Gene fusion

As an approach to the study of mammalian gene expression, the promoters and translation initiation regions of the rat preproinsulin II and the simian virus 40 early genes were fused to the structural gene of Escherichia coli 3-galactosidase, a sensitive probe for gene expression. These fusions were introduced into COS-7 cells, a simian virus 40 large tumor-antigen-producing monkey kidney cell line, where they directed the synthesis of enzymatically active hybrid 3-galactosidase proteins. Conditions for transfection were varied to optimize the expression of 13-galactosidase activity in the transfected cells. The pH optimum of this activity was found to be 7.0, the same as that of native E. coli fgalactosidase and distinct from the major lysosomal "acid" fl-galactosidase. The fused preproinsulin-fl-galactosidase was further characterized by gel electrophoresis of nondenatured cell extracts stained by a fluorogenic substrate and by immunoprecipitation and gel electrophoresis of 3H-labeled cell proteins. These results all indicate that fully active tetrameric P-galactosidase hybrids can be produced in mammalian cells. The expression of preproinsulin-,B-galactosidase activity was measured in the presence of high glucose, insulin, dexamethasone, or epidermal growth factor but no regulatory changes were observed. Gene fusions utilizing the Escherichia coli lacZ structural gene provide an especially suitable method for studying gene expression and regulation (1). Its product, f3-galactosidase, can be as

We report the display of human ciliary neurotrophic factor (hCNTF), a survival factor for neuronal cells belonging to the ~-helical cytokine superfamily, on the surface of the filamentous bacteriophage fd. The hCNTF cDNA was fused to a DNA sequence encoding the C-terminal domain of pIII, a minor coat protein exposed at one end of fd. Gene fusions were cloned into a plasmid containing the ColE1 plasmid and fd origins of replication, and were packaged into phagemid particles upon superinfection with M13KO7 helper phage. The resulting fusion phage bound specifically to anti-CNTF antibodies and to the recombinant soluble CNTF s-receptor. Moreover, phage-displayed hCNTF was found to possess biological activity at concentrations comparable to those of the soluble cytokine. These results demonstrate that CNTF can be displayed on phage in a correctly folded and functionally active form. Binding of fusion phage to immobilized CNTF ~-receptor and subsequent elution at low pH resulted in affinity purification of CNTF-displaying virions. Utilization of this technology should enable the selection of high-affinity variants from libraries of CNTF mutants displayed on phage.

Both light itself and excitation pressure have been implicated as the environmental signal that stimulates interchange of the two forms of the D1 protein of photosystem II (PS II) in Synechococcus sp. strain PCC 7942. We sought an explanation for conflicting reports regarding the role of photosynthetic electron transport in regulation of psbA expression and D1 interchange. Inhibitors that block at different points in the photosynthetic electron transport chain were administered and the effect on expression of psbAII, which encodes the high-light-induced form II of D1, was examined by measuring changes in transcript levels and in the activities of reporter enzymes. Both 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), an inhibitor of PSII, and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), an inhibitor of the cytochrome b6/f complex, prevented high-light-induced increase in β-galactosidase activity from a psbAII::lacZ gene fusion when added at a concentration that completely inhibits photosynthetic electron transport (1 μM). The same effect was observed for luciferase activity from transcriptional and translational fusions of psbAII to the luxAB genes from Vibrio harveyi. DCMU (1 μM) arrested luciferase expression at low-light levels – thus eliminating the high light response – whereas a sublethal concentration (50 nM), which reduces electron transport by 50%, had intermediate effects on psbAII-driven luciferase activity. However, psbAII transcript levels, monitored by northern blot analysis, were not altered by electron transport inhibitors, either at low-light intensity or following a high-light exposure. The suppressive effect of DCMU on expression of reporter enzymes was not restricted to the high-light response of psbAII-driven reporter systems, but was also observed using an isopropyl-(-d)-thiogalactopyranoside (IPTG)-inducible trc promoter fused to luxAB. This construct only marginally responded to IPTG addition when DCMU was present. Thus, blocking electron transport in Synechococcus affects the translation machinery in a general way, and the use of electron transport inhibitors is of limited value when focusing on specific redox regulation of D1 protein synthesis or degradation.

The chemokine receptor CXCR4 belongs to the large superfamily of G-protein-coupled receptors, and is directly involved in a number of biological processes including organogenesis, hematopoiesis, and immunity. Recent evidence has highlighted the role of CXCR4 in a variety of diseases including cancer and WHIM syndrome. Expression of CXCR4 in cancer metastasis appears to be due to dysregulation of the receptor leading to enhanced signaling. CXCR4 was also found to be a prognostic marker in various types of cancer including leukemia and breast cancer. These observations reveal that CXCR4 is an important molecule involved in several aspects of cancer progression. The SDF-1-CXCR4 axis is also involved in normal stem cell homing. Interestingly, cancer stem cells also express CXCR4 suggesting that the SDF-1-CXCR4 axis directs their trafficking/metastasis to organs that highly express SDF-1 such as the lymph nodes, lungs, liver, and bones. Here, we review what is currently known regarding the regulation of CXCR4 and how dysregulation contributes to disease progression. The Chemokine Receptor CXCR4 and Cancer The human chemokine system currently includes more than 40 chemokines and 18 chemokine receptors (Table 1). Chemokine receptors are defined by their ability to induce directional migration of cells toward a gradient of a chemotactic cytokine (chemotaxis). Chemokine receptors belong to a family of 7 transmembrane domain, G-protein-coupled cell surface receptors (GPCR) and the ligands are classified into four groups (CXC, CC, C, and CX 3 C) based on the position of the first two cysteines [1, 2]. Chemokine receptors are present on many different cell types. Initially, these receptors were identified on

Glycosyl hydrolases hydrolyze the glycosidic bond either in carbohydrates or between carbohydrate and non-carbohydrate moiety. The β-glucuronidase (beta D-glucuronoside glucuronosohydrolase; EC 3.2.1.31) enzyme belongs to the family-2 glycosyl hydrolase. The E. coli borne β-glucuronidase gene (uidA) was devised as a gene fusion marker in plant genetic transformation experiments. Recent plant transformation vectors contain a novel β-glucuronidase (gusA) derived from Staphylococcus sp. RLH1 for E. coli uidA. It is known to have a ten fold higher sensitivity compared to E. coli β-glucuronidase. The functional superiority of Staphylococcus (gusA) over E. coli (uidA) activity is not fully known. The comparison of secondary structural elements among them revealed an increased percentage of random coils in Staphylococcus β-glucuronidase. The 3D model of gusA shows catalytic site residues 396Glu, 508Glu and 471Tyr of gusA in loop regions. Accessible surface area (ASA) calculations on the 3D model showed increased ASA for active site residues in Staphylococcus β-glucuronidase. Increased random coil, the presence of catalytic residues in loops, greater solvent accessibility of active residues and increased charged residues in gusA of Staphylococcus might facilitate interaction with the solvent. This hypothesizes the enhanced catalytic activity of β-glucuronidase in Staphylococcus sp. RLH1 compared to that in E. coli.

