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.

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.

Efficient Regular Expression Pattern Matching for Network Intrusion Detection Systems using Modified Word-based Automata

Network Intrusion Detection Systems (NIDS) intercept the traffic at an organization's network periphery to thwart intrusion attempts. Signature-based NIDS compares the intercepted packets against its database of known vulnerabilities and malware signatures to detect such cyber attacks. These signatures are represented using Regular Expressions (REs) and strings. Regular Expressions, because of their higher expressive power, are preferred over simple strings to write these signatures. We present Cascaded Automata Architecture to perform memory efficient Regular Expression pattern matching using existing string matching solutions. The proposed architecture performs two stage Regular Expression pattern matching. We replace the substring and character class components of the Regular Expression with new symbols. We address the challenges involved in this approach. We augment the Word-based Automata, obtained from the re-written Regular Expressions, with counter-based states and length bound transitions to perform Regular Expression pattern matching. We evaluated our architecture on Regular Expressions taken from Snort rulesets. We were able to reduce the number of automata states between 50% to 85%. Additionally, we could reduce the number of transitions by a factor of 3 leading to further reduction in the memory requirements.

Heterologous promoter recognition leading to high-level expression of cloned foreign genes in Bombyx mori cell lines and larvae

The baculovirus expression system using the Autographa californica nuclear polyhedrosis virus (AcNPV) has been extensively utilized for high-level expression of cloned foreign genes, driven by the strong viral promoters of polyhedrin (polh) and p10 encoding genes. A parallel system using Bombyx mori nuclear polyhedrosis virus (BmNPV) is much less exploited because the choice and variety of BmNPV-based transfer vectors are limited. Using a transient expression assay, we have demonstrated here that the heterologous promoters of the very late genes polh and p10 from AcNPV function as efficiently in BmN cells as the BmNPV promoters. The location of the cloned foreign gene with respect to the promoter sequences was critical for achieving the highest levels of expression, following the order +35 > +1 > -3 > -8 nucleotides (nt) with respect to the polh or p10 start codons. We have successfully generated recombinant BmNPV harboring AcNPV promoters by homeologous recombination between AcNPV-based transfer vectors and BmNPV genomic DNA. Infection of BmN cell lines with recombinant BmNPV showed a temporal expression pattern, reaching very high levels in 60-72 h post infection. The recombinant BmNPV harboring the firefly luciferase-encoding gene under the control of AcNPV polh or p10 promoters, on infection of the silkworm larvae led to the synthesis of large quantities of luciferase. Such larvae emanated significant luminiscence instantaneously on administration of the substrate luciferin resulting in 'glowing silkworms'. The virus-infected larvae continued to glow for several hours and revealed the most abundant distribution of virus in the fat bodies. In larval expression also, the highest levels were achieved when the reporter gene was located at +35 nt of the polh.

Distress call-induced gene expression in the brain of the Indian short-nosed fruit bat, Cynopterus sphinx

Individuals in distress emit audible vocalizations to either warn or inform conspecifics. The Indian short-nosed fruit bat, Cynopterus sphinx, emits distress calls soon after becoming entangled in mist nets, which appear to attract conspecifics. Phase I of these distress calls is longer and louder, and includes a secondary peak, compared to phase II. Activity-dependent expression of egr-1 was examined in free-ranging C. sphinx following the emissions and responses to a distress call. We found that the level of expression of egr-1 was higher in bats that emitted a distress call, in adults that responded, and in pups than in silent bats. Up-regulated cDNA was amplified to identify the target gene (TOE1) of the protein Egr-1. The observed expression pattern Toe1 was similar to that of egr-1. These findings suggest that the neuronal activity related to recognition of a distress call and an auditory feedback mechanism induces the expression of Egr-1. Co-expression of egr-1 with Toe1 may play a role in initial triggering of the genetic mechanism that could be involved in the consolidation or stabilization of distress call memories.

Genome-wide expression profiling identifies deregulated miRNAs in malignant astrocytoma

Malignant astrocytoma includes anaplastic astrocytoma (grade III) and glioblastoma (grade IV). Among them, glioblastoma is the most common primary brain tumor with dismal responses to all therapeutic modalities. We performed a large-scale, genome-wide microRNA (miRNA) (n=756) expression profiling of 26 glioblastoma, 13 anaplastic astrocytoma and 7 normal brain samples with an aim to find deregulated miRNA in malignant astrocytoma. We identified several differentially regulated miRNAs between these groups, which could differentiate glioma grades and normal brain as recognized by PCA. More importantly, we identified a most discriminatory 23-miRNA expression signature, by using PAM, which precisely distinguished glioblastoma from anaplastic astrocytoma with an accuracy of 95%. The differential expression pattern of nine miRNAs was further validated by real-time RT-PCR on an independent set of malignant astrocytomas (n-72) and normal samples (n=7). Inhibition of two glioblastoma-upregulated miRNAs (miR-21 and miR-23a) and exogenous overexpression of two glioblastoma-downregulated miRNAs (miR-218 and miR-219-5p) resulted in reduced soft agar colony formation but showed varying effects on cell proliferation and chemosensitivity. Thus we have identified the miRNA expression signature for malignant astrocytoma, in particular glioblastoma, and showed the miRNA involvement and their importance in astrocytoma development. Modern Pathology (2010) 23, 1404-1417; doi:10.1038/modpathol.2010.135; published online 13 August 2010

