U12-type Spliceosome: Localization and Effects of Splicing Efficiency on Gene Expression

The removal of non-coding sequences, introns, is an essential part of messenger RNA processing. In most metazoan organisms, the U12-type spliceosome processes a subset of introns containing highly conserved recognition sequences. U12-type introns constitute less than 0,5% of all introns and reside preferentially in genes related to information processing functions, as opposed to genes encoding for metabolic enzymes. It has previously been shown that the excision of U12-type introns is inefficient compared to that of U2-type introns, supporting the model that these introns could provide a rate-limiting control for gene expression. The low efficiency of U12-type splicing is believed to have important consequences to gene expression by limiting the production of mature mRNAs from genes containing U12-type introns. The inefficiency of U12-type splicing has been attributed to the low abundance of the components of the U12-type spliceosome in cells, but this hypothesis has not been proven. The aim of the first part of this work was to study the effect of the abundance of the spliceosomal snRNA components on splicing. Cells with a low abundance of the U12-type spliceosome were found to inefficiently process U12-type introns encoded by a transfected construct, but the expression levels of endogenous genes were not found to be affected by the abundance of the U12-type spliceosome. However, significant levels of endogenous unspliced U12-type intron-containing pre-mRNAs were detected in cells. Together these results support the idea that U12-type splicing may limit gene expression in some situations. The inefficiency of U12-type splicing has also promoted the idea that the U12-type spliceosome may control gene expression, limiting the mRNA levels of some U12-type intron-containing genes. While the identities of the primary target genes that contain U12-type introns are relatively well known, little has previously been known about the downstream genes and pathways potentially affected by the efficiency of U12-type intron processing. Here, the effects of U12-type splicing efficiency on a whole organism were studied in a Drosophila line with a mutation in an essential U12-type spliceosome component. Genes containing U12-type introns showed variable gene-specific responses to the splicing defect, which points to variation in the susceptibility of different genes to changes in splicing efficiency. Surprisingly, microarray screening revealed that metabolic genes were enriched among downstream effects, and that the phenotype could largely be attributed to one U12-type intron-containing mitochondrial gene. Gene expression control by the U12-type spliceosome could thus have widespread effects on metabolic functions in the organism. The subcellular localization of the U12-type spliceosome components was studied as a response to a recent dispute on the localization of the U12-type spliceosome. All components studied were found to be nuclear indicating that the processing of U12-type introns occurs within the nucleus, thus clarifying a question central to the field. The results suggest that the U12-type spliceosome can limit the expression of genes that contain U12-type introns in a gene-specific manner. Through its limiting role in pre-mRNA processing, the U12-type splicing activity can affect specific genetic pathways, which in the case of Drosophila are involved in metabolic functions.

Type III Secretion System of Phytopathogenic Bacterium Pseudomonas syringae:From Gene to Function

The type III secretion system (T3SS) is an essential requirement for the virulence of many Gram-negative bacteria which infect plants, animals and men. Pathogens use the T3SS to deliver effector proteins from the bacterial cytoplasm to the eukaryotic host cells, where the effectors subvert host defenses. The best candidates for directing effector protein traffic are the bacterial type III-associated appendages, called needles or pili. In plant pathogenic bacteria, the best characterized example of a T3SS-associated appendage is the HrpA pilus of the plant pathogen Pseudomonas syringae pv. tomato DC3000. The components of the T3SS in plant pathogens are encoded by a cluster of hrp (hypersensitive reaction and pathogenicity) genes. Two major classes of T3SS-secreted proteins are: harpin proteins such as HrpZ which are exported into extracellular space, and avirulence (Avr) proteins such as AvrPto which are translocated directly to the plant cytoplasm. This study deals with the structural and functional characterization of the T3SS-associated HrpA pilus and the T3SS-secreted harpins. By insertional mutagenesis analysis of HrpA, we located the optimal epitope insertion site in the amino-terminus of HrpA, and revealed the potential application of the HrpA pilus as a carrier of antigenic determinants for vaccination. By pulse-expression of proteins combined with immuno-electron microscopy, we discovered the Hrp pilus assembly strategy as addition of HrpA subunits to the distal end of the growing pilus, and we showed for the first time that secretion of HrpZ occurs at the tip of the pilus. The pilus thus functions as a conduit delivering proteins to the extracellular milieu. By using phage-display and scanning-insertion mutagenesis methods we identified a conserved HrpZ-binding peptide and localized the peptide-binding site to the central domain of HrpZ. We also found that the HrpZ specifically interacts with a host bean protein. Taken together, the current results provide deeper insight into the molecular mechanism of T3SS-associated pilus assembly and effector protein translocation, which will be helpful for further studies on the pathogenic mechanisms of Gram-negative bacteria and for developing new strategies to prevent bacterial infection.

