the driving force for segregation.     

BipA: a tyrosine-phosphorylated GTPase that mediates interactions between enteropathogenic Escherichia coli (EPEC) and epithelial cells

We report the functional characterization of BipA, a GTPase that undergoes tyrosine phosphorylation in an enteropathogenic Escherichia coli (EPEC) strain. BipA⁻ mutants adhere to cultured epithelial cells but fail to trigger the characteristic cytoskeletal rearrangements found in cells infected with wild-type EPEC. In contrast, increased expression of BipA enhances actin remodelling and results in the hyperformation of pseudopods. BipA appears to be the first example of a new class of virulence regulator, as it also controls flagella-mediated cell motility and resistance to the antibacterial effects of a human host defence protein. Its striking sequence similarity to ribosome-binding elongation factors suggests that it uses a novel mechanism to modulate gene expression.     

Critical parameters influencing hyperthermia-induced apoptosis in human lymphoid cell lines

Brief mild hyperthermia is sufficient to induce apoptosis (programmed cell death) in many cell lines. Here we describe the effects of a number of factors modulating heat shock induced apoptosis outcomes. We report the effects of cell type, heat load, recovery times, cellular growth phase, and protein synthesis on the levels of apoptoses seen in heat stressed cell populations. We observe that a number of cell lines are competent to undergo heat stress induced apoptosis using both the comet assay and cellular and nuclear morphologies. Of the cell lines tested we saw a wide spectrum of sensitivities, ranging from resistant (less than 1% apoptotic after 12 h) to exquisitely sensitive (>95%). By incrementally increasing the heat load from 37–49°C, we observed a gradual increase in apoptosis with a significant change from apoptotic to necrotic death at temperatures beyond 45°C. The kinetics of the apoptotic response to heat shock were also examined. A time dependent increase in apoptotic cell death was seen after initial hyperthermic treatment with most cell types reaching a plateau at 18 h. In addition to these parameters we report that growth phase has a strong influence on the number of apoptoses induced as a result of heat stress. Cultured cells, grown to a plateau, undergo apoptosis at a much higher level than similarly treated cells taken during an exponential phase of growth. Finally, we determined the necessity of protein synthesis for apoptotic competency.     

The P140K mutant of human O(6)-methylguanine-DNA-methyltransferase (MGMT) confers resistance in vitro and in vivo to temozolomide in combination with the novel MGMT inactivator O(6)-(4-bromothenyl)guanine

The O(6)-methylguanine-DNA-methyltransferase (MGMT) inactivator O(6)-benzylguanine (O(6)-beG) is currently under clinical investigation as a potential tumour-sensitising agent. In clinical trials its use has been associated with increased myelotoxicity and a reduced maximum tolerated dose (MTD) for BCNU. Thus the concept of myeloprotection by gene therapy with an O(6)-beG-insensitive mutant of MGMT is soon to be tested. Recently, an alternative inactivator has been described (O(6)-(4-bromothenyl)guanine, PaTrin-2), which shows potential advantages over O(6)-beG in terms of higher activity against wild-type MGMT and oral formulation. The use of PaTrin-2 has also been associated with increased myelotoxicity in clinical trials and thus PaTrin-2 may also be a candidate for use in conjunction with mutant MGMT gene transfer in genetic chemoprotective strategies. However, its activity against mutant MGMTs has not been reported. We show here that the P(140)K mutant of MGMT is highly resistant to inactivation by PaTrin-2. Furthermore, we show that a human haemopoietic cell line (K562) transduced with a retroviral vector encoding MGMT(P140K) is highly resistant to the cytotoxic effects of PaTrin-2 in combination with the methylating agent temozolomide, and that cells expressing MGMT(P140K) can be effectively enriched in vitro following challenge with this drug combination. Finally, we show that animals reconstituted with bone marrow expressing MGMT(P140K) exhibit haemopoietic resistance to PaTrin-2/temozolomide, which results in in vivo selection of gene-modified cells. All of these effects were comparable to those also achieved using O(6)-beG/temozolomide. Thus our data show that MGMT(P140K) is a suitable candidate for chemoprotective gene therapy where PaTrin-2 is being used in conjunction with temozolomide.     

Video enhanced laser scanning microscopic analysis of DNA comet formation.

Laser Scanning Microscopy is a sensitive tool that provides a unique method of analyzing biological systems. Coupled with the Single Cell Gel assay, it allows for accurate and reproducible detection of DNA strand breaks. An understanding of the theory of DNA comet formation is lacking. Using dexamethasone induced apoptosis in murine thymocytes as a model for double strand breaks, we used video enhanced laser scanning microscopy to evaluate the leading edge of DNA migration in the single cell gel assay. In this system, comet length increases significantly within the first thirty seconds of electrophoresis, the greatest increase in length is completed within the first minute, and the first two minutes are important in significant increases in DNA migration during DNA comet formation.