Expression of Sonic Hedgehog During Cell Proliferation in the Human Cerebellum

The regulation of cell proliferation in the external granular layer (EGL) of the developing cerebellum is important for its normal patterning. An important signal that regulates EGL cell proliferation is Sonic hedgehog (Shh). Shh is secreted by the Purkinje cells (PC) and has a mitogenic effect on the granule cell precursors of the EGL. Deregulation of Shh signaling has been associated with abnormal development, and been implicated in medulloblastomas, which are tumors that arise from the cerebellum. Given the importance of the Shh pathway in cerebellum development and disease, there has been no systematic study of its expression pattern during human cerebellum development. In this study, we describe the expression pattern of Shh, its receptor patched, smoothened, and its effectors that belong to the Gli family of transcription factors, during normal human cerebellum development from 10 weeks of gestational age, and in medulloblastomas that represents a case of abnormal cell proliferation in the cerebellum. This expression pattern is compared to equivalent stages in the normal development of cerebellum in mouse, as well as in tumors. Important differences between human and mouse that reflect differences in the normal developmental program between the 2 species are observed. First, in humans there appears to be a stage of Shh signaling within the EGL, when the PC are not yet the source of Shh. Second, unlike in the postnatal mouse cerebellum, expression of Shh in the PC in the postnatal human cerebellum is downregulated. Finally, medulloblastomas in the human but not in patched heterozygote mouse express Shh. These results highlight cross-species differences in the regulation of the Shh signaling pathway.

Mycobacterial Stress Regulation: The Dps ``Twin Sister'' Defense Mechanism and Structure-Function Relationship

In this work, we have tried to emphasize the connection between mycobacterial growth and regulation of gene expression. Utilization of multiple carbon sources and diauxic growth helps bacteria to regulate gene expression at an optimum level so that the inhospitable conditions encountered during nutrient depletion can be circumvented. These aspects will be discussed with respect to mycobacterial growth in subsequent sections. Identification and characterization of genes induced under such conditions is helpful to understand the physiology of the bacterium. Although it is necessary to compare the total expression profile of proteins as they transit from vegetative growth to stationary phase, at times a lot of insights can be deciphered from the expression pattern of one or two proteins. We have compared the protein expression and sigma factor selectivity of two such proteins in M. smegmatis to understand the differential regulation of genes playing diverse function in the same species. Some newer insights on the structure and function of one of the Dps proteins are also explained.

Primary Structural Documentation of the Major Urinary Protein of the Indian Commensal Rat (Rattus rattus) Using a Proteomics Platform

Purpose: A number of proteome studies have been performed recently to identify pheromone-related protein expression and their molecular function using genetically modified rodents' urine. However, no such studies have used Indian commensal rodents; interestingly, in a previous investigation, we confirmed the presence of volatile molecules in commensal rodents urine and these molecules seem to be actively involved in pheromonal communication. Therefore, the present study aims to identify the major urinary protein [MUP] present in commensal rat urine, which will help us to understand the protein's expression pattern and intrinsic properties among the rodents globally. Experimental Design: Initially, the total urinary proteins were separated by 1-D and 2-D electrophoresis and then subsequently analyzed by Matrix Assisted Laser Desorption Ionization-Time of Flight and Mass Spectrometer (MALDI-TOF/MS). Furthermore, they were then fragmented with the aid of a Tandem Mass Spectrometer (TOF/TOF) and the identified sequences aligned and confirmed using similarity with the deduced primary structures of members of the lipocalin superfamily.Results: The SDS-PAGE protein profiles showed distinct proteins with molecular masses of 15, 22.4, 25, 28, 42, 50, 55, 68, and 91 kDa. Of these proteins, the 22.4 kDa protein was considered to be target candidate. When 2D electrophoresis was carried out, about similar to 50 spots were detected with different masses and various pI ranges. The 22.4 kDa protein was found to have a pI of about 4.9. This 22.4 kDa protein spot was digested and subjected to mass spectrometry; it was identified as rat MUP. The fragmented peptides from the rat MUP at 935, 1026, 1192, and 1303 m/z were further fragmented with the aid of MS/MS and generated de novo sequence and this confirmed this protein to be the MUP present in the urine of commensal rats.Conclusion: The present investigation confirms the presence of MUP with a molecular mass of 22.4 kDa in the urine of commensal rats. This protein may be involved in the binding of volatile molecules and opens up a discussion about how volatile and non-volatile molecules in the commensal rats' urine may contribute chemo-communication.