Salmonella Typhimurium: Insight into the multi-faceted role of the LysR-type transcriptional regulators in Salmonella

The LysR-type transcriptional regulators (LTTRs) are widely distributed in various genera of prokaryotes LTTRs are DNA binding proteins that can positively or negatively regulate target gene expression and can also repress their own transcription Salmonella enterica comprises a group of Gram-negative bacteria capable of causing clinical syndromes that range from self-limiting diarrhoea to severe fibrinopurulent necrotizing enteritis and life threatening systemic disease. The survival and replication of Salmonella in macrophages and in infected host is brought about by the means of various two component regulatory systems, transporters and other virulence islands In Salmonella genome the existence of 44 LTTRs has been documented These LTTRs regulate bacterial stress response. systemic virulence in mice and also many virulence determinants in vitro. Here we focus on the findings that elucidate the structure and function of the LTTRs in Salmonella and discuss the importance of these LTTRs in making Salmonella a Successful pathogen...

A brilliant breakthrough in OI type V

Interferon-induced transmembrane protein 5 or bone-restricted i ifitm-like gene (Bril) was first identified as a bone gene in 2008, although no in vivo role was identified at that time. A role in human bone has now been demonstrated with a number of recent studies identifying a single point mutation in Bril as the causative mutation in osteogenesis imperfecta type V (OI type V). Such a discovery suggests a key role for Bril in skeletal regulation, and the completely novel nature of the gene raises the possibility of a new regulatory pathway in bone. Furthermore, the phenotype of OI type V has unique and quite divergent features compared with other forms of OI involving defects in collagen biology. Currently it appears that the underlying genetic defect in OI type V may be unrelated to collagen regulation, which also raises interesting questions about the classification of this form of OI. This review will discuss current knowledge of OI type V, the function of Bril, and the implications of this recent discovery.

Nucleotide sequence and expression inE. coli of the complete P4 type VP4 from a G2 serotype human rotavirus

The complete sequence of a P4 type VP4 gene from a G2 serotype human rotavirus, IS2, isolated in India has been determined. Although the IS2 VP4 is highly homologous to the other P4 type alleles, it contained acidic amino acid substitutions at several positions that make it acidic among the P4 type alleles that are basic. Moreover, comparative sequence analysis revealed unusual polymorphism in members of the P4 type at amino acid position 393 which is highly conserved in members of other VP4 types. To date, expression of complete VP4 inE. coli has not been achieved. In this study we present successful expression inE. coli of the complete VP4 as well as VP8* and VP5* cleavage subunits in soluble form as fusion proteins of the maltose-binding protein (MBP) and their purification by single-step affinity chromatography. The hemagglutinating activity exhibited by the recombinant protein was specifically inhibited by the antiserum raised against it. Availability of pure VP4 proteins should facilitate development of polyclonal and monoclonal antibodies (MAbs) for P serotyping of rotaviruses.

