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At least 649 records · Page 36Linked to original sources
PubMed2003

Analysis of intermolecular RNA-RNA recombination by rubella virus.

To investigate whether rubella virus (RUB) undergoes intermolecular RNA-RNA recombination, cells were cotransfected with pairs of in vitro transcripts from genomic cDNA plasmid vectors engineered to contain nonoverlapping deletions: the replicative transcript maintained the 5'-proximal nonstructural (NS) ORF (which contained the replicase, making it RNA replication competent), had a deletion in the 3'-proximal structural protein (SP) ORF, and maintained the 3' end of the genome, including the putative 3' cis-acting elements (CSE), while the nonreplicative transcript consisted of the 3' half of the genome including the SP-ORF and 3' CSE. Cotransfection yielded plaque-forming virus that synthesized the standard genomic and subgenomic RNAs and thus was generated by RNA-RNA recombination. Using transcripts tagged with a 3'-terminal deletion, it was found that recombinants contained the 3' end derived from the replicative strand, indicating a cis-preference for initiation of negative-strand synthesis. In cotransfections in which the replicative transcript lacked the 3' CSE, recombination occurred, albeit at lower efficiency, indicating that initiation in trans from the NS-ORF can occur. The 3' CSE was sufficient as a nonreplicative transcript, showing that it can serve as a promoter for negative-strand RNA synthesis. While deletion mutagenesis showed that the presence of the junction untranslated region (J-UTR) between the ORFs appeared to be necessary on both transcripts for recombination in this region of the genome, analysis with transcripts tagged with restriction sites showed that the J-UTR was not a hot spot for recombination compared to neighboring regions in both ORFs. Sequence analysis of recombinants revealed that both precise (homologous) and imprecise recombination (aberrant, homologous resulting in duplications) occurred; however, imprecise recombination only involved the J-UTR or the 3' end of the NS-ORF and the J-UTR (maintaining the NS-ORF), indicating selection pressure against duplications in other regions of the genome.

3' Untranslated Regions↗
PubMed2000

Production of recombinant snakehead rhabdovirus: the NV protein is not required for viral replication.

Snakehead rhabdovirus (SHRV) affects warm water fish in Southeast Asia and belongs to the genus Novirhabdovirus by virtue of its nonvirion gene (NV). Because SHRV grows best at temperatures between 28 and 31 degrees C, we were able to use the T7 expression system to produce viable recombinant SHRV from a cloned cDNA copy of the viral genome. Expression of a positive-strand RNA copy of the 11, 550-nucleotide SHRV genome along with the viral nucleocapsid (N), phosphoprotein (P), and polymerase (L) proteins resulted in the generation of infectious SHRV in cells preinfected with a vaccinia virus vector for T7 polymerase expression. Recombinant virus production was verified by detection of a unique restriction site engineered into the SHRV genome between the NV and L genes. Since we were now able to begin examining the function of the NV gene, we constructed a recombinant virus containing a nonsense mutation located 22 codons into the coding sequence of the NV protein. The NV knockout virus was produced at a concentration as high as that of wild-type virus in cultured fish cells, and the resulting virions appeared to be identical to the wild-type virions in electron micrographs. These initial studies suggest that NV has no critical function in SHRV replication in cultured fish cells.

Amino Acid Sequence↗
PubMed1998

Generation of metastatic variants by transfection of a rat non-metastatic epithelial cell line with genomic DNA from rat prostatic carcinoma cells.

Prostate cancer is the second leading cause of male death from malignant disease in Europe and in the USA. Failure to prevent or eliminate metastatic dissemination is a fundamental problem underlying the current inadequate treatment of prostate cancer, and novel therapeutic strategies are required if this disease is to be successfully managed. No independent markers are yet available to predict the behaviour of any individual prostate cancer, particularly its potential to metastasize, and there is now an urgent prerequisite to identify and characterize genes specifically involved in determining the metastatic phenotype of prostate cancer cells before any biologically appropriate treatment modality can be devised. To identify DNA sequences that trophically promote the metastatic phenotype, we have established a new transfection assay with which to monitor activity of prostate cancer genomic DNA. Rat prostatic G and AT6.1 cell lines derived from the same original Dunning R3327 rat prostatic carcinoma exhibit, respectively, low- and high-metastatic phenotypes when grown in syngeneic Copenhagen rats. Rat mammary epithelial cell line 'Rama 37' derived originally from Wistar-Furth rats yields benign non-metastasizing adenomas when inoculated subcutaneously into syngeneic animals. In this report, the Rama 37 cell line is successfully used as the recipient cell-line for transfected DNA fragments extracted from rat prostatic carcinoma G and AT6.1 cells. New metastatic variants of Rama 37 cells have been generated. Enzymatically fragmented genomic DNA from rat metastatic prostate carcinoma cell lines was co-transfected together with plasmid pSV2neo into parental Rama 37 cells, followed by culture in the presence of Geneticin-G418 to select for the transfected cells. To enable subsequent identification of metastasis-promoting DNA sequences, the fragmented genomic DNA sequences were covalently attached to specifically engineered linker DNA molecules to flank the genomic DNA before transfection. Thereafter, the resulting transfectants were pooled and inoculated into mammary fat pads of female Wistar-Furth rats. Metastases produced by the transfectant cells in vivo were reestablished from secondary tumours and probed for the presence of the specific synthetic oligonucleotide sequences that flanked, and hence identified, the presence of the transfected DNA. These new metastatic cells are shown to provide a sensitive assay system with which to detect DNA sequences responsible for conveying the metastatic phenotype of prostate cancer when inoculated into syngeneic rats.

