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In brief: April 2005DNA repair
Conserved modes of recruitment of ATM, ATR and DNA-PKcs to sites of DNA damage.
The phosphoinositide-3-kinase-related protein kinase (PIKK)- family members ATM, ATR and DNA-PKcs are activated in response to DNA damage and recruited to sites of DNA damage by individual partner proteins. Falck et al. have now identified a conserved interaction motif in the C terminus of each partner protein — Nbs1, ATRIP and Ku80, respectively — that is essential for the recruitment of these PIKKs as well as for the downstream signalling events. Chromosome segregation
Dissociation of cohesin from chromosome arms and loss of arm cohesion during early mitosis depends on phosphorylation of SA2.
Shugoshin prevents dissociation of cohesin from centromeres during mitosis in vertebrate cells.
During mitosis, sister chromatids are held together by cohesin, which is cleaved in anaphase, triggering chromatid segregation. However, most cohesin is removed from the chromosome arms, but not from the centromere, before anaphase. One study now shows that phosphorylation of the cohesin subunit SA2 is required for cohesin removal from chromosome arms in early mitosis. So what safeguards centromeric cohesin? Shugoshin, according to a second study, as this protein seems to protect centromeric SA2 from phosphorylation to prevent premature chromatid segregation. Plant biology
RNA polymerase IV directs silencing of endogenous DNA.
Plant nuclear RNA polymerase IV mediates siRNA and DNA methylation-dependent heterochromatin formation.
Plants encode catalytic subunits of a fourth RNA polymerase, Pol IV, the function of which has remained unknown until now. Data from two studies now indicate that Pol IV causes small interfering (si)RNA-mediated gene silencing and facultative heterochromatin formation. These events are associated with RNA-directed DNA methylation, which requires siRNAs for targeting specific de novo DNA-methylation events. The production of siRNAs might be mediated by a mechanism that involves Pol IV, although its precise role is yet to be determined. Innate immunity
Dual role of
Viral immunity
Inverse correlation between IL-7 receptor expression and CD8 T cell exhaustion during persistent antigen stimulation
.
Infection with lymphocytic choriomeningitis virus is characterized by persistence of the virus and is often associated with exhaustion of CD8+ T cells. In this study, the authors show that persistent viral antigen suppressed the expression of the interleukin-7 receptor T-cell responses
The role of herpesvirus entry mediator as a negative regulator of T cell-mediated responses
.
Herpesvirus entry mediator (HVEM) has previously been described as a T-cell co-stimulatory receptor, but to the surprise of these researchers, HVEM-deficient T cells, when compared with wild-type T cells, showed increased proliferative responses after stimulation with CD3-specific antibody or exposure to concanavalin A (conA) in vitro. Furthermore, they showed that administration of conA to HVEM-deficient mice resulted in increased morbidity and mortality, owing to the development of severe T-cell-mediated hepatitis. And compared with wild-type splenocytes, conA-treated HVEM-deficient splenocytes produced higher levels of several cytokines. Finally, HVEM-deficient mice showed increased susceptibility to experimental allergic encephalomyelitis. Together, these results indicate that HVEM can function as a negative regulator of T-cell responses. B-cell development
Basal immunoglobulin signaling actively maintains developmental stage in immature B cells.
This study shows that basal signalling through the B-cell receptor (BCR) of immature B cells is crucial to suppress expression of the recombination-activating genes (RAG1 and RAG2) and to prevent 'back-differentiation' to the pro-B-cell stage. Therefore, such basal signalling is important to maintain allelic exclusion of the immunoglobulin light chains (which rearrange at this stage of development) and ensure self-tolerance. When basal IgM signalling in immature B cells was inhibited, microarray analysis and flow cytometry showed the upregulation of genes and proteins that are selectively expressed by pro-B cells. The requirement for basal signalling to maintain B-cell development could be an important quality-control mechanism to test for a functional BCR. Natural killer cells
A subset of natural killer cells achieve self-tolerance without expressing inhibitory receptors specific for self MHC molecules
.
Natural killer (NK) cells are thought to express at least one inhibitory receptor specific for a self-MHC class I molecule, and this is thought to maintain NK-cell self-tolerance. However, Fernandez et al. detected a population of NK cells that lack expression of all known inhibitory receptors specific for self-MHC class I molecules. These NK cells were hyporesponsive in vitro when cultured with either cells lacking cell-surface expression of MHC class I molecules or tumour cells expressing ligands for NK-cell activating receptors. Similar hyporesponsiveness was observed in vivo, as these NK cells were inefficient at mediating rejection of bone marrow lacking cell-surface expression of MHC class I molecules, indicating that, for some NK cells, self-tolerance is not a result of inhibitory-receptor interaction with self-MHC class I molecules but of hyporesponsiveness to self. RNA world
A potential role for RNA interference in controlling the activity of the human LINE-1 retrotransposon
.
Vigilins bind to promiscuously A-to-I-edited RNAs and are involved in the formation of heterochromatin
.
