shRNA-mediated RNA interference as a tool for genetic synthetic lethality screening in mouse embryo fibroblasts

Previously, we demonstrated the establishment of synthetic lethality screening in cultured somatic human cells, or mouse embryo fibroblasts (MEFs), for chemicals or mutant genes synergistically lethal with a mutated gene of interest. Here, we show in MEFs that the usage of RNA interference-based genetic suppressor elements encoding short hairpin RNAs (shRNAs) enables for genetic synthetic lethality screening at a frequency much higher than that achieved before with short truncated sense and antisense RNAs. These findings open up the possibility of using in mammalian cells genome-wide shRNA libraries for genetic synthetic lethality screening at the multi-gene level.     

Effects of contact-induced membrane stiffening on platelet adhesion

The adhesion of platelets plays an essential role in thrombogenesis. Adhesion occurs at sites called focal adhesions (FA), where cell-membrane receptors bind specifically to substrate proteins and couple to each other and to the cytoskeleton via various cellular proteins. The resulting molecular structure suggests that the cortex stiffens at the FA, which likely affects platelet adhesion. This hypothesis is explored by structural analysis and parametric investigation. The cortex is modeled as a shell anchored to the substrate by adhesion forces and subjected to a detachment force. Equilibrium considerations result in a non-linear, two-point boundary value problem that is solved numerically. The results show that cortex stiffening significantly influences the force required for detachment as well as the cell-membrane internal stresses. The magnitude of these effects depends on the ratio of adhesion-to-bending energies and on the inclination of the detachment force. Because the cortex stiffening depends on cellular events, these results suggest a possible mechanism by which platelets can control their adhesion and protect themselves from damage.     

Perpendicular planes of FtsZ arcs in spheroidal Escherichia coli cells

Division planes in Escherichia coli, usually restricted to one dimension of the rod-shaped cell, were induced at all possible planes by transforming the cells to spheroids with mecillinam (inactivating PbpA). Such cells displayed many nucleoids and arcs of FtsZ, genetically tagged to green fluorescent protein, that developed to rings at constriction sites all around their surface. These observations are consistent with the view (Woldringh et al., J. Bacteriol. 176 (1994) 6030-6038) that nucleoids, forced during replication to segregate in the length axis of the cell by the rigid bacillary envelope, induce assembly of FtsZ to division rings in between them.     

A Single Disulfide Bond Disruption in the β3 Integrin Subunit Promotes Thiol/Disulfide Exchange,a Molecular Dynamics Study

The integrins are a family of membrane receptors that attach a cell to its surrounding and play a crucial function in cell signaling. The combination of internal and external stimuli alters a folded non-active state of these proteins to an extended active configuration. The β3 subunit of the platelet αIIbβ3 integrin is made of well-structured domains rich in disulfide bonds. During the activation process some of the disulfides are re-shuffled by a mechanism requiring partial reduction of some of these bonds; any disruption in this mechanism can lead to inherent blood clotting diseases. In the present study we employed Molecular Dynamics simulations for tracing the sequence of structural fluctuations initiated by a single cysteine mutation in the β3 subunit of the receptor. These simulations showed that in-silico protein mutants exhibit major conformational deformations leading to possible disulfide exchange reactions. We suggest that any mutation that prevents Cys560 from reacting with one of the Cys⁵⁶⁷–Cys⁵⁸¹ bonded pair, thus disrupting its ability to participate in a disulfide exchange reaction, will damage the activation mechanism of the integrin. This suggestion is in full agreement with previously published experiments. Furthermore, we suggest that rearrangement of disulfide bonds could be a part of a natural cascade of thiol/disulfide exchange reactions in the αIIbβ3 integrin, which are essential for the native activation process.     

Tandem repeats in a new toxin gene from Bacillus thuringiensis and in other cry11-like genes

A new gene, cry11Bb2 from a field isolate of Bacillus thuringiensis, was cloned for expression in Escherichia coli. The encoded protein, with a deduced molecular mass of 89.5 kDa, exhibits 97 and 79% identities with the overlap regions of Cry11Bb1 from B. thuringiensis ssp. medellin and Cry11Ba1 from ssp. jegathesan, respectively. It is however longer than Cry11Bb1 by 42 amino acids in its carboxy-terminus, of which 32 comprise 2 tandem repeats additional to the 5 existing in the latter polypeptide. Possible roles for this recurrent motif among Cry toxins and their accessory proteins, and for their encoding genes are proposed.     