Animals↗
PubMed2000

The arterivirus replicase is the only viral protein required for genome replication and subgenomic mRNA transcription.

Equine arteritis virus (EAV) (ARTERIVIRIDAE:) encodes several structural proteins. Whether any of these also function in viral RNA synthesis is unknown. For the related mouse hepatitis coronavirus (MHV), it has been suggested that the nucleocapsid protein (N) is involved in viral RNA synthesis. As described for MHV, we established that the EAV N protein colocalizes with the viral replication complex, suggesting a role in RNA synthesis. Using an infectious cDNA clone, point mutations and deletions were engineered in the EAV genome to disrupt the expression of each of the structural genes. All structural proteins, including N, were found to be dispensable for genome replication and subgenomic mRNA transcription. We also constructed a mutant in which translation of the intraleader ORF was disrupted. This mutant had a wild-type phenotype, indicating that, at least in cell culture, the product of this ORF does not play a role in the EAV replication cycle.

Animals↗
PubMed2002

Assumptions of the tumor 'escape' hypothesis.

The reasons why cancer cells are not destroyed by the immune system are likely to be similar, in most cases, to the reasons why normal cells are not destroyed by the immune system. Unfortunately for tumor immunologists, these reasons have not yet been fully elucidated. What is known, however, is that the lack of autoimmune destruction of normal tissue after immune activation is a finely regulated, highly orchestrated sequence of events. Viewed in this light, it is interesting to conceptualize the derangement of the tumor genome not merely as an engine that enables cancer cells to dodge immune recognition. The dysregulation characteristic of the transformed genome is also what makes tumor immunity, a specialized form of autoimmunity, possible.

Antigens, Neoplasm↗
PubMed2026

Prophage landscapes in clinical MRSA: safety profiling and discovery of Lys81, a broad-spectrum bacteriolytic enzyme.

INTRODUCTION: Methicillin-resistant Staphylococcus aureus (MRSA) poses a significant threat to global healthcare, requiring novel therapeutic strategies. Prophages, latent phage genomes integrated into bacterial chromosomes, are important resources for antimicrobial development due to their genomic stability and genetic engineering potential. METHODS: In this study, we performed genomewide sequencing on 329 MRSA isolates to predict prophage sequences, followed by analyses of these prophages-including examinations of virulence genes, antibiotic resistance genes, homologous proteins of pathogenic MRSA phages, and functional predictions of these homologous proteins-to evaluate their safety and value as genetic engineering scaffolds and to screen for novel broadspectrum bacteriolytic enzymes. RESULTS: Our data indicate that 85.7% (282/329) of strains carried complete prophage sequences; 64 strains lacked virulence factors or genes, meeting the core criteria for safe vectors. Resistance screening found only 6 prophages carried msrA, confirming the biosafety of the remaining strains. A significant correlation existed between prophage virulence gene capacity and genomic structure (R2 = 0.99986684, p = 3.64e-69). High-virulence clusters (>10 factors) showed high structural similarity; 10 characteristic sequences linked to S. aureus phages and their prevalence patterns were identified via conserved motif analysis. Collinearity analysis with reference to virulent MRSA phages and 3D structural predictions of orthologous proteins identified two lysozymes and a host-recognition device. Notably, Lys81, an N-acetylmuramoyl-L-alanine amidase ortholog, was prioritized and characterized as a broad-spectrum lytic enzyme. Our data show Lys81 has key properties: (1) Broad-spectrum antibacterial activity, lysing 52.3% (23/44) of clinical S. aureus strains and cross-acting against Gram-positive bacteria such as Pseudomonas aeruginosa and Listeria; (2) Excellent environmental adaptability, maintaining activity at pH 5.0 and 0°C, with 25 mM Na+ and Ca2 + enhancing function; (3) Potent biofilm clearance, achieving 83% MRSA biofilm reduction at 50 μg/mL; and (4) Favorable in vivo safety/efficacy, eradicating MRSA infections in lung organoid models with minimal cytotoxicity. DISCUSSION: This study establishes a theoretical foundation for the clinical translation of MRSA prophages, positioning Lys81 as a novel candidate for treating drug-resistant bacterial infections.

