Search

The regulation of the nucleocytoplasmic release of herpesviral capsids is defined by the process of nuclear egress. Due to their large size, nuclear capsids are unable to traverse via nuclear pores, so that herpesviruses evolved to develop a vesicular transport pathway mediating their transition through both leaflets of the nuclear membrane. This process involves regulatory proteins, which support the local distortion of the nuclear envelope. For human cytomegalovirus (HCMV), the nuclear egress complex (NEC) is determined by the pUL50-pUL53 core that initiates multicomponent assembly with NEC-associated proteins and capsids. Hereby, pUL50 serves as a multi-interacting determinant that recruits several viral and cellular factors by direct and indirect contacts. Recently, we generated an ORF-UL50-deleted recombinant HCMV in pUL50-complementing cells and obtained first indications of putative additional functions of pUL50. In this study, we produced purified ΔUL50 particles under both complementing (ΔUL50C) and non-complementing (ΔUL50N) conditions and performed a phenotypical characterization. Findings were as follows: (i) ΔUL50N particle preparations exhibited a clear replicative defect in qPCR-based infection kinetics compared to ΔUL50C particles, (ii) immuno-EM analysis of ΔUL50C did not reveal major changes in nuclear distribution of pUL53 and lamin A/C, (iii) mass spectrometry-based quantitative proteomics showed a large concordance of protein contents in the NIEP fractions of ΔUL50C and ΔUL50N particles, but virion fraction was close to the detection limit for ΔUL50N, (iv) confocal imaging of viral marker proteins of immediate early (IE) and later phases of ΔUL50N infection indicated a very low number of cells showing an onset of viral lytic protein expression, and, finally (v) quantitative measurements of encapsidated genomes provided evidence for a substantial reduction in the DNA contents in ΔUL50N compared to ΔUL50C particles. In summary, the results point to a complex and important regulatory role of the HCMV nuclear egress protein pUL50 in the maturation of infectious virus.

Background: Regeneration of fertile plants from tissue culture is a critical bottleneck in the application of new plant breeding technologies. Ectopic overexpression of morphogenic factors is a promising workaround for this hurdle. Methods: Conditional overexpression of WUS and ARF5Δ was used to study the effect of timing the overexpression of these morphogenic factors during shoot regeneration from root explants in Arabidopsis. In addition, their effect on auxin-signaling activation was examined by visualization and cytometric quantification of the DR5:GFP auxin-signaling reporter in roots and protoplasts, respectively. Results: The induced expression of both WUS and ARF5Δ led to an activation of auxin signaling in roots. Activation of auxin signaling by WUS and ARF5Δ was further quantified by transient transformation of protoplasts. Ectopic overexpression of both WUS and ARF5Δ enhanced regeneration efficiency, but only during the shoot-induction stage of regeneration and not during the callus-induction stage. Conclusions: The overexpression of WUS and ARF5Δ both lead to activation of auxin signaling. Expression during the shoot-induction stage is critical for the enhancement of shoot regeneration by WUS and ARF5Δ.

The genetic incorporation of unnatural amino acids (Uaas) with defined properties into proteins at designated sites represents an extremely powerful tool for protein engineering. However, the efficient incorporation of Uaas in response to the amber stop codon in mammalian cells remains a substantial challenge due to the competition from release factor 1(RF1). Addressing this challenge will greatly broaden the power and scope of this technology. Here, we chose the eRF1 mutant, which can selectively enhance Uaa incorporation in response to the amber codon without increasing the readthrough of the opal and ochre codons. Then, we developed an engineered stable cell line using a tetracycline-controlled inducible lentiviral system for the conditional expression of mutant eRF1, which can minimize the potential effect on normal translation termination. Using the eRF1-engineered cells, we provided a 2-fold improvement in the yield of protein containing a Uaa incorporated at a single site, with the protein yield approaching 90% of the wild-type control without the amber codon. Moreover, we achieved the successful incorporation of Uaas at four sites in various proteins at a measured level of 20%.

Human genetic mutations that affect the N-terminal head-domain of the nonmuscle myosin-II (MyoII) molecule can result in nonsyndromic sensorineural hearing loss but the underlying mechanism is unknown. Ultimately, MyoII must be appropriately localized in order to execute endogenous functions. The aim of the current study is to determine whether the head-domain of MyoII regulates in vivo localization of the molecule in living and fixed preparations of the auditory organ.A genetic/transgenic GAL4-UAS approach was used to selectively drive the expression of zip/MyoII (Drosophila homologue of human nonmuscle MyoII) in Drosophila melanogaster auditory (Johnston's organ) sensory neurons. To follow the distribution of the full-length transgene encoded by MyoII, the N-terminus was fused to green fluorescent protein. Additionally, headless zip/MyoII molecules with and without isoleucine-glutamine or IQ motifs were also expressed in Johnston's organ neurons.Removing the entire head domain of MyoII induced localization in neuronal dendrites while removing only a portion of the head but keeping the IQ motif induced localization in the soma and axons of the neurons.The findings suggest that the head domain regulates in vivo localization of MyoII in auditory neurons.