We previously demonstrated that high glucose activates angiotensinogen (ANG) expression and that insulin inhibits this activation. The present studies aim to investigate whether insulin regulates ANG gene expression in kidney proximal tubular cells at the transcription level via interaction of the putative insulin-response element (IRE) with its binding protein(s) in the 5'-flanking region of the ANG gene. Fusion genes containing various lengths of the 5'-flanking region of the rat ANG gene fused to a human GH (hGH) gene as reporter were constructed and transiently introduced into rat immortalized renal proximal tubular cells (IRPTCs). The expression of the fusion genes was monitored by the amount of immunoreactive hGH secreted into the medium as assayed by a specific RIA for hGH. Insulin inhibited the expression of pOGH (rANG N-1498/+18), pOGH (rANG N-1120/+18) and pOGH (rANG N-882/+18) but not pOGH (rANG N-854/+18), pOGH (rANG N-820/+18), pOGH (rANG N-688/+18) and pOGH (rANG N-53/+18) in high-glucose (i.e. 25 mM) medium. Site-directed mutagenesis of nucleotides N-874 to N-867 (5' CCC GCC CT 3') in the 5'-flanking region of the rat ANG gene abolished the response to insulin. Insulin also inhibited the expression of the fusion gene containing the DNA fragment ANG N-882 to N-855 inserted upstream of the ANG gene promoter (N-53/+18), but had no effect on a mutant of N-882 to N-855. Gel mobility shift assays revealed that the labeled putative rat ANG-IRE motif (N-878 to N-864, 5' CCT TCC CGC CCT TCA 3') was bound to the nuclear proteins of IRPTCS: This binding was displaced by unlabeled ANG-IRE and IRE of human glyceraldehyde phosphate dehydrogenase but not by mutants of ANG-IRE and IRE of the rat glucagon gene. Southwestern blotting analysis revealed that the labeled putative ANG-IRE motif bound to a major nuclear protein with an apparent molecular mass of 48 kDA: Finally, high glucose levels enhanced 48-kDa nuclear protein expression and induced an additional 70-kDa nuclear protein expression in IRPTCs, as revealed by Southwestern blotting. Insulin inhibited both 48- and 70-kDa nuclear proteins expression induced by high glucose levels. Its inhibitory effect was reversed by the presence of PD98059 (an inhibitor of mitogen-activated protein kinase, MAPK) but not by wortmannin (an inhibitor of phosphatidylinositol 3- kinase). These studies demonstrate that insulin action on ANG gene expression is at the transcriptional level. The molecular mechanism (s) of insulin action is mediated, at least in part, via interaction of the functional IRE with unidentified 48- and 70- kDa nuclear proteins in the rat ANG gene and is MAPK dependent.

Benign fibrous histiocytoma (BFH) is a mesenchymal tumor that most often occurs in the skin (socalled dermatofibroma), but may also appear in soft tissues (so-called deep BFH) and in the skeleton (so-called non-ossifying fibroma). The origin of BFH is unknown, and it has been questioned whether it is a true neoplasm. Chromosome banding, fluorescence in situ hybridization, single nucleotide polymorphism arrays, RNA sequencing, RT-PCR and quantitative real-time PCR were used to search for recurrent somatic mutations in a series of BFH. BFHs were found to harbor recurrent fusions of genes encoding membrane-associated proteins (podoplanin, CD63 and LAMTOR1) with genes encoding protein kinase C (PKC) isoforms PRKCB and PRKCD. PKCs are serine-threonine kinases that through their many phosphorylation targets are implicated in a variety of cellular processes, as well as tumor development. When inactive, the amino-terminal, regulatory domain of PKCs suppresses the activity of their catalytic domain. Upon activation, which requires several steps, they typically translocate to cell membranes, where they interact with different signaling pathways. The detected PDPN-PRKCB, CD63-PRKCD and LAMTOR1-PRKCD gene fusions are all predicted to result in chimeric proteins consisting of the membrane-binding part of PDPN, CD63 or LAMTOR1 and the entire catalytic domain of the PKC. This novel pathogenetic mechanism should result in constitutive kinase activity at an ectopic location. The results show that BFH indeed is a true neoplasm, and that distorted PKC activity is essential for tumorigenesis. The findings also provide means to differentiate BFH from other skin and soft tissue tumors. This article is part of a Directed Issue entitled: Rare cancers.

A new bacterial test system for detection of genotoxic compounds was developed, based on two new Salmonella typhimurium tester strains, TGO1 and TGO2. Both strains contain a gene fusion between a strong SOS-promotor, P cda , and the gfp gene, which allows detection of genotoxic compounds that induce the SOS response. SOS induction was detected by means of flow cytometry. TGO1 showed an increased sensitivity to N-methyl-N -nitro-N-nitrosoguanidine compared with a previously developed strain, which had an Escherichia coli strain as host instead of S. typhimurium.

