Chloroplast PNPase exists as a homo-multimer enzyme complex that is distinct from the Escherichia coli degradosome.

In Escherichia coli, the exoribonuclease polynucleotide phosphorylase (PNPase), the endoribonuclease RNase E, a DEAD-RNA helicase and the glycolytic enzyme enolase are associated with a high molecular weight complex, the degradosome. This complex has an important role in processing and degradation of RNA. Chloroplasts contain an exoribonuclease homologous to E. coli PNPase. Size exclusion chromatography revealed that chloroplast PNPase elutes as a 580-600 kDa complex, suggesting that it can form an enzyme complex similar to the E. coli degradosome. Biochemical and mass-spectrometric analysis showed, however, that PNPase is the only protein associated with the 580-600 kDa complex. Similarly, a purified recombinant chloroplast PNPase also eluted as a 580-600 kDa complex after gel filtration chromatography. These results suggest that chloroplast PNPase exists as a homo-multimer complex. No other chloroplast proteins were found to associate with chloroplast PNPase during affinity chromatography. Database analysis of proteins homologous to E. coli RNase E revealed that chloroplast and cyanobacterial proteins lack the C-terminal domain of the E. coli protein that is involved in assembly of the degradosome. Together, our results suggest that PNPase does not form a degradosome-like complex in the chloroplast. Thus, RNA processing and degradation in this organelle differ in several respects from those in E. coli.     

RMI1/NCE4, a suppressor of genome instability, encodes a member of the RecQ helicase/Topo III complex

SGS1 encodes a DNA helicase whose homologues in human cells include the BLM, WRN, and RECQ4 genes, mutations in which lead to cancer-predisposition syndromes. Clustering of synthetic genetic interactions identified by large-scale genetic network analysis revealed that the genetic interaction profile of the gene RMI1 (RecQ-mediated genome instability, also known as NCE4 and YPL024W) was highly similar to that of SGS1 and TOP3, suggesting a functional relationship between Rmi1 and the Sgs1/Top3 complex. We show that Rmi1 physically interacts with Sgs1 and Top3 and is a third member of this complex. Cells lacking RMI1 activate the Rad53 checkpoint kinase, undergo a mitotic delay, and display increased relocalization of the recombination repair protein Rad52, indicating the presence of spontaneous DNA damage. Consistent with a role for RMI1 in maintaining genome integrity, rmi1Delta cells exhibit increased recombination frequency and increased frequency of gross chromosomal rearrangements. In addition, rmi1Delta strains fail to fully activate Rad53 upon exposure to DNA-damaging agents, suggesting that Rmi1 is also an important part of the Rad53-dependent DNA damage response.     

Chemical–Genetic Profiling of Imidazo[1,2-a]pyridines and -Pyrimidines Reveals Target Pathways Conserved between Yeast and Human Cells

Small molecules have been shown to be potent and selective probes to understand cell physiology. Here, we show that imidazo[1,2-a]pyridines and imidazo[1,2-a]pyrimidines compose a class of compounds that target essential, conserved cellular processes. Using validated chemogenomic assays in Saccharomyces cerevisiae, we discovered that two closely related compounds, an imidazo[1,2-a]pyridine and -pyrimidine that differ by a single atom, have distinctly different mechanisms of action in vivo. 2-phenyl-3-nitroso-imidazo[1,2-a]pyridine was toxic to yeast strains with defects in electron transport and mitochondrial functions and caused mitochondrial fragmentation, suggesting that compound 13 acts by disrupting mitochondria. By contrast, 2-phenyl-3-nitroso-imidazo[1,2-a]pyrimidine acted as a DNA poison, causing damage to the nuclear DNA and inducing mutagenesis. We compared compound 15 to known chemotherapeutics and found resistance required intact DNA repair pathways. Thus, subtle changes in the structure of imidazo-pyridines and -pyrimidines dramatically alter both the intracellular targeting of these compounds and their effects in vivo. Of particular interest, these different modes of action were evident in experiments on human cells, suggesting that chemical–genetic profiles obtained in yeast are recapitulated in cultured cells, indicating that our observations in yeast can: (1) be leveraged to determine mechanism of action in mammalian cells and (2) suggest novel structure–activity relationships.     