Peripartum management of glycemia in women with type 1 diabetes

OBJECTIVE We aimed to 1) describe the peripartum management of type 1 diabetes at an Australian teaching hospital and 2) discuss factors influencing the apparent transient insulin independence postpartum. RESEARCH DESIGN AND METHODS We conducted a retrospective review of women with type 1 diabetes delivering singleton pregnancies from 2005 to 2010. Information was collected regarding demographics, medical history, peripartum management and outcome, and breast-feeding. To detect a difference in time to first postpartum blood glucose level (BGL) >8 mmol/L between women with an early (<4 h) and late (>12 h) requirement for insulin postpartum, with a power of 80% and a type 1 error of 0.05, at least 24 patients were required. RESULTS An intravenous insulin infusion was commenced in almost 95% of women. Univariate analysis showed that increased BMI at term, lower creatinine at term, longer duration from last dose of long- or intermediate-acting insulin, and discontinuation of an insulin infusion postpartum were associated with a shorter time to first requirement of insulin postpartum (P = 0.005, 0.026, 0.026, and <0.001, respectively). There was a correlation between higher doses of insulin commenced postpartum and number of out-of-range BGLs (r[36] = 0.358, P = 0.030) and hypoglycemia (r[36] = 0.434, P = 0.007). Almost 60% had at least one BGL <3.5 mmol/L between delivery and discharge. CONCLUSIONS Changes in the pharmacodynamic profile of insulin may contribute to the transient insulin independence sometimes observed postpartum in type 1 diabetes. A dose of 50–60% of the prepregnancy insulin requirement resulted in the lowest rate of hypoglycemia and glucose excursions. These results require validation in a larger, prospective study.

Pheo-type: A diagnostic gene-expression assay for the classification of pheochromocytoma and paraganglioma

Context: Pheochromocytomas and paragangliomas (PPGLs) are heritable neoplasms that can be classified into gene-expression subtypes corresponding to their underlying specific genetic drivers. Objective: This study aimed to develop a diagnostic and research tool (Pheo-type) capable of classifying PPGL tumors into gene-expression subtypes that could be used to guide and interpret genetic testing, determine surveillance programs, and aid in elucidation of PPGL biology. Design: A compendium of published microarray data representing 205 PPGL tumors was used for the selection of subtype-specific genes that were then translated to the Nanostring gene-expression platform. A support vector machine was trained on the microarray dataset and then tested on an independent Nanostring dataset representing 38 familial and sporadic cases of PPGL of known genotype (RET, NF1, TMEM127, MAX, HRAS, VHL, and SDHx). Different classifier models involving between three and six subtypes were compared for their discrimination potential. Results: A gene set of 46 genes and six endogenous controls was selected representing six known PPGL subtypes; RTK1–3 (RET, NF1, TMEM127, and HRAS), MAX-like, VHL, and SDHx. Of 38 test cases, 34 (90%) were correctly predicted to six subtypes based on the known genotype to gene-expression subtype association. Removal of the RTK2 subtype from training, characterized by an admixture of tumor and normal adrenal cortex, improved the classification accuracy (35/38). Consolidation of RTK and pseudohypoxic PPGL subtypes to four- and then three-class architectures improved the classification accuracy for clinical application. Conclusions: The Pheo-type gene-expression assay is a reliable method for predicting PPGL genotype using routine diagnostic tumor samples.

Kinetic characterization of rat brain type IIA sodium channel alpha-subunit stably expressed in a somatic cell line

1. The rat brain type IIA Na+ channel alpha-subunit was stably expressed in Chinese hamster ovary (CHO) cells. Current through the expressed Na+ channels was studied using the whole-cell configuration of the patch clamp technique. The transient Na+ current was sensitive to TTX and showed a bell-shaped peak current vs. membrane potential relation. 2. Na+ current inactivation was better described by the sum of two exponentials in the potential range -30 to +40 mV, with. a dominating fast component and a small slower component. 3. The steady-state inactivation, h(infinity), was related to potential by a Boltzmann distribution, underlying thr ee states of the inactivation gate. 4. Recovery of the channels from inactivation at different potentials in the range -70 to -120 mV were characterized by al? initial delay which decreased with hyperpolarization. The time course was well fitted by the sum of two exponentials. In this case the slower exponential was the major component, and both time constants decreased with hyperpolarization. 5. For a working description of the Na+ channel inactivation in this preparation, with a minimal deviation from the Hodgkin-Huxley model, a three-state scheme of the form O reversible arrow I-1 reversible arrow I-2 was proposed, replacing the original two-state scheme of the Hodgkin-Huxley model, and the rate constants are reported. 6. The instantaneous current-voltage relationship showed marked deviation from linearity and was satisfactorily fitted by the constant-field equation. 7. The time course of activation was described by an m(x) model. However, the best-fitted value of x varied with the membrane potential and had a mean value of 2. 8. Effective gating charge was determined to be 4.7e from the slope of the activation plot, plotted on a logarithmic scale. 9. The rate constants of activation, alpha(m) and beta(m), were determined. Their functional dependence on the membrane potential was investigated.