The malarial parasite’s invasion is complex, active and coordinated, involving many low and high affinity interactions with receptors on target cell membrane. Proteomics analysis has described around 40 proteins in P. vivax which could be involved in reticulocyte invasion; few have been studied with the aim of elucidating how many of them establish specific interactions with their respective host cells. Given the importance of knowing which of the parasite’s protein regions are functionally important for invasion, minimum regions mediating specific interaction between Plasmodium vivax apical membrane antigen 1 (PvAMA-1) and its host cell were here elucidated. The region covering PvAMA-1 domains I and II (PvAMA-DI-II) specifically bound to the CD71+ red blood cell subpopulation. A 20 residue-long region (81EVENAKYRIPAGRCPVFGKG100) located in domain I was capable of inhibiting PvAMA-DI-II recombinant protein binding to young reticulocytes (CD71+CD45−) and rosette formation. This conserved peptide specifically interacted with high affinity with reticulocytes (CD71+) through a neuraminidase- and chymotrypsin-treatment sensitive receptor. Such results showed that, despite AMA-1 having universal functions during late Plasmodium invasion stages, PvAMA-1 had reticulocyte-preferring binding regions, suggesting that P. vivax target cell selection is not just restricted to initial interactions but maintained throughout the erythrocyte invasion cycle, having important implications for designing a specific anti-P. vivax vaccine.

The visualization of meiotic chromosomes and their associated protein structures in both wild-type and mutant cells adds valuable insight into the molecular pathways that underlie reproductive cell formation. Here we describe basic methodology for visualizing meiotic chromosomes in a long-standing model organism for investigating the molecular and cell biology of meiosis, the budding yeast, S. cerevisiae. This chapter furthermore highlights a variety of conditional expression regimes that can be used to understand the dynamics and/or developmental constraints of chromosomal protein structures; such dynamic aspects of the macromolecular structures that mediate meiotic chromosome biology are typically not obvious from standard protein visualization experiments.

In order to accurately analyze gene function in transgenic mice, as well as to generate credible murine models of human diseases, the ability to regulate temporal- and spatial-specific expression of target genes is absolutely critical. Pioneering work in inducible transgenics, begun in the 1980s and continuing to the present, has led to the development of a variety of different inducible systems dedicated to this goal, the shared basis of which is the accurate conditional expression of a given transgene. Recent advances in inducible transgene expression in mice are discussed.

International audience; Increased production of neurotrophic factors (NTFs) is one of the key responses seen following peripheral nerve injury, making them an attractive choice for pro-regenerative gene therapies. However, the downside of over-expression of certain NTFs, including glial cell line-derived neurotrophic factor (GDNF), was earlier found to be the trapping and misdirection of regenerating axons, the so-called ‘candy-store’ effect. We report a proof-of-principle study on the application of conditional GDNF expression system in injured peripheral nerve. We engineered Schwann cells (SCs) using dendrimers or lentiviral transduction with the vector providing doxycycline-regulated GDNF expression. Injection of GDNF-modified cells into the injured peripheral nerve followed by time-restricted administration of doxycycline demonstrated that GDNF expression in SCs can also be controlled locally in the peripheral nerves of the experimental animals. Cell-based GDNF therapy was shown to increase the extent of axonal regeneration, while controlled deactivation of GDNF effectively prevented trapping of regenerating axons in GDNF-enriched areas, and was associated with improved functional recovery.

The atrioventricular node controls cardiac impulse conduction and generates pacemaker activity in case of failure of the sino-atrial node. Understanding the mechanisms of atrioventricular automaticity is important for managing human pathologies of heart rate and conduction. However, the physiology of atrioventricular automaticity is still poorly understood. We have investigated the role of three key ion channel-mediated pacemaker mechanisms namely, Ca(v)1.3, Ca(v)3.1 and HCN channels in automaticity of atrioventricular node cells (AVNCs). We studied atrioventricular conduction and pacemaking of AVNCs in wild-type mice and mice lacking Ca(v)3.1 (Ca(v)3.1(-/-)), Ca(v)1.3 (Ca(v)1.3(-/-)), channels or both (Ca(v)1.3(-/-)/Ca(v)3.1(-/-)). The role of HCN channels in the modulation of atrioventricular cells pacemaking was studied by conditional expression of dominant-negative HCN4 channels lacking cAMP sensitivity. Inactivation of Ca(v)3.1 channels impaired AVNCs pacemaker activity by favoring sporadic block of automaticity leading to cellular arrhythmia. Furthermore, Ca(v)3.1 channels were critical for AVNCs to reach high pacemaking rates under isoproterenol. Unexpectedly, Ca(v)1.3 channels were required for spontaneous automaticity, because Ca(v)1.3(-/-) and Ca(v)1.3(-/-)/Ca(v)3.1(-/-) AVNCs were completely silent under physiological conditions. Abolition of the cAMP sensitivity of HCN channels reduced automaticity under basal conditions, but maximal rates of AVNCs could be restored to that of control mice by isoproterenol. In conclusion, while Ca(v)1.3 channels are required for automaticity, Ca(v)3.1 channels are important for maximal pacing rates of mouse AVNCs. HCN channels are important for basal AVNCs automaticity but do not appear to be determinant for β-adrenergic regulation.

