Open Reading Frame Of Gene Research Articles

Novel D-Galactose Isomerases from Lactobacillus Strains Isolated from the Sweet Sap of Agave atrovirens

D-tagatose is a natural monosaccharide used as a low-calorie sugar substitute in food, beverage and pharmaceutical products. Although it is a rare sugar, it can manufacture by enzymatic isomerization of D-galactose Isomerase (D-GI). In this study, a screening was carried out to search microorganism producing D-GI in aguamiel. A rich selective medium was used in arabinose extracted from gum arabic. From 98 isolates obtained aguamiel of agave pulquero (Agave atrovirens), it was obtained 4 strains of Lactobacillus that producing D-GI and 2 strains of Bacillus sp. The Podi-20 strain was identified as Lactobacillus diolivorans based on 16S rRNA analysis, biological and biochemical characteristics. Furthermore, the gene encoding D-GI from L. diolivorans Podi-20 strain. Analysis of sequence revealed that the Open Reading Frame (ORF) of the araA gene consist of 1,428 pb that encodes a protein of 476 amino acid residues. The bioconversion yield of D-galactose to D-tagatose by the purified D-GI after 14 h at 60°C was 31.4%. D-tagatose can manufacture by enzymatic isomerization. This study contributes to new knowledge on D-GI from Lactobacillus strains, in particular those isolated from artisanal functional foods from Agave.

Co-infection with human polyomavirus BK enhances gene expression and replication of human adenovirus

Immunocompromised patients are susceptible to multiple viral infections. Relevant interactions between co-infecting viruses might result from viral regulatory genes which trans-activate or repress the expression of host cell genes as well as the genes of any co-infecting virus. The aim of the current study was to show that the replication of human adenovirus 5 is enhanced by co-infection with BK polyomavirus and is associated with increased expression of proteins including early region 4 open reading frame 1 and both the large tumor antigen and small tumor antigen. Clinical samples of whole blood and urine from 156 hematopoietic stem cell transplant recipients were tested. We also inoculated adenocarcinomic human alveolar basal epithelial cells with both human adenovirus 5 and BK polyomavirus to evaluate if co-infection of viruses affected their replication. Data showed that adenovirus load was significantly higher in the plasma (mean 7.5 x 103 ± 8.5 x 102 copies/ml) and urine (mean 1.9 x 103 ± 8.0 x 102 copies/ml) of samples from patients with co-infections, in comparison to samples from patients with isolated adenovirus infection. In vitro co-infection led to an increased (8.6 times) expression of the adenovirus early region 4 open reading frame gene 48 hours post-inoculation. The expression of the early region 4 open reading frame gene positively correlated with the expression of BK polyomavirus large tumor antigen (r = 0.90, p < 0.0001) and small tumor antigen (r = 0.83, p < 0.001) genes. The enhanced expression of the early region 4 open reading frame gene due to co-infection with BK polyomavirus was associated with enhanced adenovirus, but not BK polyomavirus, replication. The current study provides evidence that co-infection of adenovirus and BK polyomavirus contributes to enhanced adenovirus replication. Data obtained from this study may have significant importance in the clinical setting.

Methods to Investigate the Molecular Basis of Progranulin Actions on Brain and Behavior In Vivo Using Knockout Mice.

Currently one of the few molecules that equally excites a neuroscientist, a cancer biologist, an immunologist, and a developmental biologist is progranulin (GRN/Grn)-a pluripotent growth factor that plays key roles in cell survival, proliferation, development, tissue regeneration, inflammation, wound healing, and angiogenesis. However, the molecular pathways associated with GRN signaling involved in these varied physiological processes are not understood. Gene inactivation has been considered as one of the best methods to delineate the biological role of a protein, and gene targeting is a direct means to disrupt a gene's open reading frame and block its expression, for instance, in a mouse. Such a gene knockout animal model also served as an in vivo disease model where loss of gene or its function is thought to be the primary disease mechanism, as is the case with progranulin loss of function in frontotemporal lobar degeneration (FTLD). It is estimated that up to half of the cases of familial, dominant FTLD might be due to GRN haploinsufficiency. To understand the molecular pathways associated with GRN loss, constitutive and conditional progranulin knockout (Grn-/-) mice have also been constructed in several laboratories, including ours. These mice show several disease-characteristic features and suggest that continued studies on the Grn-/- mice would be instructive in the understanding of complex GRN biology in health and disease.

