Structural proteins of mycobacteriophage I3: cloning, expression and sequence analysis of a gene encoding a 70-kDa structural protein

The structural proteins of mycobacteriophage I3 have been analysed by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis (SDS-PAGE), radioiodination and immunoblotting. Based on their abundance the 34- and 70-kDa bands appeared to represent the major structural proteins. Successful cloning and expression of the 70-kDa protein-encoding gene of phage I3 in Escherichia coli and its complete nucleotide sequence determination have been accomplished, A second (partial) open reading frame following the stop codon for the 70-kDa protein was also identified within the cloned fragment. The deduced amino-acid sequence of the 70-kDa protein and the codon usage patterns indicated the preponderance of codons, as predicted from the high G+C content of the genomic DNA of phage I3.

Baculovirus mediated high-level expression of luciferase in silkworm cells and larvae

Bombyx mori nuclear polyhedrosis virus (BmNPV)-based baculovirus expression system exploits silkworm larvae as an economical alternative to large-scale cell cultures for production of biomolecules. To generate recombinant BmNPV at high efficiency, we have achieved high efficiency transfection of B. mori cells, BmN, through lipofection. Optimal conditions for lipofection were standardized by quantification of the transient expression level of firefly luciferase (luc) reporter gene under control of an immediate early gene promoter of BmNPV Lipofection was 50-fold and 100-fold more efficient than the calcium phosphate method for transfecting BmN and Sf9 cells, respectively. Lipofection enabled us to generate a recombinant BmNPV (vBmluc), harboring luc under control of the strong polyhedrin promoter On infection with vBmluc, luciferase was expressed at very high levels, 170 mu g/10(6) BmN cells or 13 mg/larva. Expression of luciferase in vBmluc-infected larvae was visualized by luminescence emission instantaneously following luciferin injection generating ''glowing silkworms''.

A Ten-microRNA Expression Signature Predicts Survival in Glioblastoma

Glioblastoma (GBM) is the most common and aggressive primary brain tumor with very poor patient median survival. To identify a microRNA (miRNA) expression signature that can predict GBM patient survival, we analyzed the miRNA expression data of GBM patients (n = 222) derived from The Cancer Genome Atlas (TCGA) dataset. We divided the patients randomly into training and testing sets with equal number in each group. We identified 10 significant miRNAs using Cox regression analysis on the training set and formulated a risk score based on the expression signature of these miRNAs that segregated the patients into high and low risk groups with significantly different survival times (hazard ratio HR] = 2.4; 95% CI = 1.4-3.8; p < 0.0001). Of these 10 miRNAs, 7 were found to be risky miRNAs and 3 were found to be protective. This signature was independently validated in the testing set (HR = 1.7; 95% CI = 1.1-2.8; p = 0.002). GBM patients with high risk scores had overall poor survival compared to the patients with low risk scores. Overall survival among the entire patient set was 35.0% at 2 years, 21.5% at 3 years, 18.5% at 4 years and 11.8% at 5 years in the low risk group, versus 11.0%, 5.5%, 0.0 and 0.0% respectively in the high risk group (HR = 2.0; 95% CI = 1.4-2.8; p < 0.0001). Cox multivariate analysis with patient age as a covariate on the entire patient set identified risk score based on the 10 miRNA expression signature to be an independent predictor of patient survival (HR = 1.120; 95% CI = 1.04-1.20; p = 0.003). Thus we have identified a miRNA expression signature that can predict GBM patient survival. These findings may have implications in the understanding of gliomagenesis, development of targeted therapy and selection of high risk cancer patients for adjuvant therapy.