Genetic and chemical characterization of an EMS induced mutation in Cucumis melo CRTISO gene

In order to broaden the available genetic variation of melon, we developed an ethyl methanesulfonate mutation library in an orange-flesh ‘Charentais’ type melon line that accumulates β-carotene. One mutagenized M₂ family segregated for a novel recessive trait, a yellow–orange fruit flesh (‘yofI’). HPLC analysis revealed that ‘yofI’ accumulates pro-lycopene (tetra-cis-lycopene) as its major fruit pigment. The altered carotenoid composition of ‘yofI’ is associated with a significant change of the fruit aroma since cleavage of β-carotene yields different apocarotenoids than the cleavage of pro-lycopene. Normally, pro-lycopene is further isomerized by CRTISO (carotenoid isomerase) to yield all-trans-lycopene, which is further cyclized to β-carotene in melon fruit. Cloning and sequencing of ‘yofI’ CRTISO identified two mRNA sequences which lead to truncated forms of CRTISO. Sequencing of the genomic CRTISO identified an A–T transversion in ‘yofI’ which leads to a premature STOP codon. The early carotenoid pathway genes were up regulated in yofI fruit causing accumulation of other intermediates such as phytoene and ζ-carotene. Total carotenoid levels are only slightly increased in the mutant. Mutants accumulating pro-lycopene have been reported in both tomato and watermelon fruits, however, this is the first report of a non-lycopene accumulating fruit showing this phenomenon.     

Strigolactones interact with ethylene and auxin in regulating root-hair elongation in Arabidopsis

Strigolactones (SLs) or derivatives thereof have been identified as phytohormones, and shown to act as long-distance shoot-branching inhibitors. In Arabidopsis roots, SLs have been suggested to have a positive effect on root-hair (RH) elongation, mediated via the MAX2 F-box. Two other phytohormones, auxin and ethylene, have been shown to have positive effects on RH elongation. Hence, in the present work, Arabidopsis RH elongation was used as a bioassay to determine epistatic relations between SLs, auxin, and ethylene. Analysis of the effect of hormonal treatments on RH elongation in the wild type and hormone-signalling mutants suggested that SLs and ethylene regulate RH elongation via a common regulatory pathway, in which ethylene is epistatic to SLs, whereas the effect of SLs on RH elongation requires ethylene synthesis. SL signalling was not needed for the auxin response, whereas auxin signalling was not necessary, but enhanced RH response to SLs, suggesting that the SL and auxin hormonal pathways converge for regulation of RH elongation. The ethylene pathway requirement for the RH response to SLs suggests that ethylene forms a cross-talk junction between the SL and auxin pathways.     

Strigolactones affect lateral root formation and root-hair elongation in Arabidopsis

Strigolactones (SLs) have been proposed as a new group of plant hormones, inhibiting shoot branching, and as signaling molecules for plant interactions. Here, we present evidence for effects of SLs on root development. The analysis of mutants flawed in SLs synthesis or signaling suggested that the absence of SLs enhances lateral root formation. In accordance, roots grown in the presence of GR24, a synthetic bioactive SL, showed reduced number of lateral roots in WT and in max3-11 and max4-1 mutants, deficient in SL synthesis. The GR24-induced reduction in lateral roots was not apparent in the SL signaling mutant max2-1. Moreover, GR24 led to increased root-hair length in WT and in max3-11 and max4-1 mutants, but not in max2-1. SLs effect on lateral root formation and root-hair elongation may suggest a role for SLs in the regulation of root development; perhaps, as a response to growth conditions.     

MARCH8 Ubiquitinates the Hepatitis C Virus Nonstructural 2 Protein and Mediates Viral Envelopment

1. Download high-res image (195KB)
2. Download full-size image