DNA repair is essential to maintain genome integrity, and genes with roles in DNA repair are frequently mutated in a variety of human diseases. Repair via homologous recombination typically restores the original DNA sequence without introducing mutations, and a number of genes that are required for homologous recombination DNA double-strand break repair (HR-DSBR) have been identified. However, a systematic analysis of this important DNA repair pathway in mammalian cells has not been reported. Here, we describe a genome-scale endoribonuclease-prepared short interfering RNA (esiRNA) screen for genes involved in DNA double strand break repair. We report 61 genes that influenced the frequency of HR-DSBR and characterize in detail one of the genes that decreased the frequency of HR-DSBR. We show that the gene KIAA0415 encodes a putative helicase that interacts with SPG11 and SPG15, two proteins mutated in hereditary spastic paraplegia (HSP). We identify mutations in HSP patients, discovering KIAA0415/SPG48 as a novel HSP-associated gene, and show that a KIAA0415/SPG48 mutant cell line is more sensitive to DNA damaging drugs. We present the first genome-scale survey of HR-DSBR in mammalian cells providing a dataset that should accelerate the discovery of novel genes with roles in DNA repair and associated medical conditions. The discovery that proteins forming a novel protein complex are required for efficient HR-DSBR and are mutated in patients suffering from HSP suggests a link between HSP and DNA repair.

The analysis of microcircuitry (the connectivity at the level of individual neuronal processes and synapses), which is indispensable for our understanding of brain function, is based on serial transmission electron microscopy (TEM) or one of its modern variants. Due to technical limitations, most previous studies that used serial TEM recorded relatively small stacks of individual neurons. As a result, our knowledge of microcircuitry in any nervous system is very limited. We applied the software package TrakEM2 to reconstruct neuronal microcircuitry from TEM sections of a small brain, the early larval brain of Drosophila melanogaster. TrakEM2 enables us to embed the analysis of the TEM image volumes at the microcircuit level into a light microscopically derived neuro-anatomical framework, by registering confocal stacks containing sparsely labeled neural structures with the TEM image volume. We imaged two sets of serial TEM sections of the Drosophila first instar larval brain neuropile and one ventral nerve cord segment, and here report our first results pertaining to Drosophila brain microcircuitry. Terminal neurites fall into a small number of generic classes termed globular, varicose, axiform, and dendritiform. Globular and varicose neurites have large diameter segments that carry almost exclusively presynaptic sites. Dendritiform neurites are thin, highly branched processes that are almost exclusively postsynaptic. Due to the high branching density of dendritiform fibers and the fact that synapses are polyadic, neurites are highly interconnected even within small neuropile volumes. We describe the network motifs most frequently encountered in the Drosophila neuropile. Our study introduces an approach towards a comprehensive anatomical reconstruction of neuronal microcircuitry and delivers microcircuitry comparisons between vertebrate and insect neuropile.

Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) by overexpression of the transcription factors OCT4, SOX2, KLF4, and c-Myc holds great promise for the development of personalized cell replacement therapies. In an attempt to minimize the risk of chromosomal disruption and to simplify reprogramming, several studies demonstrated that a reduced set of reprogramming factors is sufficient to generate iPSC. We recently showed that a reduction of reprogramming factors in murine cells not only reduces reprogramming efficiency but also may worsen subsequent differentiation. To prove whether this is also true for human cells, we compared the efficiency of neuronal differentiation of iPSC generated from fetal human neural stem cells with either one (OCT4; hiPSC1F-NSC) or two (OCT4, KLF4; hiPSC2F-NSC) reprogramming factors with iPSC produced from human fibroblasts using three (hiPSC3F-FIB) or four reprogramming factors (hiPSC4F-FIB). After four weeks of coculture with PA6 stromal cells, neuronal differentiation of hiPSC1F-NSC and hiPSC2F-NSC was as efficient as iPSC3F-FIB or iPSC4F-FIB. We conclude that a reduction of reprogramming factors in human cells does reduce reprogramming efficiency but does not alter subsequent differentiation into neural lineages. This is of importance for the development of future application of iPSC in cell replacement therapies.