Background To analyze gene function in mammalian cells tetracycline inducible expression of a gene-of-interest at a specific genomic location (Flp-In T-REx™) is most attractive. However, leakiness of basal transgene expression and artificially high expression level upon tetracycline addition may be disadvantageous. Findings To solve these problems, we developed two different approaches to improve our pancreatic β-cell line INS-1 Flp-In T-REx™ expressing the tissue restricted transcription factor HNF4α under control of tetracycline. On the one hand we replaced the strong full length CMV promoter (CMV-Wt) with a weaker 5'-deleted CMV promoter fragment of 138 nucleotides in length (CMV-138). On the other hand we extended our INS-1 Flp-In T-REx™ cell lines with a Shield-1 dependent conditional control system of protein stability. Therefore, we fused HNF4α to the destabilization domain (DD) deduced from human FKBP12 protein. As a result in both approaches basal transgene expression level was markedly reduced, but HNF4α induction could still be maintained. Additionally, we could show that a low increase in HNF4α induces caspase activity indicating an apoptotic effect of HNF4α in these cells. Conclusion In the present study we considerably improved our INS-1 Flp-In T-REx™ cell lines conditionally expressing HNF4α to reduce leakiness and to optimize exogenous HNF4α protein expression to a physiological level. As an important result we could extend our previous results that HNF4α induces apoptosis in the pancreatic β-cell line INS-1 with the new aspect that an expression level of the HNF4α transgene marginally exceeding the endogenous level is sufficient to trigger apoptosis.

Mammalian development is highly sensitive to Tbx1 gene dosage reduction. Gene function insights can also be learned from increased or ectopic expression. The authors generated a novel mouse transgenic line, named COET, which expresses Tbx1 upon Cre-mediated recombination. The authors crossed this transgenic line with Tbx1(Cre) animals to activate expression in the Tbx1-expression domain. Compound mutant COET;Tbx1(Cre/+) animals died after birth and showed heart enlargement. At E18.5, compound mutants showed ventricular septal defects and thymic abnormalities. The authors crossed compound mutants into a Tbx1 null background to understand whether this phenotype is caused by gene overdosage. Results showed that gene dosage reduction at the endogenous locus could not rescue heart and thymic defects, although the transgene rescued the loss of function phenotype. Thus, the transgenic phenotype appears to be due to gain of function. Resultant data demonstrate that Tbx1 expression must be tightly regulated to be compatible with normal embryonic development. genesis 47:188-195, 2009. (c) 2009 Wiley-Liss, Inc.

In this issue of Blood, Nakajima-Takagi et al use human embryonic stem (ES) and induced pluripotent stem (iPS) cells to identify Sox17 as a regulator of hemogenic endothelium, and use conditional expression of Sox17 to capture endothelial cells at the critical moment of hemogeneic transition.

International audience; The UNUSUAL FLORAL ORGANS (UFO) gene is required for several aspects of floral development in Arabidopsis including specification of organ identity in the second and third whorls and the proper pattern of primordium initiation in the inner three whorls. UFO is expressed in a dynamic pattern during the early phases of flower development. Here we dissect the role of UFO by ubiquitously expressing it in ufo loss-of-function flowers at different developmental stages and for various durations using an ethanol-inducible expression system. The previously known functions of UFO could be separated and related to its expression at specific stages of development. We show that a 24- to 48-hour period of UFO expression from floral stage 2, before any floral organs are visible, is sufficient to restore normal petal and stamen development. The earliest requirement for UFO is during stage 2, when the endogenous UFO gene is transiently expressed in the centre of the wild-type flower and is required to specify the initiation patterns of petal, stamen and carpel primordia. Petal and stamen identity is determined during stages 2 or 3, when UFO is normally expressed in the presumptive second and third whorl. Although endogenous UFO expression is absent from the stamen whorl from stage 4 onwards, stamen identity can be restored by UFO activation up to stage 6. We also observed floral phenotypes not observed in loss-of-function or constitutive gain-of-function backgrounds, revealing additional roles of UFO in outgrowth of petal primordia.

SUMMARYIn order to investigate the importance of the nuclear genotype during morphogenesis of the cortex inParamecium aurelia, a search was made for conditional and unconditional mutants affecting cell shape. Ten different mutants were readily recovered following mutagenesis withN-methyl-N-nitro-N-nitrosoguanidine on stock 51s of syngen 4. The detailed pattern of surface units remained unchanged in these mutants but each one can be described as resembling a normal cell twisted about its long axis. Nine of the mutants complement each other and show no linkage. The remaining mutant, a ‘chain former’, has conditional expression, and is only recognizable at 35 °C. The penetrance of this mutant is affected by the food organism used. The conditional mutant also interacts with seven of the other mutants in pairwise homozygous combinations so that in these genotypes it can be classified after growth at 25 °C. The mutants are discussed in relation to the search for genie effects on the cortical pattern.