Expression of the Japanese encephalitis virus NS3 and NS2b proteins as glutathione S-transferase fusions

Flaviviruses generate their structural and nonstructural proteins by proteolytic processing of a single large polyprotein precursor. These proteolytic events are brought about both by host cell signalase and a virally encoded protease. The virally encoded proteolytic activity has been shown to reside within the nonstructural protein 3 (NS3) and requires the product of the nonstructural 2b (NS2b) gene. In order to obtain sufficient quantities of pure NS2b and NS3 proteins for kinetic analysis, we have expressed both these proteins in recombinant systems as fusions to glutathione S-transferase (GST). The fusion constructs were driven by the strong bacteriophage T7 promoter. Transfection of these constructs into the African green monkey kidney cell line CV-1 previously infected with a recombinant vaccinia virus expressing the T7 RNA polymerase resulted in synthesis of the fusion proteins. Both the fusion proteins could be purified to homogeneity in a single step using a glutathione agarose affinity matrix.

Cloning, expression, purification, crystallization and preliminary X-ray studies of the mannose-binding lectin domain of MSMEG_3662 from Mycobacterium smegmatis

The mannose-binding lectin domain of MSMEG_3662 from Mycobacterium smegmatis has been cloned, expressed, purified and crystallized and the crystals have been characterized using X-ray diffraction. The Matthews coefficient suggests the possibility of two lectin domains in the triclinic cell. The amino-acid sequence of the domain indicates structural similarity to well characterized beta-prism II fold lectins.

Scheduling expression trees with reusable registers on delayed-load architectures

In this paper, we look at the problem of scheduling expression trees with reusable registers on delayed load architectures. Reusable registers come into the picture when the compiler has a data-flow analyzer which is able to estimate the extent of use of the registers. Earlier work considered the same problem without allowing for register variables. Subsequently, Venugopal considered non-reusable registers in the tree. We further extend these efforts to consider a much more general form of the tree. We describe an approximate algorithm for the problem. We formally prove that the code schedule produced by this algorithm will, in the worst case, generate one interlock and use just one more register than that used by the optimal schedule. Spilling is minimized. The approximate algorithm is simple and has linear complexity.

Unwinding of heterologous DNA by RecA protein during the search for homologous sequences

The search for homologous sequences promoted by RecA protein in vitro involves a presynaptic filament and naked duplex DNA, the multiple contacts of which produce nucleoprotein networks or coaggregates. The single-stranded DNA within the presynaptic filaments, however, is extended to an axial spacing 1.5 times that of B-form DNA. To investigate this paradoxical difference between the spacing of bases in the RecA presynaptic filament versus the target duplex DNA, we explored the effect of heterologous contacts on the conformation of DNA, and vice versa. In the presence of wheat germ topoisomerase I, RecA presynaptic filaments induced a rapid, limited reduction in the linking number of heterologous circular duplex DNA. This limited unwinding of heterologous duplex DNA, termed heterologous unwinding, was detected within 30 seconds and reached a steady state within a few minutes. Presynaptic filaments that were formed in the presence of ATP?S and separated from free RecA protein by gel filtration also generated a ladder of topoisomers upon incubation with relaxed duplex DNA and topoisomerase. The inhibition of heterologous contacts by 60 mImage -NaCl or 5 mImage -ADP resulted in a corresponding decrease in heterologous unwinding. In reciprocal fashion, the stability or number of heterologous contacts with presynaptic filaments was inversely related to the linking number of circular duplex DNA. These observations show that heterologous contacts with the presynaptic filament cause a limited unwinding of the duplex DNA, and conversely that the ability of the DNA to unwind stabilizes transient heterologous contacts.

Progesterone receptor expression in the human placenta

The presence of progesterone receptors (PR) in the human placenta has been demonstrated using the reverse transcriptase-polymerase chain reaction technique. It was observed that the amount of PR in the human placenta is less during late gestation. Electrophoretic mobility shift assays with nuclear extract isolated from the first trimester and term placenta revealed three complexes when incubated with [P-32]dCTP-labelled progesterone response element, and, in competition with unlabelled progesterone response element, the formation of all three complexes was inhibited. When supershift analysis of these complexes was carried out using antibodies which cross-react with both the A and B types of the PR or only with the B type receptor, only the A-form of PR was detected in the human placenta.