The microphthalmia with linear skin defects syndrome (MLS) is an X-linked dominant disorder with male lethality. In the majority of the patients reported, the MLS syndrome is caused by segmental monosomy of the Xp22.3 region. To date, five male patients with MLS and 46,XX karyotype (“XX males”) have been described. Here we report on the first male case with MLS and an XY complement. The patient showed agenesis of the corpus callosum, histiocytoid cardiomyopathy, and lactic acidosis but no microphthalmia, and carried a mosaic subtle inversion of the short arm of the X chromosome in 15% of his peripheral blood lymphocytes, 46,Y,inv(X)(p22.13∼22.2p22.32∼22.33)[49]/46,XY[271]. By fluorescence in situ hybridization (FISH), we showed that YAC 225H10 spans the breakpoint in Xp22.3. End-sequencing and database analysis revealed a YAC insert of at least 416 kb containing the genes HCCS and AMELX, and exons 2–16 of ARHGAP6. Molecular cytogenetic data suggest that the Xp22.3 inversion breakpoint is located in intron 1 of ARHGAP6, the gene encoding the Rho GTPase activating protein 6. Future molecular studies in karyotypically normal female MLS patients to detect submicroscopic rearrangements including the ARHGAP6 gene as well as mutation screening of ARHGAP6 in patients with no obvious chromosomal rearrangements will clarify the role of this gene in MLS syndrome. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Tocopherols, collectively known as vitamin E, are lipophilic antioxidants, which are synthesized only by photosynthetic organisms. Due to their enormous potential to protect cells from oxidative damage, tocopherols are used e.g. as nutraceuticals and additives in pharmaceuticals. The most biologically active form of vitamin E is α-tocopherol. Most tocopherols are currently produced via chemical synthesis. Nevertheless, this always results in a racemic mixture of different and less effective stereoisomers because the natural isomer has the highest biological activity. Therefore, tocopherols synthesized in natural sources are preferred for medical purposes. The annual sunflower (Helianthus annuus L.) is a well-known source for α-tocopherol. Within the presented work, sunflower callus and suspension cultures were established growing under photomixotrophic conditions to enhance α-tocopherol yield. The most efficient callus induction was achieved with sunflower stems cultivated on solid Murashige and Skoog medium supplemented with 30 g l-1 sucrose, 0.5 mg l-1 of the auxin 1-naphthalene acetic acid and 0.5 mg l-1 of the cytokinin 6-benzylaminopurine. Photomixotrophic sunflower suspension cultures were induced by transferring previously established callus into liquid medium. The effects of light intensity, sugar concentration and culture age on growth rate and α-tocopherol synthesis rate were characterized. A considerable increase (max. 230 %) of α-tocopherol production in the cells was obtained within the photomixotrophic cell culture compared to a heterotrophic cell culture. These results will be useful for improving α-tocopherol yields of plant in vitro cultures.

Transgenic plants expressing a foreign gene are a suitable system for the production of relevant immunogens in high amounts that can be used for the development of a new generation of vaccines against a variety of infectious diseases. In the present study, the expression of the nucleocapsid (N) protein of hantavirus serotype Puumala in tobacco and potato plants was investigated. Transgenic tobacco and potato plants were generated and established. These transgenic plants expressed the N protein of Puumala virus strain CG-1820. No major differences were observed when the phenotype and growth rates of transgenic plants were compared to those of normal plants. However, it was found that the leaves of transgenic tobacco plants were more slender and the tubers of transgenic potato plants were smaller than those in normal plants. In order to investigate the distribution of the expression of the foreign gene in transgenic plants, the proteins of leaves and roots of the individual transgenic tobacco and potato plants were examined by Western blot analyses. It was found that all transgenic tobacco and potato plants expressed the N protein in the leaves, whereas transgenic potato plants are able to significantly express the viral proteins also in the tubers and roots. The antigens were expressed at a level of 1 ng of protein/5 μg of dried leaves. The hantaviral recombinant N proteins obtained from transgenic tobacco and potato plants were able to elicit specific humoral and mucosal immune responses when administered intraperitoneally or orally to rabbits and mice. The expression of viral proteins in plants has two major advantages compared to other expression systems: firstly, there is no risk of contamination with mammalian viruses or other pathogens, and secondly, the production of high amounts of antigens is cheap and therefore of great economic interest. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