Characterization of the gene encoding the envelope fusion glycoprotein GP64 from Bombyx mori nucleopolyhedrovirus

We describe here the characterization of the gene gp64 encoding the envelope fusion protein GP64 (open reading frame) ORF 105 from Bombyx mori nucleopolyhedrovirus (BmNPV). gp64 was transcribed from the early to late stages of infection and the transcripts were seen from 6 to 72 h post infection (hpi). The early transcripts initiated from a consensus CAGT motif while the late transcripts arose from three conserved TAAG motifs, all of which were located in the near upstream region of the coding sequence. Both early and late transcripts terminated at a run of T residues following the second polyadenylation signal located 31 nt downstream of the translation termination codon. BmGP64 protein was detectable from 6 hpi and was present in larger quantities throughout the infection process from 12 hpi, in BmNPV-infected BmN cells. The persistent presence of GP64 in BmN cells differed from the protein expression pattern of GP64 in Autographa californica multinucleocapsid nucleopolyhedrovirus infection, where the protein levels decreased significantly by late times (48 hpi). BmGP64 was located in the membrane and cytoplasm of the infected host cells and as a component of the budded virions. The production of infectious budded virus and the fusion activity were reduced when glycosylation of GP64 was inhibited. (C) 2003 Elsevier Science B.V. All rights reserved.

Genome wide chromatin occupancy of mrhl RNA and its role in gene regulation in mouse spermatogonial cells

Mrhl RNA is a nuclear lncRNA encoded in the mouse genome and negatively regulates Wnt signaling in spermatogonial cells through p68/Ddx5 RNA helicase. Mrhl RNA is present in the chromatin fraction of mouse spermatogonial Gc1-Spg cells and genome wide chromatin occupancy of mrhl RNA by ChOP (Chromatin oligo affinity precipitation) technique identified 1370 statistically significant genomic loci. Among these, genes at 37 genomic loci also showed altered expression pattern upon mrhl RNA down regulation which are referred to as GRPAM (Genes Regulated by Physical Association of Mrhl RNA). p68 interacted with mrhl RNA in chromatin at these GRPAM loci. p68 silencing drastically reduced mrhl RNA occupancy at 27 GRPAM loci and also perturbed the expression of GRPAM suggesting a role for p68 mediated mrhl RNA occupancy in regulating GRPAM expression. Wnt3a ligand treatment of Gc1-Spg cells down regulated mrhl RNA expression and also perturbed expression of these 27 GRPAM genes that included genes regulating Wnt signaling pathway and spermatogenesis, one of them being Sox8, a developmentally important transcription factor. We also identified interacting proteins of mrhl RNA associated chromatin fraction which included Pc4, a chromatin organizer protein and hnRNP A/B and hnRNP A2/B1 which have been shown to be associated with lincRNA-Cox2 function in gene regulation. Our findings in the Gc1-Spg cell line also correlate with the results from analysis of mouse testicular tissue which further highlights the in vivo physiological significance of mrhl RNA in the context of gene regulation during mammalian spermatogenesis.

High-level expression of biologically active glycoprotein hormones in Pichia pastoris strains—selection of strain GS115, and not X-33, for the production of biologically active N-glycosylated 15N-labeled phCG

The methylotrophic yeast Pichia pastoris is widely used for the production of recombinant glycoproteins. With the aim to generate biologically active 15N-labeled glycohormones for conformational studies focused on the unravelling of the NMR structures in solution, the P. pastoris strains GS115 and X-33 were explored for the expression of human chorionic gonadotropin (phCG) and human follicle-stimulating hormone (phFSH). In agreement with recent investigations on the N-glycosylation of phCG, produced in P. pastoris GS115, using ammonia/glycerol-methanol as nitrogen/carbon sources, the N-glycosylation pattern of phCG, synthesized using NH4Cl/glucose–glycerol–methanol, comprised neutral and charged, phosphorylated high-mannose-type N-glycans (Man8–15GlcNAc2). However, the changed culturing protocol led to much higher amounts of glycoprotein material, which is of importance for an economical realistic approach of the aimed NMR research. In the context of these studies, attention was also paid to the site specific N-glycosylation in phCG produced in P. pastoris GS115. In contrast to the rather simple N-glycosylation pattern of phCG expressed in the GS115 strain, phCG and phFSH expressed in the X-33 strain revealed, besides neutral high-mannose-type N-glycans, also high concentrations of neutral hypermannose-type N-glycans (Manup-to-30GlcNAc2). The latter finding made the X-33 strain not very suitable for generating 15N-labeled material. Therefore, 15N-phCG was expressed in the GS115 strain using the new optimized protocol. The 15N-enrichment was evaluated by 15N-HSQC NMR spectroscopy and GLC-EI/MS. Circular dichroism studies indicated that 15N-phCG/GS115 had the same folding as urinary hCG. Furthermore, 15N-phCG/GS115 was found to be similar to the unlabeled protein in every respect as judged by radioimmunoassay, radioreceptor assays, and in vitro bioassays.