Gene Expression Profiling of Preovulatory Follicle in the Buffalo Cow: Effects of Increased IGF-I Concentration on Periovulatory Events

The preovulatory follicle in response to gonadotropin surge undergoes dramatic biochemical, and morphological changes orchestrated by expression changes in hundreds of genes. Employing well characterized bovine preovulatory follicle model, granulosa cells (GCs) and follicle wall were collected from the preovulatory follicle before, 1, 10 and 22 h post peak LH surge. Microarray analysis performed on GCs revealed that 450 and 111 genes were differentially expressed at 1 and 22 h post peak LH surge, respectively. For validation, qPCR and immunocytochemistry analyses were carried out for some of the differentially expressed genes. Expression analysis of many of these genes showed distinct expression patterns in GCs and the follicle wall. To study molecular functions and genetic networks, microarray data was analyzed using Ingenuity Pathway Analysis which revealed majority of the differentially expressed genes to cluster within processes like steroidogenesis, cell survival and cell differentiation. In the ovarian follicle, IGF-I is established to be an important regulator of the above mentioned molecular functions. Thus, further experiments were conducted to verify the effects of increased intrafollicular IGF-I levels on the expression of genes associated with the above mentioned processes. For this purpose, buffalo cows were administered with exogenous bGH to transiently increase circulating and intrafollicular concentrations of IGF-I. The results indicated that increased intrafollicular concentrations of IGF-I caused changes in expression of genes associated with steroidogenesis (StAR, SRF) and apoptosis (BCL-2, FKHR, PAWR). These results taken together suggest that onset of gonadotropin surge triggers activation of various biological pathways and that the effects of growth factors and peptides on gonadotropin actions could be examined during preovulatory follicle development.

Expression of the receptor guanylyl cyclase C and its ligands in reproductive tissues of the rat: A potential role for a novel signaling pathway in the epididymis

Guanylyl cyclase C (GC-C) is a membrane-associated form of guanylyl cyclase and serves as the receptor for the heat-stable enterotoxin (ST) peptide and endogenous ligands guanylin, uroguanylin, and lymphoguanylin. The major site of expression of GC-C is the intestinal epithelial cell, although GC-C is also expressed in extraintestinal tissue such as the kidney, airway epithelium, perinatal liver, stomach, brain, and adrenal glands. Binding of ligands to GC-C leads to accumulation of intracellular cGMP, the activation of protein kinases G and A, and phosphorylation of the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel that regulates salt and water secretion. We examined the expression of GC-C and its ligands in various tissues of the reproductive tract of the rat. Using reverse transcriptase and the polymerase chain reaction, we demonstrated the presence of GC-C, uroguanylin, and guanylin mRNA in both male and female reproductive organs. Western blot analysis using a monoclonal antibody to GC-C revealed the presence of differentially glycosylated forms of GC-C in the caput and cauda epididymis. Exogenous addition of uroguanylin to minced epididymal tissue resulted in cGMP accumulation, suggesting an autocrine or endocrine activation of GC-C in this tissue. Immunohistochemical analyses demonstrated expression of GC-C in the tubular epithelial cells of both the caput epididymis and cauda epididymis. Our results suggest that the GC-C signaling pathway could converge on CFTR in the epididymis and perhaps control fluid and ion balance for optimal sperm maturation and storage in this tissue.