The bush dog (Speothos venaticus) is a South American canid, included in the IBAMA (Brazilian Institute of Environment and Renewable Natural Resources) official list of animals threatened with extinction, in the vulnerable category. As a preservation and conservation strategy, specimens kept in captivity by Brazilian Institutions are monitored by a management plan. In order to characterize and analyze the genetic variability of bush dog specimens, a cytogenetic analysis was carried out, and microsatellite data were also obtained through the use of 15 primers, originally developed for the domestic dog (Canis familiaris). All tested primers showed transferability and amplified fragment sizes similar to those described for the canine genome. From the total number of primers, eight were tested, and presented two polymorphic regions. Regarding cytogenetic analysis, one of the animals had chromosomal mosaicism, disqualifying it as a reproducer to form stocks. Thus, we concluded that the genetic evaluation of wild animals kept in captivity provides data that can help with the practice of exchange between different institutions, avoiding problems in the reproductive capacity of the breeding stock.

The demands of structural and functional genomics for large quantities of soluble, properly folded proteins in heterologous hosts have been aided by advancements in the Weld of protein production and puriWcation. Escherichia coli, the preferred host for recombinant protein expression, presents many challenges which must be surmounted in order to over-express heterologous proteins. These challenges include the proteolytic degradation of target proteins, protein misfolding, poor solubility, and the necessity for good puri-Wcation methodologies. Gene fusion technologies have been able to improve heterologous expression by overcoming many of these challenges. The ability of gene fusions to improve expression, solubility, puriWcation, and decrease proteolytic degradation will be discussed in this review. The main disadvantage, cleaving the protein fusion, will also be addressed. Focus will be given to the newly described SUMO fusion system and the improvements that this technology has advanced over traditional gene fusion systems.  2005 Published by Elsevier Inc.

Lead is a non-essential element that exhibits a high degree of toxicity, especially in children. Most research on lead has focused on its effects on organ systems such as the nervous system, the red blood cells, and the kidneys which are considered to be the primary targets of lead toxicity. However, the molecular mechanisms by which it induces toxicity, and carcinogenesis remain to be elucidated. In this research, we performed the MTT assay to assess the cytotoxicity, and the CAT-Tox assay to assess the transcriptional responses associated with lead exposure to thirteen different recombinant cell lines generated from human liver carcinoma cells (HepG2), by creating stable transfectants of mammalian promoter chloramphenicol (CAT) gene fusions. Study results indicated that lead nitrate is cytotoxic to HepG2 cells, showing LD50 values of 49.0 ± 18.0 mgr;g/mL, 37.5 ± 9.2 μg/mL, and 3.5 ± 0.7 μg/mL for cell mortality upon 24, 48 and 72 h of exposure, respectively; indicating a dose- and time-dependent response with regard to the cytotoxic effect of lead nitrate. A dose-response relationship was also recorded with respect to the induction of stress genes in HepG2 cells exposed to lead nitrate. Overall, six out of the thirteen recombinant cell lines tested showed inductions to statistically significant levels (p < 0.05). At 50 μg/mL of lead nitrate, the average fold inductions were: 2.1 ± 1.0, 5.4 ± 0.4, 12.1 ± 6.2, 5.0 ± 1.7, 2.5 ± 1.3, and 4.8 ± 4.5 for XRE, HSP70, CRE, GADD153, and GRP78, respectively. These results indicate the potential for lead nitrate to undergo biotransformation in the liver (XRE), to cause cell proliferation (c-fos), protein damage (HSP70, GRP78), metabolic perturbation (CRE), and growth arrest and DNA damage (GADD153). Marginal but not significant inductions were also obtained with the GSTYa (1.5 ± 0.8), and GADD45 (5.7 ± 8.1) promoters, and the NF-κB (2.0 ± 1.7) response element, indicating the potential for oxidative stress. No significant inductions (p > 0.05) were recorded for CYP1A1, HMTIIA, p53RE, and RARE.

Inoculation of plants with beneficial microbes can be traced back for centuries. Although bacteria were not proven to exist until in 1683 von Leeuwenhoek discovered microscopic ‘animals’ under the lens of his microscope, their utilisation to stimulate plant growth in agriculture has been exploited since ancient times. Theophrastus (372-287 BC) suggested the mixing of different soils as a means of ‘remedying defects and adding heart to the soil’ (Vessey 2003). Colonisation of plant root system is the very first step in nearly all interactions between plants and soil borne microbes. The importance of this compartment for plant growth and soil microbiology had already been realised in the very pioneer times of microbiology in the late 19th century. In 1888, Hellriegel and Wilfarth proved the special case of nitrogen nutrition of legumes through their root nodule bacteria, which Beijerinck finally isolated in 1889. In 1887–1888 Hiltner was certainly fascinated by the findings of Hellriegel and Wilfarth about the special case of nitrogen nutrition in legumes. Together with Professor Nobbe, intensive studies were conducted about the nature of the symbiotic interaction of nodule bacteria and legume roots (Nobbe and Hiltner 1896). Apparently during these studies, Hiltner became aware of the importance of the ecological interactions in the root zone. Together with his teacher he developed the first inoculant based on root nodule bacteria for agricultural practice which they called ‘Nitragin’ in 1890. As agricultural practice was in close contact to the new achievements of basic sciences, the challenge quickly arose whether at all, and how, these discoveries of the — at that time — very young science ‘soil bacteriology’ could be applied in the field. Region of contact between root and soil where soil is affected by roots was designated as “rhizosphere” by Hiltner (1904).

The genetic modification of Coffea arabica fruits is an important tool for the investigation of physiological characteristics and functional validation of genes related to coffee bean quality traits. In this work, plants of C. arabica cultivar Catuaí Vermelho were successfully genetically modified by bombardment of embryogenic calli. Calli were obtained from 90% of the leaf explants cultivated in a callogenesis-inducing medium modified with 20 μM 2, 4-dichlorophenoxyacetic acid (2,4-D). The resulting calli were bombarded with the pBI426 vector containing a uidA and nptII gene fusion that was driven by the double CaMV35s promoter. Kanamycin-selected embryos were positive for β-glucuronidase (GUS) activity in histochemical assays and for target gene amplification by polymerase chain reaction. Integration of the nptII gene was confirmed by Southern blot and showed a low copy number (one to three) of insertions. Transformed plants showed normal development and settled fruits. GUS expression was assessed in the flower and fruit organs demonstrating the capacity of the double CaMV35s promoter to drive long-term stable expression of uidA in C. arabica fruit tissues. Moreover, we obtained a T 1 progeny presenting 3:1 Mendelian segregation of the uidA gene. This investigation is the first to report exogenous gene expression in coffee fruits and transgenic inheritance in C. arabica plants.