The reproducibility of acquiring three dimensional gait and plantar pressure data using established protocols in participants with and without type 2 diabetes and foot ulcers

Background Several prospective studies have suggested that gait and plantar pressure abnormalities secondary to diabetic peripheral neuropathy contributes to foot ulceration. There are many different methods by which gait and plantar pressures are assessed and currently there is no agreed standardised approach. This study aimed to describe the methods and reproducibility of three-dimensional gait and plantar pressure assessments in a small subset of participants using pre-existing protocols. Methods Fourteen participants were conveniently sampled prior to a planned longitudinal study; four patients with diabetes and plantar foot ulcers, five patients with diabetes but no foot ulcers and five healthy controls. The repeatability of measuring key biomechanical data was assessed including the identification of 16 key anatomical landmarks, the measurement of seven leg dimensions, the processing of 22 three-dimensional gait parameters and the analysis of four different plantar pressures measures at 20 foot regions. Results The mean inter-observer differences were within the pre-defined acceptable level (<7 mm) for 100 % (16 of 16) of key anatomical landmarks measured for gait analysis. The intra-observer assessment concordance correlation coefficients were > 0.9 for 100 % (7 of 7) of leg dimensions. The coefficients of variations (CVs) were within the pre-defined acceptable level (<10 %) for 100 % (22 of 22) of gait parameters. The CVs were within the pre-defined acceptable level (<30 %) for 95 % (19 of 20) of the contact area measures, 85 % (17 of 20) of mean plantar pressures, 70 % (14 of 20) of pressure time integrals and 55 % (11 of 20) of maximum sensor plantar pressure measures. Conclusion Overall, the findings of this study suggest that important gait and plantar pressure measurements can be reliably acquired. Nearly all measures contributing to three-dimensional gait parameter assessments were within predefined acceptable limits. Most plantar pressure measurements were also within predefined acceptable limits; however, reproducibility was not as good for assessment of the maximum sensor pressure. To our knowledge, this is the first study to investigate the reproducibility of several biomechanical methods in a heterogeneous cohort.

Serum bilirubin concentration is modified by UGT1A1 Haplotypes and influences risk of type-2 diabetes in the Norfolk Island Genetic Isolate

Background Located in the Pacific Ocean between Australia and New Zealand, the unique population isolate of Norfolk Island has been shown to exhibit increased prevalence of metabolic disorders (type-2 diabetes, cardiovascular disease) compared to mainland Australia. We investigated this well-established genetic isolate, utilising its unique genomic structure to increase the ability to detect related genetic markers. A pedigree-based genome-wide association study of 16 routinely collected blood-based clinical traits in 382 Norfolk Island individuals was performed. Results A striking association peak was located at chromosome 2q37.1 for both total bilirubin and direct bilirubin, with 29 SNPs reaching statistical significance (P < 1.84 × 10−7). Strong linkage disequilibrium was observed across a 200 kb region spanning the UDP-glucuronosyltransferase family, including UGT1A1, an enzyme known to metabolise bilirubin. Given the epidemiological literature suggesting negative association between CVD-risk and serum bilirubin we further explored potential associations using stepwise multivariate regression, revealing significant association between direct bilirubin concentration and type-2 diabetes risk. In the Norfolk Island cohort increased direct bilirubin was associated with a 28 % reduction in type-2 diabetes risk (OR: 0.72, 95 % CI: 0.57-0.91, P = 0.005). When adjusted for genotypic effects the overall model was validated, with the adjusted model predicting a 30 % reduction in type-2 diabetes risk with increasing direct bilirubin concentrations (OR: 0.70, 95 % CI: 0.53-0.89, P = 0.0001). Conclusions In summary, a pedigree-based GWAS of blood-based clinical traits in the Norfolk Island population has identified variants within the UDPGT family directly associated with serum bilirubin levels, which is in turn implicated with reduced risk of developing type-2 diabetes within this population.