The use of reporter gene fusions to assess cellular processes such as protein targeting and regulation of transcription or translation is established technology in archaeal, bacterial, and eukaryal genetics. Fluorescent proteins or enzymes resulting in chromogenic substrate turnover, like β-galactosidase, have been particularly useful for microscopic and screening purposes. However, application of such methodology is of limited use for strictly anaerobic organisms due to the requirement of molecular oxygen for chromophore formation or color development. We have developed β-lactamase from Escherichia coli (encoded by bla) in conjunction with the chromogenic substrate nitrocefin into a reporter system usable under anaerobic conditions for the methanogenic archaeon Methanosarcina acetivorans. By using a signal peptide of a putative flagellin from M. acetivorans and different catabolic promoters, we could demonstrate growth substrate-dependent secretion of β-lactamase, facilitating its use in colony screening on agar plates. Furthermore, a series of fusions comprised of a constitutive promoter and sequences encoding variants of the synthetic tetracycline-responsive riboswitch (tc-RS) was created to characterize its influence on translation initiation in M. acetivorans. One tc-RS variant resulted in more than 11-fold tetracycline-dependent regulation of bla expression, which is in the range of regulation by naturally occurring riboswitches. Thus, tc-RS fusions represent the first solely cis-active, that is, factor-independent system for controlled gene expression in Archaea.

Objective: To analyze cartilage gene expression of patients with osteoarthritis (OA) in correlation with radiographic and histological findings. Materials and Methods: Twenty-one patients with OA of the knee admitted for total knee replacement were analyzed clinically and radiographically by the Kellgren and Lawrence system. During surgery, cartilage samples from the medial and lateral condyles and tibial plateaus were harvested separately. Specimens were analyzed histologically (Mankin score) and total RNA was extracted directly from cartilage tissue. Steady state levels of stromelysin (MMP-3), aggrecan (AGG) and the house-keeping gene β-actin were measured using quantitative PCR. Results: Histology of medial and lateral knee compartments corresponded to radiographic changes (Spearman correlation coefficient: r = 0.7 (p < 0.01)). There was a positive correlation between MMP-3 and AGG gene expression (r = 0.4; p < 0.01). We found considerable variation of expression levels of MMP-3 and AGG and no correlation of gene expression with histological or radiographic scoring. Conclusion: The positive correlation between AGG and MMP-3 suggests a common regulation of anabolic and catabolic metabolism. There was no simple dependency between gene expression and histological and radiological findings in cartilage. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Over the past decades, quantitative methods linking theory and observation became increasingly important in many areas of life science. Subsequently, a large number of mathematical and computational models has been developed. The BioModels database alone lists more than 140,000 Systems Biology Markup Language (SBML) models. However, while the exchange within specific model classes has been supported by standardisation and database efforts, the generic application and especially the re-use of models is still limited by practical issues such as easy and straight forward model execution. MAGPIE, a Modeling and Analysis Generic Platform with Integrated Evaluation, closes this gap by providing a software platform for both, publishing and executing computational models without restrictions on the programming language, thereby combining a maximum on flexibility for programmers with easy handling for non-technical users. MAGPIE goes beyond classical SBML platforms by including all models, independent of the underlying programming language, ranging from simple script models to complex data integration and computations. We demonstrate the versatility of MAGPIE using four prototypic example cases. We also outline the potential of MAGPIE to improve transparency and reproducibility of computational models in life sciences. A demo server is available at magpie.imb.medizin.tu-dresden.de.

Plastid genomes of photosynthetic flowering plants are usually highly conserved in both structure and gene content. However, the plastomes of parasitic and mycoheterotrophic plants may be released from selective constraint due to the reduction or loss of photosynthetic ability. Here we present the greatly reduced and highly divergent, yet functional, plastome of the nonphotosynthetic holoparasite Hydnora visseri (Hydnoraceae, Piperales). The plastome is 27 kb in length, with 24 genes encoding ribosomal proteins, ribosomal RNAs, tRNAs and a few non-bioenergetic genes, but no genes related to photosynthesis. The inverted repeat and the small single copy region are only ~1.5 kb, and intergenic regions have been drastically reduced. Despite extreme reduction, gene order and orientation are highly similar to the plastome of Piper cenocladum, a related photosynthetic plant in Piperales. Gene sequences in Hydnora are highly divergent and several complementary approaches using the highest possible sensitivity were required for identification and annotation of this plastome. Active transcription is detected for all of the protein coding genes in the plastid genome, and one of two introns is appropriately spliced out of rps12 transcripts. The whole genome shotgun read depth is 1,400X coverage for the plastome, while the mitochondrial genome is covered at 40X and the nuclear genome at 2X. Despite the extreme reduction of the genome and high sequence divergence, the presence of syntenic, long transcriptionally-active open reading frames with distant similarity to other plastid genomes and a high plastome stoichiometry relative to the mitochondrial and nuclear genomes suggests that the plastome remains functional in Hydnora visseri. A four stage model of gene reduction, including the potential for complete plastome loss, is proposed to account for the range of plastid genomes in nonphotosynthetic plants.