A key step in mitosis is the sister-chromatid separation at the metaphase-anaphase (M/A) transition. Several earlier studies had suggested that Ca 2þ signal is involved in regulating this process in somatic cells. The detailed mechanisms, however, are not yet well understood. In this study, we used the GFP-gene fusion method and a living-cell imaging technique to examine the effects of suppressing cytosolic Ca 2þ level on the mitotic process in HeLa and PtK2 cells. We observed that application of the Ca 2þ chelator BAPTA/AM can block or severely delay the M/A transition. This blockage was caused by a failure in activating the anaphasepromoting complex (APC), since both cyclin B and securin could not be degraded under this situation. Furthermore, using YFPlabeled tubulin, we found that the mitotic spindle structure in most of the BAPTA-treated cells gradually deformed with time. Other Ca 2þ signal blockers, such as heparin, also produced a similar effect. These results suggest that one pathway for the blockage of M/A transition by suppressing cytosolic Ca 2þ level is due to its interference with the mitotic spindle checkpoint.

Background: TMPRSS2-ERG gene fusions occur in about 50% of all prostate cancer cases and represent promising markers for molecular subtyping. Although TMPRSS2-ERG fusion seems to be a critical event in prostate cancer, the precise functional role in cancer development and progression is still unclear.

Mini-transposon is the generic name given to the members of a collection of genetic assets derived from transposons Tn10 and Tn5, in which the naturally occurring functional segments of DNA have been rearranged artificially to originate shorter mobile elements. In the most widespread design (that known as the pUT system), any heterologous DNA segment can be conveniently cloned within the boundaries of a mini-Tn5 vector and finally inserted into the chromosome of target Gramnegative bacteria after a few simple genetic manipulations. The large variety of antibiotic, non-antibiotic and excisable selection markers available has been combined at ease with DNA fragments encoding one or more phenotypes of interest for ecological or biotechnological applications. These include the tagging of specific strains in a community with selectable and/or optical marker genes, the production of stable gene fusions for monitoring transcriptional regulation in single cells, the metabolic engineering of strains destined for bioremediation, the non-disruptive monitoring of gene transfer and the assembly of gene containment and strain containment circuits for genetically manipulated microorganisms. z

The presence of the TMPRSS2-ERG fusion gene in prostate tumors has recently been associated with an aggressive phenotype, as well as recurrence and death from prostate cancer. These associations suggest the hypothesis that the gene fusion may be used as a prognostic indicator for prostate cancer.

The Escherichia coli cytoplasmic protein thioredoxin 1 can be efficiently exported to the periplasmic space by the signal sequence of the DsbA protein (DsbAss) but not by the signal sequence of alkaline phosphatase (PhoA) or maltose binding protein (MBP). Using mutations of the signal recognition particle (SRP) pathway, we found that DsbAss directs thioredoxin 1 to the SRP export pathway. When DsbAss is fused to MBP, MBP also is directed to the SRP pathway. We show directly that the DsbAss-promoted export of MBP is largely cotranslational, in contrast to the mode of MBP export when the native signal sequence is utilized. However, both the export of thioredoxin 1 by DsbAss and the export of DsbA itself are quite sensitive to even the slight inhibition of SecA. These results suggest that SecA may be essential for both the slow posttranslational pathway and the SRP-dependent cotranslational pathway. Finally, probably because of its rapid folding in the cytoplasm, thioredoxin provides, ...

Background: Here we present the first paired-end sequencing of tumors from genetically engineered mouse models of cancer to determine how faithfully these models recapitulate the landscape of somatic rearrangements found in human tumors. These were models of Trp53-mutated breast cancer, Brca1-and Brca2-associated hereditary breast cancer, and E-cadherin (Cdh1) mutated lobular breast cancer.

All known P450-containing monooxygenase systems share common structural and functional domain architecture. Apart from P450 itself, these systems can comprise several fundamentally different protein components or domains, all of which are shared by other multicomponent/multidomain enzyme systems with various functions: FAD flavoprotein or domain, FMN domain, Fe2S2 ferredoxin, Fe3S4 ferredoxin, and cytochrome b5. Either FMN domain, ferredoxins or cytochrome bs serve as the electron transport intermediate between the FAD domain and P450. The molecular evolution of both P450-containing systems and of each particular component does not follow phylogeny in general. Gene fusion and horizontal gene transfer events can lead to the appearance of novel redox chains in the same manner that artificial chimeric proteins can be constructed by humans. Recent studies using genetic and protein engineering techniques to investigate the separate domains and their interaction are described.

Pyruvate carboxylase (PYC) is a biotin-dependent enzyme catalyzing the anaplerotic conversion of pyruvate to oxaloacetate in Rhizobium etli strain CE3. A pyc::Tn5 mutant had severely reduced growth, or failed to grow on sugars, three-carbon organic acids or glycerol, consistent with these substrates being metabolized via pyruvate. Transconjugants expressing a pyc::β-glucuronidase gene fusion had slightly increased apparent pyc transcription during growth on pyruvate as compared to succinate, similar to the modest carbon source dependent changes in PYC activity reported previously. Biotin supplementation of cultures growing on pyruvate dramatically increased PYC activity but not apparent pyc transcription. Bacteroids isolated from bean nodules did not contain detectable PYC activity while apparent pyc transcription occurred at a moderate level.