Molecular cloning and expression pattern of a Cubitus interruptus homologue from the mulberry silkworm Bombyx mori

A homologue of the segment polarity gene Cubitus interruptus from Bombyx Mori, (BmCi) has been cloned and characterized. This region harbouring Zn2+ finger motif is highly conserved across species. In B. Mori, BmCi RNA expression was first detected at stage 6 of embryogenesis, which reached maximum levels at stage 21C and was maintained until larval hatching. The segmentally reiterated striped pattern of transcript distribution in stage 21C embryos was in conformity with its predicted segment polarity nature. BmCi was expressed in the fore- and hind-wing discs, ovaries, testes and gut during fifth larval intermolt, reminiscent of its expression domains in Drosophila. Besides, BmCi expression was seen in the. anterior part of the middle silkglands in late embryonic stages, and this pattern was maintained during larval development. The transition from third to fourth and fifth larval intermolts was accompanied by an increase in the transcript levels in the middle silkglands. Our results demonstrate the presence of a novel expression domain for Ci in Bombyx. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.

Scheduling expression trees for delayed-load architectures

In this paper we consider the problem of scheduling expression trees on delayed-load architectures. The problem tackled here takes root from the one considered in [Proceedings of the ACM SIGPLAN '91 Conf. on Programming Language Design and Implementation, 1991. p. 256] in which the leaves of the expression trees all refer to memory locations. A generalization of this involves the situation in which the trees may contain register variables, with the registers being used only at the leaves. Solutions to this generalization are given in [ACM Trans. Prog. Lang. Syst. 17 (1995) 740, Microproc. Microprog. 40 (1994) 577]. This paper considers the most general case in which the registers are reusable. This problem is tackled in [Comput. Lang, 21 (1995) 49] which gives an approximate solution to the problem under certain assumptions about the contiguity of the evaluation order: Here we propose an optimal solution (which may involve even a non-contiguous evaluation of the tree). The schedule generated by the algorithm given in this paper is optimal in the sense that it is an interlock-free schedule which uses the minimum number of registers required. An extension to the algorithm incorporates spilling. The problem as stated in this paper is an instruction scheduling problem. However, the problem could also be rephrased as an operations research problem with a difference in terminology. (C) 2002 Elsevier Science B.V. All rights reserved.

Cloning, expression, purification and preliminary X-ray diffraction studies of a putative Mycobacterium smegmatis thiolase

Thiolases are important in fatty-acid degradation and biosynthetic pathways. Analysis of the genomic sequence of Mycobacterium smegmatis suggests the presence of several putative thiolase genes. One of these genes appears to code for an SCP-x protein. Human SCP-x consists of an N-terminal domain (referred to as SCP2 thiolase) and a C-terminal domain (referred as sterol carrier protein 2). Here, the cloning, expression, purification and crystallization of this putative SCP-x protein from M. smegmatis are reported. The crystals diffracted X-rays to 2.5 angstrom resolution and belonged to the triclinic space group P1. Calculation of rotation functions using X-ray diffraction data suggests that the protein is likely to possess a hexameric oligomerization with 32 symmetry which has not been observed in the other six known classes of this enzyme.

Expression pattern of Cubitus interruptus from the mulberry silkworm Bombyx mori in late developmental stages

A partial genomic clone of Bombyx mori homologue of the segment polarity gene Cubitus interruptus (BmCi), encoding the conserved zinc finger domain and harbouring two introns, has been characterized. BmCi was expressed in the silkglands of B. mori from embryonic to the late larval stages(3rd, 4th and 5th intermoults). The expression was confined to the anterior region of the middle silkglands, overlapping with the domain of sericin-2 expression and excluding the domains of Bm invected expression, namely the middle and posterior regions of the middle silkglands. In the wing discs, the expression was restricted to the anterior compartment, which increased from 4th to 5th larval intermoults and declined later in the pupal wing buds. In gonadal tissues (both ovaries and testes) BmCi was expressed from the larval to pupal stages. The transcripts were localized to the sperm tubes containing spermatogonia in the testis of Bombyx larvae. BmCi expression, however, was not detected in any of these tissues during the moulting stages. Expression of Ci in the wing discs and gonads is evolutionarily conserved, while the silkgland represents a novel domain. Our results imply that BmCi is involved in the specification and maintenance of micro-compartment identity within the middle silkglands.