Bioinformatics and expression analysis of NAC transcription factor genes in Scutellaria baicalensis

Background: NAC, as a unique transcription factor to plants, plays important roles in multiple biological functions, such as regulation of plant growth and development, hormone levels, and responses to various kinds of stresses. However, there is a lack of research of NAC genes in Chinese herbs. Objective: The study aimed to evaluate the potential functions of NAC genes in Scutellaria baicalensis by bioinformatics and expression analysis, and provide evidence of the molecular regulation mechanism involved in flavonoid biosynthesis in S. baicalensis. Methods: The genes of NAC transcription factors in S. baicalensis were obtained from cDNA library and their functions were explored using bioinformatic methods. The NAC genes were screened from the cDNA library of S. baicalensis using BLAST comparison software. Then, the open reading frame (ORF) finder online tool was used to predict the full-length ORFs of NAC genes and their protein characteristics were explored by bioinformatic methods. The expression of NAC genes was then detected by quantitative polymerase chain reaction in different parts of S. baicalensis and different leaves treated by gibberellin GA3 treatment. Results: Six genes of NAC transcription factors were cloned, two of which had complete ORFs. NAC genes cloned in this study were mainly expressed in the flowers of S. baicalensis. The expression levels of NAC2, NAC3, NAC4, NAC5, NAC6 were increased firstly and then decreased gradually after 100 μM GA3 treatment. Meanwhile, some NACs and PAL2 in S. baicalensis showed strong correlation. Conclusion: This study suggested that NACs cloned in this study were mainly regulated the flavonoid biosynthesis in the flowers of S. baicalensis; NAC6 in S. baicalensis might be involved in the regulation of PAL2 transcription and affected the accumulation of flavonoids in the root of S. baicalensis. Our results provided a basis for further understanding the molecular regulation mechanism of flavonoid biosynthesis in S. baicalensis.

Histopathology and Detection of Cyprinid Herpesvirus Infection in Two Koi Farms in Tianjin City (China)

Koi herpesvirus (KHV) is the etiologic agent of koi herpesvirus disease (KHVD) causing mass mortalities in common carp (Cyprinus carpio carpio), koi (Cyprinus carpio koi), and ghost carp (Cyprinus carpio goi) populations. In this study, we report new outbreaks of the koi herpesvirus (KHV) disease in a koi carp farm in Tianjin city, northern China. From June to September 2017, severe mortalities of koi juvenile and broodstock occurred in ornamental fish, and infected koi carp which showed signs of lethargy and loss of appetite, and clinical signs including sunken eyes, gill necrosis, and excessive secretion of mucus. Histopathologically, the gills showed hyperplasia and degeneration of epithelial cells and fusion of the lamellae, and foci necrosis was found in gills, liver, kidney, and spleen. In addition, KHV and Carp edema virus (CEV) were detected in the diseased koi fish by diagnostic PCR tests for the viruses. The results showed that the nucleotide sequences of thymidine kinase (TK), DNA polymerase (SphI-5), and four open reading frame (ORF 25, ORF 56, ORF 72, ORF 81) genes confirmed the highest identity with other KHV strains such as KHV-I, KHV-U and KHV-J, but the CEV was negative. The phylogenetic tree showed KHV-TJ1708 and KHV-TJ1709 clustered with the Asian strain KHV-J (AP008984). Results of the present study confirmed the prevalence of Carp herpes disease (KHVD) in China koi populations by means of clinical examination, histopathology and molecular technique. These data will provide a reference for diagnosis, quarantine, and surveillance of KHV in China.

Varicella-Zoster Virus Expresses Multiple Small Noncoding RNAs.