Lys81↗
PubMed2001

The color of mice: in the light of GFP-variant reporters.

The mouse currently represents the premier model organism for mammalian genetic studies. Over the past decade the production of targeted and transgenic lines of mice has become commonplace, with current technology allowing the creation of mutations at base pair resolution. Such genome modifications are becoming increasingly elaborate and often incorporate gene-based reporters for tagging different cellular populations. Until recently, lacZ, the bacterial beta-galactosidase gene has been the marker of choice for most studies in the mouse. However, over the past 3 years another valuable reporter has emerged, and its attractiveness is reflected by an explosion in its use in mice. Green fluorescent protein (GFP), a novel autofluorescent genetic reporter derived from the bioluminescent jellyfish Aequorea victoria, currently represents a unique alternative to other gene-based reporters in that its visualization is non-invasive and so can be monitored in real-time in vitro or in vivo. It has the added advantage that it can be quantified by, for example, flow cytometry, confocal microscopy, and fluorometric assays. Several mutants of the original wild-type GFP gene that improve thermostability and fluorescence have been engineered. Enhanced GFP is one such variant, which has gained popularity for use in transgenic or targeted mice. Moreover, various GFP spectral variants have also been developed, and two of these novel color variants, enhanced yellow fluorescent protein (EYFP) and enhanced cyan fluorescent protein (ECFP), can also be used in mice. Since the spectral profiles of the ECFP and EYFP color variants are distinct and non-overlapping, these two reporters can be co-visualized, and are therefore ideal for in vivo double-labeling or fluorescent energy transfer analyses. The use of GFP and its color variants as reporters provides an unprecedented level of sophistication and represents the next step in mouse genome engineering technology by opening up the possibility of combinatorial non-invasive reporter usage within a single animal.

Animals↗
PubMed2016

Controlling AAV Tropism in the Nervous System with Natural and Engineered Capsids.

More than one hundred naturally occurring variants of adeno-associated virus (AAV) have been identified, and this library has been further expanded by an array of techniques for modification of the viral capsid. AAV capsid variants possess unique antigenic profiles and demonstrate distinct cellular tropisms driven by differences in receptor binding. AAV capsids can be chemically modified to alter tropism, can be produced as hybrid vectors that combine the properties of multiple serotypes, and can carry peptide insertions that introduce novel receptor-binding activity. Furthermore, directed evolution of shuffled genome libraries can identify engineered variants with unique properties, and rational modification of the viral capsid can alter tropism, reduce blockage by neutralizing antibodies, or enhance transduction efficiency. This large number of AAV variants and engineered capsids provides a varied toolkit for gene delivery to the CNS and retina, with specialized vectors available for many applications, but selecting a capsid variant from the array of available vectors can be difficult. This chapter describes the unique properties of a range of AAV variants and engineered capsids, and provides a guide for selecting the appropriate vector for specific applications in the CNS and retina.

Animals↗
PubMed1999

The minimal genome concept.

Complete genome sequences are becoming available for a large number of diverse species. Quantification of gene content, of gene family expansion, of orthologous gene conservation, as well as their displacement, are now possible - laying the ground for the estimation of the minimal set of proteins sufficient for cellular life. The consensus of computational results suggests a set close to 300 genes. These predictions will be evaluated by engineering of small bacterial genomes.

Bacterial Proteins↗
PubMed2002

The future of nanofabrication and molecular scale devices in nanomedicine.

Nanotechnology is engineering and manufacturing at the molecular scale, and the application of nanotechnology to medicine is called nanomedicine. Nanomedicine subsumes three mutually overlapping and progressively more powerful molecular technologies. First, nanoscale-structured materials and devices that can be fabricated today hold great promise for advanced diagnostics and biosensors, targeted drug delivery and smart drugs, and immunoisolation therapies. Second, biotechnology offers the benefits of molecular medicine via genomics, proteomics, and artificial engineered microbes. Third, in the longer term, molecular machine systems and medical nanorobots will allow instant pathogen diagnosis and extermination, chromosome replacement and individual cell surgery in vivo, and the efficient augmentation and improvement of natural physiological function. Current research is exploring the fabrication of designed nanostructures, nanoactuators and nanomotors, microscopic energy sources, and nanocomputers at the molecular scale, along with the means to assemble them into larger systems, economically and in great numbers.

