Genome-wide analysis reveals extensive functional interaction between DNA replication initiation and transcription in the genome of Trypanosoma brucei.

Identification of replication initiation sites, termed origins, is a crucial step in understanding genome transmission in any organism. Transcription of the Trypanosoma brucei genome is highly unusual, with each chromosome comprising a few discrete transcription units. To understand how DNA replication occurs in the context of such organization, we have performed genome-wide mapping of the binding sites of the replication initiator ORC1/CDC6 and have identified replication origins, revealing that both localize to the boundaries of the transcription units. A remarkably small number of active origins is seen, whose spacing is greater than in any other eukaryote. We show that replication and transcription in T. brucei have a profound functional overlap, as reducing ORC1/CDC6 levels leads to genome-wide increases in mRNA levels arising from the boundaries of the transcription units. In addition, ORC1/CDC6 loss causes derepression of silent Variant Surface Glycoprotein genes, which are critical for host immune evasion.

NUP-1 Is a large coiled-coil nucleoskeletal protein in trypanosomes with lamin-like functions.

A unifying feature of eukaryotic nuclear organization is genome segregation into transcriptionally active euchromatin and transcriptionally repressed heterochromatin. In metazoa, lamin proteins preserve nuclear integrity and higher order heterochromatin organization at the nuclear periphery, but no non-metazoan lamin orthologues have been identified, despite the likely presence of nucleoskeletal elements in many lineages. This suggests a metazoan-specific origin for lamins, and therefore that distinct protein elements must compose the nucleoskeleton in other lineages. The trypanosomatids are highly divergent organisms and possess well-documented but remarkably distinct mechanisms for control of gene expression, including polycistronic transcription and trans-splicing. NUP-1 is a large protein localizing to the nuclear periphery of Trypanosoma brucei and a candidate nucleoskeletal component. We sought to determine if NUP-1 mediates heterochromatin organization and gene regulation at the nuclear periphery by examining the influence of NUP-1 knockdown on morphology, chromatin positioning, and transcription. We demonstrate that NUP-1 is essential and part of a stable network at the inner face of the trypanosome nuclear envelope, since knockdown cells have abnormally shaped nuclei with compromised structural integrity. NUP-1 knockdown also disrupts organization of nuclear pore complexes and chromosomes. Most significantly, we find that NUP-1 is required to maintain the silenced state of developmentally regulated genes at the nuclear periphery; NUP-1 knockdown results in highly specific mis-regulation of telomere-proximal silenced variant surface glycoprotein (VSG) expression sites and procyclin loci, indicating a disruption to normal chromatin organization essential to life-cycle progression. Further, NUP-1 depletion leads to increased VSG switching and therefore appears to have a role in control of antigenic variation. Thus, analogous to vertebrate lamins, NUP-1 is a major component of the nucleoskeleton with key roles in organization of the nuclear periphery, heterochromatin, and epigenetic control of developmentally regulated loci.

Heterotopic ossification in patients with hip osteoarthritis below forty years old after total hip replacement.

BACKGROUND: Even though heterotopic ossification (HO) is most often asymptomatic in patients after total hip replacement (THR), it remains a serious problem in orthopaedics as it is observed in nearly all operated patients. MATERIALS AND METHODS: The article presents a pre- and post-operative retrospective analysis of heterotopic ossification based on radiographic evidence. The study involved 77 patients below forty years old who underwent THR due to degenerative changes. Heterotopic ossification was evaluated using Brooker's basic four-grade scale. RESULTS: Heterotopic ossification was diagnosed more often in women. The study confirmed once again that non-steroidal anti-inflammatory drug (NSAID) prophylaxis reduces the risk of ossification. We propose that the hydroxyapatite coating of implants may also contribute to a higher incidence of heterotopic ossification in operated patients. CONCLUSIONS: A history of surgical procedures and a lack of NSAID prophylaxis contributed to heterotopic ossification in the region of the replaced hip joint in the study group.

The TIR domain of TIR-NB-LRR resistance proteins is a signaling domain involved in cell death induction.

In plants, the TIR (toll interleukin 1 receptor) domain is found almost exclusively in nucleotide-binding (NB) leucine-rich repeat resistance proteins and their truncated homologs, and has been proposed to play a signaling role during resistance responses mediated by TIR containing R proteins. Transient expression in Nicotiana benthamiana leaves of "TIR + 80", the RPS4 truncation without the NB-ARC domain, leads to EDS1-, SGT1-, and HSP90-dependent cell death. Transgenic Arabidopsis plants expressing the RPS4 TIR+80 from either dexamethasone or estradiol-inducible promoters display inducer-dependent cell death. Cell death is also elicited by transient expression of similarly truncated constructs from two other R proteins, RPP1A and At4g19530, but is not elicited by similar constructs representing RPP2A and RPP2B proteins. Site-directed mutagenesis of the RPS4 TIR domain identified many loss-of-function mutations but also revealed several gain-of function substitutions. Lack of cell death induction by the E160A substitution suggests that amino acids outside of the TIR domain contribute to cell death signaling in addition to the TIR domain itself. This is consistent with previous observations that the TIR domain itself is insufficient to induce cell death upon transient expression.

[Early clinical and radiological results following cervical disc arthroplasty]. / Wczesne wyniki kliniczne i radiologiczne protezoplastyki krazka miedzykregowego w odcinku szyjnym.