Many herpesviruses express small noncoding RNAs (sncRNAs), including microRNAs (miRNAs), that may play roles in regulating lytic and latent infections. None have yet been reported in varicella-zoster virus (VZV; also known as human herpesvirus 3 [HHV-3]). Here we analyzed next-generation sequencing (NGS) data for small RNAs in VZV-infected fibroblasts and human embryonic stem cell-derived (hESC) neurons. Two independent bioinformatics analyses identified more than 20 VZV-encoded 20- to 24-nucleotide RNAs, some of which are predicted to have stem-loop precursors potentially representing miRNAs. These sequences are perfectly conserved between viruses from three clades of VZV. One NGS-identified sequence common to both bioinformatics analyses mapped to the repeat regions of the VZV genome, upstream of the predicted promoter of the immediate early gene open reading frame 63 (ORF63). This miRNA candidate was detected in each of 3 independent biological repetitions of NGS of RNA from fibroblasts and neurons productively infected with VZV using TaqMan quantitative PCR (qPCR). Importantly, transfected synthetic RNA oligonucleotides antagonistic to the miRNA candidate significantly enhanced VZV plaque growth rates. The presence of 6 additional small noncoding RNAs was also verified by TaqMan qPCR in productively infected fibroblasts and ARPE19 cells. Our results show VZV, like other human herpesviruses, encodes several sncRNAs and miRNAs, and some may regulate infection of host cells.IMPORTANCE Varicella-zoster virus is an important human pathogen, with herpes zoster being a major health issue in the aging and immunocompromised populations. Small noncoding RNAs (sncRNAs) are recognized as important actors in modulating gene expression, and this study demonstrates the first reported VZV-encoded sncRNAs. Many are clustered to a small genomic region, as seen in other human herpesviruses. At least one VZV sncRNA was expressed in productive infection of neurons and fibroblasts that is likely to reduce viral replication. Since sncRNAs have been suggested to be potential targets for antiviral therapies, identification of these molecules in VZV may provide a new direction for development of treatments for painful herpes zoster.

The complete chloroplast genome sequence of Oryza sativa aus-type variety Nagina-22 (Poaceae)

Rice (Oryza sativa) is the predominant staple food crop belonging to the Poaceae family. In this study, complete chloroplast genome sequence of O. sativa aus-type variety Nagina-22 was characterized through de novo assembly. The genome is a circular DNA molecule of 134,503 bp and has typical quadripartite structures including large single copy region (80,548 bp), small single copy region (12,347 bp), and a pair of inverted repeats (20,804 bp). A total of 120 genes were predicted in the genome, including 77 protein-coding genes, 8 open reading frame genes, 31 tRNA genes, and 4 rRNA genes. Phylogenetic analysis confirmed a close taxonomical relationship with O. sativa ssp. Indica species.

Completed sequence and corrected annotation of the genome of maize Iranian mosaic virus.

Maize Iranian mosaic virus (MIMV) is a negative-sense single-stranded RNA virus that is classified in the genus Nucleorhabdovirus, family Rhabdoviridae. The MIMV genome contains six open reading frames (ORFs) that encode in 3΄ to 5΄ order the nucleocapsid protein (N), phosphoprotein (P), putative movement protein (P3), matrix protein (M), glycoprotein (G) and RNA-dependent RNA polymerase (L). In this study, we determined the first complete genome sequence of MIMV using Illumina RNA-Seq and 3'/5' RACE. MIMV genome ('Fars' isolate) is 12,426 nucleotides in length. Unexpectedly, the predicted N gene ORF of this isolate and of four other Iranian isolates is 143 nucleotides shorter than that of the MIMV coding-complete reference isolate 'Shiraz 1' (Genbank NC_011542), possibly due to a minor error in the previous sequence. Genetic variability among the N, P, P3 and G ORFs of Iranian MIMV isolates was limited, but highest in the G gene ORF. Phylogenetic analysis of complete nucleorhabdovirus genomes demonstrated a close evolutionary relationship between MIMV, maize mosaic virus and taro vein chlorosis virus.