Spitz tumors represent a group of melanocytic neoplasms that typically affect young individuals. Microscopically, the lesions are composed of cytologically distinct spindle and epithelioid melanocytes, with a range in the architectural display or the cells, their nuclear features, and secondary epidermal or stromal changes. Recently, kinase fusions have been documented in a subset of Spitz tumors, but there is limited information on the clinical and pathologic features associated with those lesions. Here, we report a series of 17 patients (9 male, 8 female) with spitzoid neoplasms showing ALK fusions (5 Spitz nevi and 12 atypical Spitz tumors). The patients' ages ranged from 2 years to 35 years (mean = 17 y; median = 16 y). Most lesions were located on the lower extremities and presented clinically as polypoid nodules. All tumors were compound melanocytic proliferations with a predominant intradermal growth. Tumor thickness ranged from 1.1 to 6 mm (mean = 2.9 mm; median = 2.5 mm). The most characteristic histopathologic feature of the tumors (seen in all but 2 lesions) was a plexiform dermal growth of intersecting fascicles of fusiform melanocytes. All but 2 tumors were amelanotic. All tumors were strongly immunoreactive for ALK. The ALK rearrangements were confirmed in all cases by fluorescence in situ hybridization (FISH), and the fusion partner was determined by quantitative polymerase chain reaction as TPM3 (tropomyosin 3) in 11 cases and DCTN1 (dynactin 1) in 6 cases. None of the 8 tumors that were analyzed by FISH for copy number changes of 6p, 6q, 9p, or 11q met criteria for melanoma. Two patients underwent a sentinel lymph node biopsy, and in both cases melanocyte nests were found in the subcapsular sinus of the node. Array comparative genomic hybridization of these 2 tumors revealed no chromosomal gains or losses. In conclusion, our study revealed that Spitz nevi/tumors with ALK rearrangement show a characteristic plexiform morphology and that ALK im-munohistochemistry and FISH enable the accurate identification of this morphologic and genetic distinct subset of spitzoid neoplasms.

Lung cancer is emerging as a paradigm for disease molecular subtyping, facilitating targeted therapy based on driving somatic alterations. Here we perform transcriptome analysis of 153 samples representing lung adenocarcinomas, squamous cell carcinomas, large cell lung cancer, adenoid cystic carcinomas and cell lines. By integrating our data with The Cancer Genome Atlas and published sources, we analyse 753 lung cancer samples for gene fusions and other transcriptomic alterations. We show that higher numbers of gene fusions is an independent prognostic factor for poor survival in lung cancer. Our analysis confirms the recently reported CD74-NRG1 fusion and suggests that NRG1, NF1 and Hippo pathway fusions may play important roles in tumours without known driver mutations. In addition, we observe exon-skipping events in c-MET, which are attributable to splice site mutations. These classes of genetic aberrations may play a significant role in the genesis of lung cancers lacking known ...

Morphological, cytogenetic, and biological evidence supports a relationship between congenital (infantile) fibrosarcoma (CFS) and congenital mesoblastic nephroma (CMN). These tumors have a very similar histological appearance, and they are both associated with polysomies for chromosomes 8, 11, 17, and 20. Recently, CFS was shown to contain a novel t(12; 15)(p13;q25) translocation resulting in ETV6-NTRK3 gene fusion. The aims of this study were to determine whether congenital mesoblastic nephroma contains the t(12;15)(p13;q25) translocation and ETV6-NTRK3 gene fusion and whether ETV6-NTRK3 fusions, in CMN and CFS, antedate acquisition of nonrandom chromosome polysomies. To address these aims, we evaluated 1) ETV6-NTRK3 fusion transcripts by reverse transcriptase polymerase chain reaction and sequence analysis, 2) genomic ETV6-region chromosomal rearrangement by fluorescence in situ hybridization, and 3) chromosomal polysomies by karyotyping and fluorescence in situ hybridization. We report ETV6-NTRK3 fusion transcripts and/or ETV6-region rearrangement in five of six CMNs and in five of five CFSs. The ETV6-NTRK3 fusion transcripts and/or ETV-region chromosome rearrangements were demonstrated in two CMNs and one CFS that lacked chromosome polysomies. These findings demonstrate that t(12;15) translocation, and the associated ETV6-NTRK3 fusion, can antedate acquisition of chromosome polysomies in CMN and CFS. CMN and CFS are pathogenetically related, and it is likely that they represent a single neoplastic entity, arising in either renal or soft tissue locations.

Promoter::gene fusions which differed only in the presumed polyadenylation signals attached to the 3' end of the gene, have been used to examine the effect of these signals on expression of the gene in protoplasts and in transgenic plants. The gene constructs were comprised of the 35S promoter, the chloramphenicol acetyl transferase (CAT) gene and DNA sequences carrying the presumed polyadenylation signals of the nopaline synthase (nos) gene or of transcript 7 (T-7) from the T-DNA of Agrobacterium. Our results show that levels of gene expression were not significantly affected by the orientation or absence of these sequences. We therefore suggest that the practice of cloning presumptive polyadenylation sequences at the 3' end of a gene for expression in plants may be unnecessary.

Inoculation of plants with beneficial microbes can be traced back for centuries. Although bacteria were not proven to exist until in 1683 von Leeuwenhoek discovered microscopic ‘animals’ under the lens of his microscope, their utilisation to stimulate plant growth in agriculture has been exploited since ancient times. Theophrastus (372-287 BC) suggested the mixing of different soils as a means of ‘remedying defects and adding heart to the soil’ (Vessey 2003). Colonisation of plant root system is the very first step in nearly all interactions between plants and soil borne microbes. The importance of this compartment for plant growth and soil microbiology had already been realised in the very pioneer times of microbiology in the late 19th century. In 1888, Hellriegel and Wilfarth proved the special case of nitrogen nutrition of legumes through their root nodule bacteria, which Beijerinck finally isolated in 1889. In 1887–1888 Hiltner was certainly fascinated by the findings of Hellriegel and Wilfarth about the special case of nitrogen nutrition in legumes. Together with Professor Nobbe, intensive studies were conducted about the nature of the symbiotic interaction of nodule bacteria and legume roots (Nobbe and Hiltner 1896). Apparently during these studies, Hiltner became aware of the importance of the ecological interactions in the root zone. Together with his teacher he developed the first inoculant based on root nodule bacteria for agricultural practice which they called ‘Nitragin’ in 1890. As agricultural practice was in close contact to the new achievements of basic sciences, the challenge quickly arose whether at all, and how, these discoveries of the — at that time — very young science ‘soil bacteriology’ could be applied in the field. Region of contact between root and soil where soil is affected by roots was designated as “rhizosphere” by Hiltner (1904).