These papers provide new insights into cellular responses to double-stranded RNA (dsRNA). LINE-1 elements pose a threat to genome integrity in human cells through their ability to move around by retrotransposition. Soifer et al. present the first evidence that LINE-1 dsRNAs are targets of the RNAi machinery, indicating that this pathway is one form of defence that the human genome uses against retrotransposition. dsRNAs from various sources also undergo promiscuous adenosine-to-inosine editing, and Wang et al. provide evidence that this leads to the formation of silent heterochromatin at the corresponding genomic sequences. RNAi and RNA editing therefore provide alternative and perhaps overlapping pathways to RNA-mediated silencing. Technology
MAGIC, an in vivo genetic method for the rapid construction of recombinant DNA molecules
.
Conventional cloning is an expensive and time-consuming process that involves multiple steps from the initial DNA plasmid preparation to transformation. The need to achieve high-throughput recombinant-DNA production led Li and Elledge to design a new cloning method that involves only three steps. The approach relies on bacterial conjugation, in vivo site-specific endonuclease cleavage and homologous recombination to place the DNA fragment of interest under the control of new regulatory elements in the desired vector. It's cheap and easy — it's MAGIC. Technology
Ubiquitous GFP expression in transgenic chickens using a lentiviral vector
.
Production of transgenic chickens is an attractive goal for both pharmaceutical and developmental biologists, but has been held back owing to technical challenges. Chapman and colleagues now show that ubiquitous GFP expression in the chicken embryo can be achieved using a lentiviral vector. With the chicken genome sequence now available, this method will be useful for investigating gene expression during embryonic development — an important tool given that the chicken is an established developmental model. It also provides a model for a new generation of transgenics for studying the expression of pharmaceutical products in egg albumen. Developmental genetics
Genetic programs activated by proneural proteins in the developing Drosophila PNS
.
As their name suggests, proneural transcription factors promote neurogenesis. Their role is well established, unlike their mode of action, because few of their targets are known. Using whole-genome microarray analysis and in situ hybridization in proneural cell clusters, the authors identified a set of genes that are preferentially expressed in these cells. Loss of function of two of the candidates confirmed their role in PNS development. Sequence analysis and reporter studies allowed the authors to describe cis-regulatory elements that direct proneural gene expression. Human disease
Inactivation of TGFB This paper provides a link between transforming growth factor- Functional genomics
A universal plasmid library encoding all permutations of small interfering RNA
.
Small interfering RNA (siRNA) libraries are limited to targeting predicted or known genes, restricting their usefulness in species for which the transcriptome is uncharacterized. Chen et al. circumvented this problem by constructing a library of 5 Cardiovascular disease
Circulating transcriptome reveals markers of atherosclerosis
.
Circulating monocytes, which mediate inflammation in atherosclerosis, might serve as accessible reporters of disease. Patino et al. compared the in vivo transcriptomes of monocytes from patients with atherosclerosis and normal patients, and provide data that the FOS gene is a marker and mediator of atherosclerosis. Similar approaches examining the circulating transcriptome in other conditions might also be valuable. Kinases
Chemical genomic profiling to identify intracellular targets of a multiplex kinase inhibitor
.
Identifying which kinases are targeted by protein kinase inhibitors is a key challenge in validating their use as therapeutic agents or chemical tools to probe biology. The authors describe a strategy to address this challenge that uses a direct comparison between microarray transcriptional signatures elicited by an inhibitor of unknown specificity and those elicited by highly specific pharmacological inhibition of engineered kinases, which they use to identify the targets of a cyclin-dependent kinase inhibitor of previously unknown specificity. Imaging and Visualization Near infrared–emissive polymersomes: self-assembled soft matter for in vivo optical imaging The development of a versatile and stable reagent with near-infrared fluorescence would be a considerable boon for imaging studies that require deep penetration of living tissues. Ghoroghchian et al. describe the generation of self-assembling polymersomes that incorporate an oligo(porphyrin)-based near-infrared fluorophore and demonstrate the potential of these bodies for in vivo imaging applications.
Ghoroghchian, P.P.
et al.
Proteomics Interaction network containing conserved and essential protein complexes in Escherichia coli By adapting the yeast-based tandem affinity purification (TAP) technique for use in E. coli, Butland et al. have assembled a detailed interaction network that describes the associations between nearly 650 different bacterial proteins, confirming several predicted associations and identifing many interaction 'hubs' that seem to be broadly conserved among prokaryotes.
Butland, G.
et al.
RNA Interference A universal plasmid library encoding all permutations of small interfering RNA (siRNA) Many current RNA interference (RNAi) studies begin with a target gene and attempt to identify a suitable siRNA. Chen et al. present a tool for researchers who want to work in the reverse direction—a library encoding nearly every possible 19-nucleotide siRNA sequence—and describe how such libraries could prove valuable for conducting phenotype-driven RNA interference studies.
Chen, M.
et al.
Microbiology Screening for quorum-sensing inhibitors (QSIs) by use of a new genetic system, the QSI selector Genes involved in quorum sensing, the process by which many bacteria organize their pathological progression in response to population density, are now recognized as an important potential antibiotic target. Rasmussen et al. demonstrate several novel genetic screens for the identification of QSIs.
Rasmussen, T.B.
et al.
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-defensin-1 in anti-HIV-1 innate immunity
. 
107 plasmids containing all permutations of siRNA sequences. The library should enable large-scale RNAi screens to be carried out in any organism or cell type.