Cloning and Expression of a Secretory form of Truncated ORF2 (aa 112-607) from Hepatitis E Virus in the pVAX1 Vector

Background: The hepatitis E virus (HEV) accounts for the hepatitis E infection with a high mortality rate in pregnant women. Therefore, the design of the novel and effective vaccines seems essential and the DNA vaccine approach could be useful to achieve this anticipated goal. Objectives: The aim of this study was the cloning of a secretory form of truncated open reading frame 2 (ORF2) of HEV containing amino acids (aa) 112 - 607 into the eukaryotic expression pVAX1 Vector and evaluating of the expression of this recombinant protein in eukaryotic cells. Methods: The truncated ORF2 gene (aa 112 - 607) was cloned in the pVAX1 plasmid by restriction enzyme digest and confirmed by digestion and sequencing. Then, the recombinant plasmid was transfected into eukaryotic cells to express the recombinant protein. The expressed protein in the cell lysate and supernatant was evaluated by immunofluorescence assay (IFA) and western blot assay. Results: Colony polymerase chain reactions (PCR), restriction enzyme digestion, along with DNA sequencing of the recombinant plasmid were performed for confirmation of cloning tPA-PADRE-truncated ORF2 gene (aa 112 - 607) into pVAX1 eukaryotic expression vector. The appearance of the truncated ORF2 protein (56 kDa) in the eukaryotic cells was accepted by the western blot assay, reverse transcription polymerase chain reaction (RT-PCR) method, as well as IFA. Conclusions: All outcomes of the present research showed that pVAX1-tPA-PADRE-truncated ORF2 (aa 112 - 607, 56 kDa) recombinant plasmid was able to express truncated ORF2 from HEV as a potential candidate vaccine.

Molecular cloning and expression analysis of major intrinsic protein gene in Chlamydomonas sp. ICE-L from Antarctica.

Major intrinsic proteins (MIPs) form channels facilitating the passive transport of water and other small polar molecules across membranes. In this study, the complete open reading frame (ORF) of CiMIP1 (GenBank ID KY316061) encoding one kind of MIPs in the Antarctic ice microalga Chlamydomonas sp. ICE-L is successfully cloned using RACE. In addition, the expression patterns of CiMIP1 gene under different conditions of temperature and salinity are determined by qRT-PCR. The ORF of CiMIP1 gene encodes 308 amino acids, and the deduced amino acid sequence shows 74% homology with Chlamydomonas reinhardtii CrMIP1 (GenBank number 159471952). Phylogenetic analysis reveals that algal MIPs are divided into seven groups, and it is speculated that CiMIP1 most likely belongs to the MIPD subfamily. In addition, we are surprised to find that a third NPA motif exists at the carboxy terminus of the target protein except for two highly conserved ones. Expression analysis shows that the transcriptional levels of CiMIP1 gene are upregulated under either lower temperature or higher temperature and high salinity. In summary, the results together have provide new insights into the newly discovered gene in green algae and lay the foundation for further studies on the adaptation mechanism of Chlamydomonas sp. ICE-L to abiotic stresses.

The Genomic Architecture of Novel Simulium damnosum Wolbachia Prophage Sequence Elements and Implications for Onchocerciasis Epidemiology.