Biomedical Technology↗
PubMed2004

Oncogene expression and genetic background influence the frequency of DNA copy number abnormalities in mouse pancreatic islet cell carcinomas.

Quantitative measurements of tumor genome composition show remarkable heterogeneity in tumors arising from the same anatomical location and/or histopathological class and stage. The factors that contribute to genomic heterogeneity are not clear, but germ-line allelic variation and timing of initiating oncogenic events are likely candidates. We investigated these factors by using array comparative genomic hybridization to measure genomic aberrations in genetically engineered mouse models of pancreatic islet cell carcinoma, in which oncogenic transformation is elicited by the SV40 T antigens expressed under the control of the rat insulin promoter (RIP-Tag). Two distinct transgenic RIP-Tag lines, and three polymorphic sublines of one, enabled us to investigate the effects of genetic background and differing age of oncogene induction. Both parameters were found to bias the spectrum of genomic copy number abnormalities. Specifically, the frequency of losing portions of chromosomes 9 and 16 was significantly modulated by genetic background, with the former being lost at highest rates in the FVB/N background and the latter being lost to greatest extent in both FVB/N and C57Bl/6 tumors compared with C3HeB/Fe tumors. The frequency of losing a region of chromosome 6 varied according to the age when tumorigenesis was initiated; loss of chromosome 6 was significantly higher when oncogene expression was first activated in adulthood. These studies illustrate the utility of transgenic animal models for investigation of factors influencing genomic heterogeneity despite the commonalty of target cell type and initiating oncogene.

Animals↗
PubMed2000

Genome-wide search for loss of heterozygosity using laser capture microdissected tissue of breast carcinoma: an implication for mutator phenotype and breast cancer pathogenesis.

Breast cancer is considered to display a high degree of intratumor heterogeneity, without any obvious morphological and pathological steps to define sequential evolution, and its progression may vary among individual tumors. In an attempt to elucidate these etiological and phenotypic complexities, the present study, based on the fundamental concept that genomic instability is the engine of both tumor progression and tumor heterogeneity, was conducted to test the hypothesis that breast cancer pathogenesis is driven by double-strand break (DSB)-initiated chromosome instability (CIN). The rationale underlying this hypothesis is derived from the clues provided by family breast cancer syndromes, in which susceptibility genes, including p53, ATM, BRCA1 and BRCA2, are involved within the common functional pathway of DSB-related checkpoint/ repair. Because genomic deletion caused by DSB is reflected in the genetic mechanism of loss of heterozygosity (LOH), this genome-wide LOH study was conducted, using 100 tumors and 400 microsatellite markers. To minimize the effect of heterogeneity within tumors, the experimental technique of laser capture microdissection was used to ensure that genetic and phenotypic examinations were based on the same tumor cells. Support for our hypothesis comes from the observations that: (a) the extent of DSB-initiated CIN in tumors significantly increased as tumors progressed to poorer grades or later stages; (b) in the sequential steps toward CIN, the loci of p53 and ATM, the key checkpoint genes against DSB, were lost at the earliest stage; and (c) many loci identified to be important in breast tumorigenesis were the genomic sites possibly harboring the genes involved in DSB-related checkpoint/repair (including RAD51, RAD52, and BRCA1) or CIN (including FA-A, FA-D, and WRN), and a higher number of these loci showing LOH was significantly associated with increased level of DSB-initiated CIN (P < 0.0001). Breast cancers are thus considered to be sequentially progressive with CIN. However, CIN might also cause genetic heterogeneity, which was revealed by the findings that LOH at some markers was observed only in the component of ductal carcinoma in situ but not in the invasive component of the same tumors. In addition, some markers were found to preferentially lose at specific tumor grades, implying their contribution to genetic heterogeneity during tumor development. Therefore, this study suggests that breast cancer progression is clonal with regard to CIN, but different breast cancers would present distinct molecular profiles resulting from genetic heterogeneity caused by CIN.

Adult↗
PubMed2006

Replication of murine hepatitis virus is regulated by papain-like proteinase 1 processing of nonstructural proteins 1, 2, and 3.