High-yield bacterial expression and structural characterization of recombinant human insulin-like growth factor binding protein-2

The diverse biological activities of the insulin-like growth factors (IGF-1 and IGF-2) are mediated by the IGF-1 receptor (IGF-1R). These actions are modulated by a family of six IGF-binding proteins (ICFBP-1-6; 22-31 kDa) that via high affinity binding to the IGFs (K-D similar to 300-700 pM) both protect the IGFs in the circulation and attenuate IGF action by blocking their receptor access In recent years, IGFBPs have been implicated in a variety of cancers However, the structural basis of their interaction with IGFs and/or other proteins is not completely understood A critical challenge in the structural characterization of full-length IGFBPs has been the difficulty in expressing these proteins at levels suitable for NMR/X-ray crystallography analysis Here we describe the high-yield expression of full-length recombinant human IGFBP-2 (rhIGFBP-2) in Eschericha coli Using a single step purification protocol, rhIGFBP-2 was obtained with >95% purity and structurally characterized using NMR spectroscopy. The protein was found to exist as a monomer at the high concentrations required for structural studies and to exist in a single conformation exhibiting a unique intra-molecular disulfide-bonding pattern The protein retained full biologic activity. This study represents the first high-yield expression of wild-type recombinant human IGFBP-2 in E coli and first structural characterization of a full-length IGFBP (C) 2010 Elsevier Inc. All rights reserved

Differential expression of estrogen receptor alpha in the embryonic adrenal-kidney-gonadal complex of the oviparous lizard, Calotes versicolor (Daud.)

Estrogen signalling is critical for ovarian differentiation in reptiles with temperature-dependent sex determination (TSD). To elucidate the involvement of estrogen in this process, adrenal-kidney-gonadal (AKG) expression of estrogen receptor (ER alpha) was studied at female-producing temperature (FPT) in the developing embryos of the lizard, Calotes versicolor which exhibits a distinct pattern of TSD. The eggs of this lizard were incubated at 31.5 +/- 0.5 degrees C (100% FPT). The torso of embryos containing adrenal-kidney-gonadal complex (AKG) was collected during different stages of development and subjected to Western blotting and immunohistochemistry analysis. The ER alpha, antibody recognized two protein bands with apparent molecular weight similar to 55 and similar to 45 kDa in the total protein extracts of embryonic AKG complex of C. versicolor. The observed results suggest the occurrence of isoforms of ER alpha. The differential expression of two different protein isoforms may reveal their distinct role in cell proliferation during gonadal differentiation. This is the first report to reveal two isoforms of the ER alpha in a reptile during development. Immunohistochemical studies reveal a weak, but specific, cytoplasmic ER alpha immunostaining exclusively in the AKG during late thermo-sensitive period suggesting the responsiveness of AKG to estrogens before gonadal differentiation at FPT. Further, cytoplasmic as well as nuclear expression of ER alpha in the medulla and in oogonia of the cortex (faint activity) at gonadal differentiation stage suggests that the onset of gonadal estrogen activity coincides with sexual differentiation of gonad. Intensity and pattern of the immunoreactions of ER alpha in the medullary region at FPT suggest endogenous production of estrogen which may act in a paracrine fashion to induce neighboring cells into ovarian differentiation pathway. (C) 2014 Elsevier Inc. All rights reserved.

Divergence in Patterns of Leaf Growth Polarity Is Associated with the Expression Divergence of miR396

Lateral appendages often show allometric growth with a specific growth polarity along the proximo-distal axis. Studies on leaf growth in model plants have identified a basipetal growth direction with the highest growth rate at the proximal end and progressively lower rates toward the distal end. Although the molecular mechanisms governing such a growth pattern have been studied recently, variation in leaf growth polarity and, therefore, its evolutionary origin remain unknown. By surveying 75 eudicot species, here we report that leaf growth polarity is divergent. Leaf growth in the proximo-distal axis is polar, with more growth arising from either the proximal or the distal end; dispersed with no apparent polarity; or bidirectional, with more growth contributed by the central region and less growth at either end. We further demonstrate that the expression gradient of the miR396-GROWTH-REGULATING FACTOR module strongly correlates with the polarity of leaf growth. Altering the endogenous pattern of miR396 expression in transgenic Arabidopsis thaliana leaves only partially modified the spatial pattern of cell expansion, suggesting that the diverse growth polarities might have evolved via concerted changes in multiple gene regulatory networks.