Research interest in Wolbachia is growing as new discoveries and technical advancements reveal the public health importance of both naturally occurring and artificial infections. Improved understanding of the Wolbachia bacteriophages (WOs) WOcauB2 and WOcauB3 [belonging to a sub-group of four WOs encoding serine recombinases group 1 (sr1WOs)], has enhanced the prospect of novel tools for the genetic manipulation of Wolbachia. The basic biology of sr1WOs, including host range and mode of genomic integration is, however, still poorly understood. Very few sr1WOs have been described, with two such elements putatively resulting from integrations at the same Wolbachia genome loci, about 2 kb downstream from the FtsZ cell-division gene. Here, we characterize the DNA sequence flanking the FtsZ gene of wDam, a genetically distinct line of Wolbachia isolated from the West African onchocerciasis vector Simulium squamosum E. Using Roche 454 shot-gun and Sanger sequencing, we have resolved >32 kb of WO prophage sequence into three contigs representing three distinct prophage elements. Spanning ≥36 distinct WO open reading frame gene sequences, these prophage elements correspond roughly to three different WO modules: a serine recombinase and replication module (sr1RRM), a head and base-plate module and a tail module. The sr1RRM module contains replication genes and a Holliday junction recombinase and is unique to the sr1 group WOs. In the extreme terminal of the tail module there is a SpvB protein homolog—believed to have insecticidal properties and proposed to have a role in how Wolbachia parasitize their insect hosts. We propose that these wDam prophage modules all derive from a single WO genome, which we have named here sr1WOdamA1. The best-match database sequence for all of our sr1WOdamA1-predicted gene sequences was annotated as of Wolbachia or Wolbachia phage sourced from an arthropod. Clear evidence of exchange between sr1WOdamA1 and other Wolbachia WO phage sequences was also detected. These findings provide insights into how Wolbachia could affect a medically important vector of onchocerciasis, with potential implications for future control methods, as well as supporting the hypothesis that Wolbachia phages do not follow the standard model of phage evolution.

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast.

We describe a PCR- and homologous recombination-based system for generating targeted mutations in histone genes in budding yeast cells. The resulting mutant alleles reside at their endogenous genomic sites and no exogenous DNA sequences are left in the genome following the procedure. Since in haploid yeast cells each of the four core histone proteins is encoded by two non-allelic genes with highly homologous open reading frames (ORFs), targeting mutagenesis specifically to one of two genes encoding a particular histone protein can be problematic. The strategy we describe here bypasses this problem by utilizing sequences outside, rather than within, the ORF of the target genes for the homologous recombination step. Another feature of this system is that the regions of DNA driving the homologous recombination steps can be made to be very extensive, thus increasing the likelihood of successful integration events. These features make this strategy particularly well-suited for histone gene mutagenesis, but can also be adapted for mutagenesis of other genes in the yeast genome.

UspA Gene Based Characterization of Escherichia coli Strains Isolated from Different Disease Conditions in Goats

Escherichia coli (E. coli) carry six universal stress protein (usp) genes: A, C, D, E, F and G, and the expression of these genes are triggered by various environmental stresses. The uspA gene is important for survival of E. coli during cellular growth, adhesion and motility. The present study was conducted to characterize three pathogenic E. coli strains isolated from the cases of diarrhoea, pneumonia and mastitis in goats. A polymerase chain reaction (PCR) was performed to amplify 884 bp open reading frame (ORF) of the uspA gene from the E. coli strains. The uspA amplicons of the three E. coli strains were sequenced, and compared with the published sequences in NCBI GenBank, and their phylogenetic relationships were analysed. The diarrheic strain showed significant variation in the nucleotide composition as compared to pneumonia and mastitis associated strains. In the ORF of uspA gene, silent mutations were noticed in the nucleotide sequence positions 27, 33, 207 and 316, which were not reflected phenotypically. Among the peptides, ‘KHILIAVDLS’ could be a putative candidate for use as epitope in diagnostics. Further, comprehensive studies on sequence analysis of the uspA gene will help us to understand the distribution, variability, and phylogenetic relationships of different pathogenic E. coli isolated from different disease conditions in goats.

Molecular Cloning and Characterization of Two Key Enzymes involved in the Diterpenoid Biosynthesis Pathway of Isodon rubescens