DCL is a plant-specific protein required for plastid ribosomal RNA processing and embryo development

The defective chloroplast and leaf-mutable (dcl-m) mutation of tomato blocks chloroplast differentiation in leaf mesophyll cells and a signaling system that appears to be required for morphogenesis of palisade cells during leaf growth. To dissect the function of DCL, mutants with stable dcl alleles (dcl-s) were generated and examined for their phenotype. DCL/dcl-s plant produce dcl-s/dcl-s seeds with embryos arrested at the globular stage of development. The levels of several chloroplast- and nuclear-encoded proteins are strongly reduced in dcl-m mutant leaf sectors without significant changes in their corresponding mRNAs. The 4.5S rRNA fails to be processed efficiently, however, suggesting that DCL has a direct or indirect function in rRNA processing or correct ribosome assembly. Accordingly, chloroplasts in dcl-m sectors are impaired in polysome assembly, which can explain the reduced accumulation of chloroplast-encoded proteins. These results suggest that DCL is required for chloroplast rRNA processing, and emphasize the importance of plastid function during embryogenesis.     

Elg1 forms an alternative RFC complex important for DNA replication and genome integrity

Genome-wide synthetic genetic interaction screens with mutants in the mus81 and mms4 replication fork-processing genes identified a novel replication factor C (RFC) homolog, Elg1, which forms an alternative RFC complex with Rfc2-5. This complex is distinct from the DNA replication RFC, the DNA damage checkpoint RFC and the sister chromatid cohesion RFC. As expected from its genetic interactions, elg1 mutants are sensitive to DNA damage. Elg1 is redundant with Rad24 in the DNA damage response and contributes to activation of the checkpoint kinase Rad53. We find that elg1 mutants display DNA replication defects and genome instability, including increased recombination and mutation frequencies, and minichromosome maintenance defects. Mutants in elg1 show genetic interactions with pathways required for processing of stalled replication forks, and are defective in recovery from DNA damage during S phase. We propose that Elg1-RFC functions both in normal DNA replication and in the DNA damage response.     

Low-copy-number molecules are produced by recombination, actively maintained and can be amplified in the mitochondrial genome of Brassicaceae: relationship to reversion of the male sterile phenotype in some cybrids

A PCR analysis of mitochondrial (mt) genomes of cybrid rapeseed plants revealed substoichiometric concentrations of molecules bearing different configurations of the gene (orf138) responsible for Ogura cytoplasmic male sterility (CMS). These sub-stoichiometric molecules are also present in plants bearing the unmodified Ogura cytoplasm. In one cybrid family, which shows reversion of the male sterile phenotype, we observed changes in the respective proportions of these molecules. The phenotypic (sterility-fertility) reversion occurs as a result of a modification of the equilibrium state between the different forms of the orf138 gene and is very probably determined by the level of expression of this gene. Stable situations are always characterized by one predominant form; the others, when present, exist in substoichiometric amounts. We report results indicating that the different forms of the orf138 gene are continuously interconverted by recombination and that an active mechanism is involved in the maintenance of some substoichiometric molecules.     

Low-copy-number molecules are produced by recombination, actively maintained and can be amplified in the mitochondrial genome of Brassicaceae: relationship to reversion of the male sterile phenotype in some cybrids

A PCR analysis of mitochondrial (mt) genomes of cybrid rapeseed plants revealed substoichiometric concentrations of molecules bearing different configurations of the gene (orf138) responsible for Ogura cytoplasmic male sterility (CMS). These sub-stoichiometric molecules are also present in plants bearing the unmodified Ogura cytoplasm. In one cybrid family, which shows reversion of the male sterile phenotype, we observed changes in the respective proportions of these molecules. The phenotypic (sterility-fertility) reversion occurs as a result of a modification of the equilibrium state between the different forms of the orf138 gene and is very probably determined by the level of expression of this gene. Stable situations are always characterized by one predominant form; the others, when present, exist in substoichiometric amounts. We report results indicating that the different forms of the orf138 gene are continuously interconverted by recombination and that an active mechanism is involved in the maintenance of some substoichiometric molecules. Received: 14 July 1997 / Accepted: 16 September 1997     

Intérêt de l’imagerie hybride TEMP-TDM dans l’histiocytose langerhansienne osseuse. À propos d’un cas

Langerhans’ cell histiocytosis is a non-malignant proliferative disease of unknown etiology. It is a rare illness affecting mainly children and young adults with a male predominance. It can affect one or many organs. The bone locations are the most frequent. We present one case of multifocal Langerhans’ cell histiocytosis in a 22-year-old male patient followed for diabetes insipidus with an enlargement of the pituitary stalk at the magnetic resonance imaging (MRI), and a lytic bone lesion revealed in lumbar spine (L2) by ^99mTc-HMDP scintigraphy conducted in planar mode, and then in Single Photon Emission Computed Tomography coupled to Computed Tomography (SPECT/CT) in hybrid mode. To this end, we propose to highlight diagnostic gain of SPECT/CT compared with SPECT and planar scintigraphy to characterize radiotracer uptake abnormalities in bone in Langerhans’ cell histiocytosis.