Coronaviruses are positive-strand RNA viruses that translate their genome RNA into polyproteins that are co- and posttranslationally processed into intermediate and mature replicase nonstructural proteins (nsps). In murine hepatitis virus (MHV), nsps 1, 2, and 3 are processed by two papain-like proteinase activities within nsp3 (PLP1 and PLP2) to yield nsp1, an nsp2-3 intermediate, and mature nsp2 and nsp3. To determine the role in replication of processing between nsp2 and nsp3 at cleavage site 2 (CS2) and PLP1 proteinase activity, mutations were engineered into the MHV genome at CS2, at CS1 and CS2, and at the PLP1 catalytic site, alone and in combination. Mutant viruses with abolished cleavage at CS2 were delayed in growth and RNA synthesis but grew to wild-type titers of >10(7) PFU/ml. Mutant viruses with deletion of both CS1 and CS2 exhibited both a delay in growth and a decrease in peak viral titer to approximately 10(4) PFU/ml. Inactivation of PLP1 catalytic residues resulted in a mutant virus that did not process at either CS1 or CS2 and was severely debilitated in growth, achieving only 10(2) PFU/ml. However, when both CS1 and CS2 were deleted in the presence of inactivated PLP1, the growth of the resulting mutant virus was partially compensated, comparable to that of the CS1 and CS2 deletion mutant. These results demonstrate that interactions of PLP1 with CS1 and CS2 are critical for protein processing and suggest that the interactions play specific roles in regulation of the functions of nsp1, 2, and 3 in viral RNA synthesis.

Animals↗
PubMed2005

Evolutionary programming as a platform for in silico metabolic engineering.

BACKGROUND: Through genetic engineering it is possible to introduce targeted genetic changes and hereby engineer the metabolism of microbial cells with the objective to obtain desirable phenotypes. However, owing to the complexity of metabolic networks, both in terms of structure and regulation, it is often difficult to predict the effects of genetic modifications on the resulting phenotype. Recently genome-scale metabolic models have been compiled for several different microorganisms where structural and stoichiometric complexity is inherently accounted for. New algorithms are being developed by using genome-scale metabolic models that enable identification of gene knockout strategies for obtaining improved phenotypes. However, the problem of finding optimal gene deletion strategy is combinatorial and consequently the computational time increases exponentially with the size of the problem, and it is therefore interesting to develop new faster algorithms. RESULTS: In this study we report an evolutionary programming based method to rapidly identify gene deletion strategies for optimization of a desired phenotypic objective function. We illustrate the proposed method for two important design parameters in industrial fermentations, one linear and other non-linear, by using a genome-scale model of the yeast Saccharomyces cerevisiae. Potential metabolic engineering targets for improved production of succinic acid, glycerol and vanillin are identified and underlying flux changes for the predicted mutants are discussed. CONCLUSION: We show that evolutionary programming enables solving large gene knockout problems in relatively short computational time. The proposed algorithm also allows the optimization of non-linear objective functions or incorporation of non-linear constraints and additionally provides a family of close to optimal solutions. The identified metabolic engineering strategies suggest that non-intuitive genetic modifications span several different pathways and may be necessary for solving challenging metabolic engineering problems.

Algorithms↗
PubMed2003

Switching species tropism: an effective way to manipulate the feline coronavirus genome.

Feline infectious peritonitis virus (FIPV), a coronavirus, is the causative agent of an invariably lethal infection in cats. Like other coronaviruses, FIPV contains an extremely large positive-strand RNA genome of ca. 30 kb. We describe here the development and use of a reverse genetics strategy for FIPV based on targeted RNA recombination that is analogous to what has been described for the mouse hepatitis virus (MHV) (L. Kuo et al., J. Virol. 74:1393-1406, 2000). In this two-step process, we first constructed by targeted recombination a mutant of FIPV, designated mFIPV, in which the ectodomain of the spike glycoprotein was replaced by that of MHV. This switch allowed for the selection of the recombinant virus in murine cells: mFIPV grows to high titers in these cells but has lost the ability to grow in feline cells. In a second, reverse process, mFIPV was used as the recipient, and the reintroduction of the FIPV spike now allowed for selection of candidate recombinants by their regained ability to grow in feline cells. In this fashion, we reconstructed a wild-type recombinant virus (r-wtFIPV) and generated a directed mutant FIPV in which the initiation codon of the nonstructural gene 7b had been disrupted (FIPV Delta 7b). The r-wtFIPV was indistinguishable from its parental virus FIPV 79-1146 not only for its growth characteristics in tissue culture but also in cats, exhibiting a highly lethal phenotype. FIPV Delta 7b had lost the expression of its 7b gene but grew unimpaired in cell culture, confirming that the 7b glycoprotein is not required in vitro. We establish the second targeted RNA recombination system for coronaviruses and provide a powerful tool for the genetic engineering of the FIPV genome.

Amino Acid Sequence↗