Isodon rubescens, an important medical plant, contains various terpenoids. This plant’s active compounds are primarily oridonin with antitumor properties. As the precursor for oridonin biosynthesis, are synthesized by MEP pathway. On the basis of our earlier studies, we isolated and cloned two important genes catalyzing diterpenoid biosynthesis in the MEP pathway. 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase and 4-hydroxy-3- methylbut-2-enyl diphosphate reductase are the fifth enzymes and the last step key enzyme for the methylerythritol phosphate (MEP) pathway, respectively, which is important for the regulation of isoprenoid biosynthesis. Sequence analysis revealed that DcIspF (accession no. KT948057) was 966 bp, contains a gene open reading frame (ORF) of 708 bp belonging to the MECDP-synthase superfamily and DcIspH (accession no KT948058) contains a 1389 bp ORF encoding a predicted 462 amino acid polypeptides as a member of the lytB_ispH superfamily. The deduced DcIspF and DcIspH amino acid sequences shared high similarity with DcIspF and DcIspH of other plant respectively, each of them exhibiting an N-terminal transit peptide and conserved amino acid sites. Quantitative real-time PCR analysis showed that the expression of DcIspF was considerably higher in leaves, the lowest in callus. These results indicate that we have identified functional DcIspF and DcIspH enzymes, which may play a pivotal role in the biosynthesis of diterpenoid in I. rubescens.

Molecular Cloning, Expression Profiling, and Marker Validation of the Chicken Myoz3 Gene.

Myozenin3 (Myoz3) has been reported to bind multiple Z-disc proteins and hence play a key role in signal transduction and muscle fiber type differentiation. The purpose of current study is to better understand the basic characteristics of Myoz3. Firstly, we cloned the ORF (open reading frame) of the Myoz3 gene. AA (amino acid) sequence analysis revealed that the Myoz3 gene encodes a 26 kDa protein which have 97% identities with that of turkey. Expression profiling showed that Myoz3 mRNA is mainly expressed in leg muscle and breast muscle. Furthermore, we investigated Myoz3 gene polymorphisms in two broiler breeds, the Yellow Bantam (YB) and the Avian. Five SNPs (single nucleotide polymorphisms) were identified in the YB breed and 3 were identified in the Avian breed. Genotypes and haplotype were constructed and their associations with carcass traits were analyzed. In the YB breed, c.516 C>T had a strong effect on both shank bone length and the value of breast muscle, and the H1H3 diplotype had the highest FC compared to other diplotypes. The markers identified in this study may serve as useful targets for the marker-assisted selection (MAS) of growth and meat quality traits in chickens.

Recombinant Proteins and Monoclonal Antibodies.

The human genome has become a subject of public interest, whilst the proteome remains the province of specialists. Less appreciated is the human glycoprotein (GP) repertoire (proteoglycome!); however, some 50% of open reading frame genes encode for proteins (P) that may accept the addition of N-linked and/or O-linked sugar chains (oligosaccharides). It is established that the attachment of defined oligosaccharide structures impacts mechanisms of action (MoAs), pharmacokinetics, pharmacodynamics, etc., and is a critical quality attribute (CQA) for recombinant GP therapeutics. The oligosaccharide structure attached at a given site may exhibit structural heterogeneity, and individual structures (glycoforms) may modulate MoAs. The biopharmaceutical industry is challenged, therefore, to produce recombinant GP therapeutics that have structural fidelity to the natural (endogenous) molecule, in non-human cells. Multiple production platforms have been developed that, in addition to the natural glycoform, may produce unnatural glycoforms, including sugar residues that can be immunogenic in human subjects. Following a general introduction to the field, this review discusses glycosylation of recombinant monoclonal antibodies (mAbs), the contribution of glycoforms to MoAs and the development of customised mAb therapeutic glycoforms to optimise MoAs for individual disease indications.

Inhibition of JCPyV infection mediated by targeted viral genome editing using CRISPR/Cas9.

Progressive multifocal leukoencephalopathy (PML) is a debilitating disease resulting from infection of oligodendrocytes by the JC polyomavirus (JCPyV). Currently, there is no anti-viral therapeutic available against JCPyV infection. The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system (CRISPR/Cas9) is a genome editing tool capable of introducing sequence specific breaks in double stranded DNA. Here we show that the CRISPR/Cas9 system can restrict the JCPyV life cycle in cultured cells. We utilized CRISPR/Cas9 to target the noncoding control region and the late gene open reading frame of the JCPyV genome. We found significant inhibition of virus replication and viral protein expression in cells recipient of Cas9 together with JCPyV-specific single-guide RNA delivered prior to or after JCPyV infection.