The t(9;22)(q34;q11) translocation occurs in chronic myeloid leukemia (CML) and adult B-cell acute lymphoblastic leukemia (ALL), leading to fusion of BCR to ABL1 and constitutive activation of ABL1 tyrosine kinase activity. The main BCR-ABL1 breakpoints result in P190 BCR-ABL1 or P210 BCR-ABL1 fusion proteins. The latter is found in almost all cases of CML and in one third of the cases of t(9;22)-positive adult B-ALL. P190 BCR-ABL1 is found in the remaining two thirds of t(9;22)-positive adult B-ALL cases but only exceptionally in CML. We describe here the first case of t(9;22)(q34;q11) associated with t(10;11)(p13;q14) in acute monocytic leukemia. The recurrent t(10;11)(p13;q14) translocation, usually found in acute myeloid leukemia (AML) and T-ALL, merges PICALM to MLLT10. RT-PCR enabled identification of PICALM-MLLT10 and BCR-ABL1 e1-a2 fusion transcripts; in the context of chronic and acute myeloid leukemia, the latter usually has a monocytic presentation. We also identified ove...

Vibrio anguillarum, a fish pathogen, produces a 38 kDa major outer-membrane porin, which may be involved in environmental adaptation. The gene encoding the 38 kDa porin was cloned and deleted. The deduced protein sequence was 75 % identical to that of the major outer-membrane protein (OMP), OmpU, from Vibrio cholerae. LacZ expression from an ompU : : lacZ transcriptional gene fusion was increased 1?5-fold in the presence of bile salts and was decreased 50-to 100-fold in a toxR mutant compared to that in the wild-type, showing that ompU expression is positively regulated by ToxR and induced by bile salts. Similar to a toxR mutant, an ompU mutant showed a slight decrease in motility, an increased sensitivity to bile salts and a thicker biofilm with better surface area coverage compared to that of the wild-type. When ompU was expressed under a ToxRindependent promoter in the toxR mutant, the phenotypes for bile resistance and biofilm formation, but not motility were complemented to that of the wild-type. In rainbow trout, the ompU mutant showed wild-type virulence via immersion into infected seawater and intraperitoneal injection. The ompU mutant produced two colony morphologies: opaque, which did not grow at 0?2 % bile, and translucent, which grew at 2 % bile. The translucent ompU mutant strain produced a second major OMP that was induced by bile. All ompU mutants showed variations in the amount and length of smooth LPS. In V. anguillarum, OmpU is not required for virulence, possibly due to a second OMP also critical for resistance to bile; however, outside of the fish host, OmpU limits the progression of biofilm formation. 3Present address:

Two simple, rapid and efficient protocols for the regeneration of transformed tetraploid lines of alfalfa (Medicago falcata L.) have been developed and compared. Leaf explants from embryogenic lines 47/1-150 and 47/1-5 were inoculated with Agrobacterium tumefaciens containing constructs carrying the nptII selectable marker gene and promoter: gusA gene fusions under the control of the CaMV 35S or Arabidopsis cdc2a, CycB1 and CycA2 promoters. In the first regeneration system (the MSH system), inoculated leaf explants were incubated on MS medium supplemented with 2,4-D and kinetin and then subcultured onto plant growth regulator-free MS medium in order to induce direct somatic embryogenesis. In the second regeneration system (the B5h system), the inoculated explants were incubated on B5h medium to induce indirect production of somatic embryos via embryogenic callus. In both systems, an effective kanamycin selection regime was employed and was maintained when the embryos were subcultured onto a recovery medium (Boi2Y) to promote further embryo development. The use of Boi2Y medium was particularly important for shortening the regeneration time and promoting a higher frequency of healthy plantlet production from the somatic embryos. The mature somatic embryos were finally transferred to plant growth regulator-free MS medium for plantlet formation. Transgenic plantlets were produced within 10-14 weeks in the MSH system and 12-16 weeks in the B5h system. The MSH system appears to be the fastest transformation system reported for leguminous species to date. Confirmation of transformation was obtained using a re-callusing assay on kanamycin and subsequent Southern blot hybridisation and PCR analysis. The ability to induce expression of GUS activity in leaf explants containing the cell division cycle gene promoter:gusA constructs by 2,4-D treatment also proved to be a reliable indicator of transformation.

Motivation: Next generation sequencing technology allows the detection of genomic structural variations, novel genes and transcript isoforms from the analysis of high throughput data. In this work, we propose a new framework for the detection of fusion transcripts through short paired-end reads which integrates splicing-driven alignment and abundance estimation analysis, producing a more accurate set of reads supporting the junction discovery and taking into account also not annotated transcripts. Bellerophontes performs a selection of putative junctions on the basis of a match to an accurate gene fusion model. Results: We report the fusion genes discovered by the proposed framework on experimentally validated biological samples of Chronic Myelogenous Leukemia (CML) and on public NCBI datasets, for which Bellerophontes is able to detect the exact junction sequence. With respect to state-of-art approaches Bellerophontes detects the same experimentally validated fusions, however it is more selective on the total number of detected fusions and provides a more accurate set of spanning reads supporting the junctions. We finally report the fusions involving non-annotated transcripts found in CML samples.

The d-globin gene (HBD) of eutherian mammals exhibits a propensity for recombinational exchange with the closely linked b-globin gene (HBB) and has been independently converted by the HBB gene in multiple lineages. Here we report the presence of a chimeric b/d fusion gene in the African elephant (Loxodonta africana) that was created by unequal crossing-over between misaligned HBD and HBB paralogs. The recombinant chromosome that harbors the b/d fusion gene in elephants is structurally similar to the ''anti-Lepore'' duplication mutant of humans (the reciprocal exchange product of the hemoglobin Lepore deletion mutant). However, the situation in the African elephant is unique in that the chimeric b/d fusion gene supplanted the parental HBB gene and is therefore solely responsible for synthesizing the b-chain subunits of adult hemoglobin. A phylogenetic survey of b-like globin genes in afrotherian and xenarthran mammals revealed that the origin of the chimeric b/d fusion gene and the concomitant inactivation of the HBB gene predated the radiation of ''Paenungulata,'' a clade of afrotherian mammals that includes three orders: Proboscidea (elephants), Sirenia (dugongs and manatees), and Hyracoidea (hyraxes). The reduced fitness of the human Hb Lepore deletion mutant helps to explain why independently derived b/d fusion genes (which occur on an anti-Lepore chromosome) have been fixed in a number of mammalian lineages, whereas the reciprocal d/b fusion gene (which occurs on a Lepore chromosome) has yet to be documented in any nonhuman mammal. This illustrates how the evolutionary fates of chimeric fusion genes can be strongly influenced by their recombinational mode of origin.