The aim of this study was to assess early results of anterior cervical decompression and arthroplasty for cervical disc disease. The results of operations of 32 patients (43 instrumented levels) were examined to assess clinical outcome, the restoration of disc height, postoperative lordosis, mobility of instrumented segment and complication rate. Clinical outcome evaluated with VAS score improved from 7.5 preoperatively to 2.7 post operation. There was improvement in disc height measured by Tureyen method of 48%, 5 degree restoration of lordosis per segment and average 5 degree mobility of operated segment. In two cases spontaneous fusion occurred. There were no loosening of prosthesis observed. Overall the study shows that early results of arthroplasty are clinically efficient and safe as fusion. Assessment of biomechanical advantages of arthroplasty demands further observation to compare if long-term results are superior to fusion.

The Arabidopsis thaliana TIR-NB-LRR R-protein, RPP1A; protein localization and constitutive activation of defence by truncated alleles in tobacco and Arabidopsis.

Specific recognition of Hyaloperonospora parasitica isolate Cala2 by Arabidopsis thaliana Ws-0 is mediated by the resistance gene RPP1A. Transient expression of different truncations of RPP1A in tobacco leaves revealed that its TIR-NB-ARC portion is sufficient to induce an elicitor-independent cell death. In stable transgenic lines of Arabidopsis, overexpression of the RPP1A TIR-NB-ARC domains (E12) using the 35S promoter leads to broad-spectrum resistance to virulent strains of H. parasitica and Pseudomonas syringae DC3000. The TIR-NB-ARC-mediated constitutive immunity is due to activation of the salicylic acid-dependent resistance pathway and is relieved by either a mutation in EDS1 or the presence of the salicylate hydroxylase gene, NahG. Growth of 35S::E12 plants is reduced, a phenotype observed in many constitutively resistant mutants. RPP1A carries a hydrophobic peptide at its N-terminus that directs the RPP1A protein into membranes, though it may not be the sole determinant mediating membrane association of RPP1A. Two-phase partitioning and sucrose density gradient sedimentation established that RPP1A resides in the endoplasmic reticulum and/or Golgi apparatus.

Expression of RPS4 in tobacco induces an AvrRps4-independent HR that requires EDS1, SGT1 and HSP90.

The Arabidopsis RPS4 gene belongs to the Toll/interleukin-1 receptor/nucleotide-binding site/leucine-rich repeat (TIR-NB-LRR) class of plant resistance (R) genes. It confers resistance to Pseudomonas syringae carrying the avirulence gene avrRps4. Transient expression of genomic RPS4 driven by the 35S promoter in tobacco leaves induces an AvrRps4-independent hypersensitive response (HR). The same phenotype is seen after expression of a full-length RPS4 cDNA. This indicates that alternative splicing of RPS4 is not involved in this HR. The extent of HR is correlated with RPS4 protein levels. Deletion analyses of RPS4 domains show the TIR domain is required for the HR phenotype. Mutations in the P-loop motif of the NB domain abolish the HR. Using virus-induced gene silencing, we found that the cell death resulting from RPS4 expression is dependent on the three plant signalling components EDS1, SGT1 and HSP90. All these data suggest that heterologous expression of an R gene can result in activation of cell death even in the absence of its cognate avirulence product, and provides a system for studying the RPS4 domains required for HR.

Low sequence similarity and gene content of symbiotic clusters of Bradyrhizobium sp. WM9 (Lupinus) indicate early divergence of "lupin" lineage in the genus Bradyrhizobium.

Two sequenced nodulation regions of lupin Bradyrhizobium sp. WM9 carried the majority of genes involved in the Nod factor production. The nod region I harbored: nolA, nodD, nodA, nodB, nodC, nodS, nodI, nodJ, nolO, nodZ, fixR, nifA, fixA, nodM, nolK and noeL. This gene arrangement resembled that found in the nodulation region of Bradyrhizobium japonicum USDA110, however strain WM9 harbored only one nodD gene copy, while the nodM, nolK and noeL genes had no counterparts in the 410 kb symbiotic region of strain USDA110. Region II harbored nolL and nodW, but lacked an nodV gene. Both regions carried ORFs that lacked similarity to the published USDA110 sequences, though they had homologues in symbiotic regions of Rhizobium etli, Sinorhizobium sp. NGR234 and Mesorhizobium loti. These differences in gene content, as well as a low average sequence identity (70%) of symbiotic genes with respect to B. japonicum USDA110 were in contrast with the phylogenetic relationship of USDA110 and WM9 revealed by the analysis of 16S rDNA and dnaK sequences. This most likely reflected an early divergence of symbiotic loci, and possible co-speciation with distinct legumes. During this process the loss of a noel gene and the acquisition of a nolL gene could be regarded as an adaptation towards these legumes that responded to Nod factors carrying 4-O-acetylfucose rather than 2-O-methylfucose. This explained various responses of lupins and serradella plants to infection by mutants in nodZ and nolL genes, knowing that serradella is a stringent legume while lupins are more promiscuous legumes.

Yellow lupine gene encoding stearoyl-ACP desaturase--organization, expression and potential application.

A gene for the delta9 desaturase specific to stearoyl-ACP (acyl carrier protein) was identified from yellow lupine (Lupinus luteus) cDNA and genomic libraries through the differential display method. The desaturase transcript appears in plants infected with Bradyrhizobium sp. (Lupinus) as revealed by Northern hybridization, RT-PCR and expression of beta-glucuronidase under the desaturase promoter. A small amount of desaturase transcript was also detected in uninfected plants, which suggests that the gene does not belong to the strict nodule-specific sequences. The desaturase provides unsaturated fatty acids for additional cell membrane synthesis. During nodule and symbiosome development a peribacteroid membrane is formed and the requirement for membrane surface increases, thus the level of desaturase expression is also higher. Transgenic plants of Nicotiana tabacum with overexpression of the full-length lupine stearoyl-ACP desaturase sequence were obtained. They revealed higher content of unsaturated fatty acids (especially oleic acid) in comparison with control plants.