Identification of two new keratinolytic proteases from a Bacillus pumilus strain using protein analysis and gene sequencing.

The Bacillus strain (CCUG 66887) has a high capacity to excrete keratinase with the ability to degrade both alpha- and beta keratin. In this study we aimed to show the characteristics of the keratinolytic protease and to identify its gene by using liquid chromatography–electrospray ionization tandem mass spectrometry methods (nanoHPLC–ESI–MS/MS) followed by Mascot data base search. The results showed that the enzyme in fact consists of two different keratinases, both with a molecular mass of 38 kDa. Further, DNA sequencing generated the open reading frame (ORF) of one of the genes (Ker1), and de novo genome sequencing identified the ORF of the second gene (Ker2). The two keratinase genes contain 1153 base pairs each and have a gene similarity of 67 %. In addition, the Bacillus strain was classified as Bacillus pumilus and its genes were annotated in the GeneBank at NCBI (accession: CP011109.1). Amino acid sequences alignment with known B. pumilus proteases indicated that the two keratinases of B. pumilus strain C4 are subtilisin-like serine proteases belonging to the Protease S8 family. Taken together, these result suggest the two keratinases as promising candidates for enzymatic processing of keratinous wastes in waste refinery.

Tumour-specific triple-regulated oncolytic herpes virus to target glioma.

Oncolytic herpes simplex virus type 1 (oHSV-1) therapy is an emerging treatment modality that selectively destroys cancer. Here we report use of a glioma specific HSV-1 amplicon virus (SU4-124 HSV-1) to selectively target tumour cells. To achieve transcriptional regulation of the SU4-124 HSV-1 virus, the promoter for the essential HSV-1 gene ICP4 was replaced with a tumour specific survivin promoter. Translational regulation was achieved by incorporating 5 copies of microRNA 124 target sequences into the 3′UTR of the ICP4 gene. Additionally, a 5′UTR of rat fibroblast growth factor -2 was added in front of the viral ICP4 gene open reading frame. Our results confirmed enhanced expression of survivin and eIF4E in different glioma cells and increased micro-RNA124 expression in normal human and mouse brain tissue. SU4-124 HSV-1 had an increased ICP4 expression and virus replication in different glioma cells compared to normal neuronal cells. SU4-124 HSV-1 exerted a strong antitumour effect against a panel of glioma cell lines. Intracranial injection of SU4-124 HSV-1 did not reveal any sign of toxicity on day 15 after the injection. Moreover, a significantly enhanced antitumour effect with the intratumourally injected SU4-124 HSV-1 virus was demonstrated in mice bearing human glioma U87 tumours, whereas viral DNA was almost undetectable in normal organs. Our study indicates that incorporation of multiple cancer-specific regulators in an HSV-1 system significantly enhances both cancer specificity and oncolytic activity.

Cloning and molecular characterization of a putative voltage-gated sodium channel gene in the crayfish.

Voltage-gated sodium channel genes and associated proteins have been cloned and studied in many mammalian and invertebrate species. However, there is no data available about the sodium channel gene(s) in the crayfish, although the animal has frequently been used as a model to investigate various aspects of neural cellular and circuit function. In the present work, by using RNA extracts from crayfish abdominal ganglia samples, the complete open reading frame of a putative sodium channel gene has firstly been cloned and molecular properties of the associated peptide have been analyzed. The open reading frame of the gene has a length of 5793 bp that encodes for the synthesis of a peptide, with 1930 amino acids, that is 82% similar to the α-peptide of a sodium channel in a neighboring species, Cancer borealis. The transmembrane topology analysis of the crayfish peptide indicated a pattern of four folding domains with several transmembrane segments, as observed in other known voltage-gated sodium channels. Upon analysis of the obtained sequence, functional regions of the putative sodium channel responsible for the selectivity filter, inactivation gate, voltage sensor, and phosphorylation have been predicted. The expression level of the putative sodium channel gene, as defined by a qPCR method, was measured and found to be the highest in nervous tissue.