Synovial sarcomas are high-grade malignant mesenchymal tumors that account for 10% of all softtissue sarcomas. Almost 95% of these tumors are characterized by a nonrandom chromosomal abnormality, t(X;18)(p11.2;q11.2), that is observed in both biphasic and monophasic variants. In this article, we present the case of a 57-year-old woman diagnosed with high-grade biphasic synovial sarcoma in which conventional cytogenetic analysis revealed the constant presence of a unique t(18;22)(q12;q13), in addition to trisomy 8. The rearrangement was confirmed by fluorescence in situ hybridization. The use of the whole chromosome painting probes WCPX did not detect any rearrangements involving chromosome X, although reverse-transcriptase polymerase chain reaction (PCR) analysis demonstrated the conspicuous presence of a SYT/SXX1 fusion gene. Spectral karyotyping (SKY) was also performed and revealed an insertion of material from chromosome 18 into one of the X chromosomes at position Xp11.2. Thus, the karyotype was subsequently interpreted as 47,X,der(X)ins(X;18) (p11.2;q11.2q11.2),der(18)del(18)(q11.2q11.2)t(18;22)(q12;q13),der(22)t(18;22). Real-time PCR analysis of BCL2 expression in the tumor sample showed a 433-fold increase. This rare finding exemplifies that thorough molecularecytogenetic analyses are required to elucidate complex and/or cryptic tumor-specific translocations. Ó

We have developed a versatile Escherichia coli expression system based on the use of E. coli thioredoxin (trxA) as a gene fusion partner. The broad utility of the system is illustrated by the production of a variety of mammalian cytokines and growth factors as thioredoxin fusion proteins. Although many of these cytokines previously have been produced in E. coli as insoluble aggregates or &amp;quot;inclusion bodies&amp;quot;, we show here that as thioredoxin fusions they can be made in soluble forms that are biologically active. In general we find that linkage to thioredoxin dramatically increases the solubility of heterologous proteins synthesized in the E. coli cytoplasm, and that thioredoxin fusion proteins usually accumulate to high levels. Two additional properties of E. coli thioredoxin, its ability to be specifically released from the E. coli cytoplasm by osmotic shock or freeze/thaw treatments and its intrinsic thermal stability, are retained by some fusions and provide convenient purification steps. We also find that the active-site loop of E. coli thioredoxin can be used as a general site for small peptide insertions, allowing for the high level production of soluble peptides in the E. coli cytoplasm.

We localized the chromosomal targets of several of the regulatory controls of expression of the CAR1 gene. Fusion to lacZ of several fragments of the 5' non-coding region showed that induction of CAR1 by arginine is positively regulated by the products of the ARGR genes. The target lies upstream of another site where repression by the CARGRI molecule occurs. The latter control is not specific to arginine catabolism since it also affects CYC-1 and indeed does not appear to involve arginine. The primary target of the two other regulatory alleles CARGRH and CARGRIIIis not situated in the 5' non-coding region. Deletion analysis supports the fusion data and confirms the order of the regulatory regions: 5' -nitrogen catabolite repression -activation by arginine -CARGRI-mediated repression-CAR1.

Background. Gene fusions arising from chromosomal translocations have been implicated in cancer. RNA-seq has the potential to discover such rearrangements generating functional proteins (chimera/fusion). Recently, many methods for chimeras detection have been published. However, specificity and sensitivity of those tools were not extensively investigated in a comparative way. Results. We tested eight fusion-detection tools (FusionHunter, FusionMap, FusionFinder, MapSplice, deFuse, Bellerophontes, ChimeraScan, and TopHat-fusion) to detect fusion events using synthetic and real datasets encompassing chimeras. The comparison analysis run only on synthetic data could generate misleading results since we found no counterpart on real dataset. Furthermore, most tools report a very high number of false positive chimeras. In particular, the most sensitive tool, ChimeraScan, reports a large number of false positives that we were able to significantly reduce by devising and applying two filters to remove fusions not supported by fusion junction-spanning reads or encompassing large intronic regions. Conclusions. The discordant results obtained using synthetic and real datasets suggest that synthetic datasets encompassing fusion events may not fully catch the complexity of RNA-seq experiment. Moreover, fusion detection tools are still limited in sensitivity or specificity; thus, there is space for further improvement in the fusion-finder algorithms.

Congenital (or infantile) fibrosarcoma (CFS) is a relatively rare pediatric malignancy, usually affecting the extremities. Metastasis is rare although there is a high local recurrence rate. The majority of CFS karyotypes and/or interphase fluorescence in situ hybridization (FISH) analyses have shown various combinations of recurrent trisomies but few structural anomalies. In 1998, a novel cryptic translocation, der(15)t(12;15)(p13;q25), was added to the list of chromosomal aberrations seen in this tumor. The breakpoints were cloned, and a new gene fusion, ETV6-NTRK3, was demonstrated [1]. It was suggested that the ETV6-NTRK3 translocation, when present, predicts more benign behavior of the neoplasm, in contrast to that seen in the highly malignant form of adult-type fibrosarcoma (ATFS), which lacks the translocation [2,3]. The same der(15)t(12;15)(p13;q25)associated ETV6-NTRK3 gene fusion has recently been found in cellular congenital mesoblastic nephroma (CMN) , another usually benign tumor affecting the infantile kidney. This was construed as indicative of a histogenic link between CFS and CMN. It also lent further credence to ETV6-NTRK3 as a predictor of benign behavior. Our experience with a baby with CFS leads us to question the significance of the translocation-associated gene fusion as a major prognostic predictor.

Development of the vertebral column is controlled by a complex of pleiotropic and polygenetic phenomena, in the mouse and chick regulating formation of different parts of individual vertebrae and morphological identity along the column ('Hox code'). In mouse and chick, experimental misexpression, including upstream and downstream genes, results in shifts in vertebral identity, loss of particular parts of individual vertebrae or vertebral fusion. Axial skeleton homologies across the Vertebrata allow these observations to be extended to taxa such as Homo sapiens, Chondrichthyes and Placodermi, the latter an entirely fossil group. Misexpression phenotypes among fossil taxa illuminate the phylogenetic history of these regulatory mechanisms. Phenotypes associated with genes originating via genomic duplication can determine the historical depth for these duplication events. Analysis of an ontogenetic sequence for the occipital-synarcual complex in the placoderm Cowralepis mclachlani provides the basis for comparison of this early gnathostome with other placoderms, chondrichthyans and amniotes. The occipital-synarcual patterns in placoderms parallel the phenotypic misexpression in mice and chicks (fusion and homeotic mutation) and the varying degrees of fusion in the Type I-III human Klippel-Feil syndrome. The association of these phenotypes to Hoxd regulatory complexes indicates that the gnathostome genomic duplication occurred at the base of the gnathostome stem group. Given the conservative nature of regulatory genes and the homology of vertebral elements, the presence of fusion in stem gnathostomes implies that the mechanism of fusion in mouse and chick models can be extrapolated to extant chondrichthyans (testable) and accounts for the phenotypic similarity across gnathostomes. The presence of these phenotypes in fossils indicates the antiquity of these regulatory mechanisms and of genomic duplication.

Renal cell carcinomas (RCCs) are rare in children and studies of their subtypes and clinicopathologic associations are limited to small series. We identified 8 patients with RCC treated at our institution between 1981 and 2003, reviewed their clinicopathologic features, cytogenetics findings, and evaluated the status of TFE3 expression by immunohistochemistry and numerical chromosomal alterations by interphase fluorescent in situ hybridization on paraffin-embedded tissue. These 8 patients (5 female and 3 male) had diploidy, and 5 had morphologic features compatible with the recently described RCC associated with Xp11.2 translocations/TFE3 gene fusions and demonstrated nuclear labeling for TFE3 protein by immunohistochemistry. The translocation was confirmed in 2 of these 5 patients by conventional cytogenetics. One case was a high-grade nonpapillary RCC and the other was compatible with type 2 papillary RCC. Four patients showed at least 1 chromosomal gain including trisomy 7 and/or trisomy 17. None of the tumors from male patients showed evidence of loss of the Y chromosome, but 2 patients showed numerical abnormalities of X chromosome +add(X). Two patients had sickle cell disease, and 1 of these also had stage IV-S neuroblastoma. This study suggests that many cases of RCC in children reported under the terms “papillary” and “clear cell” likely represent Xp11.2 translocation/TFE3 gene fusion-associated RCC. It also emphasizes the unusual associations of RCC with neuroblastoma and sickle cell hemoglobinopathy, which need further study.

An improved method for integration of exogenous DNA fragments at a defined site within the genome of Pseudomonas aeruginosa was developed. The method relies on two integration-proficient vectors, mini-CTX1 and mini-CTX2. These two vectors contain (1) a tetracycline (tet) selectable marker, (2) an oriT for conjugation-mediated plasmid transfer, (3) the pMB1-derived origin of replication, (4) a modified CTX integrase (int) gene, (5) a versatile multiple cloning site (MCS) flanked by T4 transcriptional termination sequences (⍀ elements), and (6) the CTX attachment site. The MCS and ⍀ elements are flanked by yeast Flp recombinase target sites that allow in vivo excision of unwanted plasmid backbone sequences, including tet and int, from the genome of integrants by Flp recombinase. In the mini-CTX2 vector int transcription is driven from the strong trc promoter, which is regulated by the Lac repressor that is encoded by lacI q also contained on the plasmid. Upon conjugal transfer, mini-CTX1 and mini-CTX2 integrated at frequencies of 10 Ϫ8 and 10 Ϫ7 , respectively. The usefulness of the integration vectors for gene fusion analyses was demonstrated by chromosomal insertion of autoinducer (AI)-regulated lasB-lacZ and rhlA-lacZ fusions into wild-type and AI synthase mutants. In wild-type, the fusions responded in a cell density-dependent manner and expression of both fusions was either greatly reduced or abolished in AI synthase mutants. Finally, an expression cassette containing the T7 polymerase gene under Lac repressor control was constructed, integrated into the P. aeruginosa chromosome, and used to express the hexahistidine-tagged P. aeruginosa AI synthase RhlI.

Chromosome translocations in the common epithelial cancers are abundant, yet little is known about them. They have been thought to be almost all unbalanced and therefore dismissed as mostly mediating tumour suppressor loss. We present a comprehensive analysis by array painting of the chromosome translocations of breast cancer cell lines HCC1806, HCC1187 and ZR-75-30. In array painting, chromosomes are isolated by flow cytometry, amplified and hybridized to DNA microarrays. A total of 200 breakpoints were identified and all were mapped to 1Mb resolution on BAC arrays, then 40 selected breakpoints, including all balanced breakpoints, were further mapped on tiling-path BAC arrays or to around 2kb resolution using oligonucleotide arrays. Many more of the translocations were balanced at 1Mb resolution than expected, either reciprocal (eight in total) or balanced for at least one participating chromosome (19 paired breakpoints). Secondly, many of the breakpoints were at genes that are plausible targets of oncogenic translocation, including balanced breaks at CTCF, EP300/p300, and FOXP4. Two gene fusions were demonstrated, TAX1BP1-AHCY and RIF1-PKD1L1. Our results support the idea that chromosome rearrangements may play an important role in common epithelial cancers such as breast cancer.

Glutamate receptors are well-known actors in the central and peripheral nervous systems, and altered glutamate signaling is implicated in several neurological and psychiatric disorders. It is increasingly recognized that such receptors may also have a role in tumor growth. Here we provide direct evidence of aberrant glutamate signaling in the development of a locally aggressive bone tumor, chondromyxoid fibroma (CMF). We subjected a series of CMFs to whole-genome mate-pair sequencing and RNA sequencing and found that the glutamate receptor gene GRM1 recombines with several partner genes through promoter swapping and gene fusion events. The GRM1 coding region remains intact, and 18 of 20 CMFs (90%) showed a more than 100-fold and up to 1,400-fold increase in GRM1 expression levels compared to control tissues. Our findings unequivocally demonstrate that direct targeting of GRM1 is a necessary and highly specific driver event for CMF development.