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At least 1,189 records · Page 66Linked to original sources
PubMed1994

Recent advances in brain tumor molecular biology.

Work in the field of molecular neuro-oncology has evolved from a purely descriptive catalogue of regional mutations to the implication of specific genes in the malignant process. Reverse genetic strategies have resulted in the cloning of the neurofibromatosis-1 and neurofibromatosis-2 genes, the molecular physiology of the p53 gene, and work is in progress toward identifying specific genes in each of the other major genomic regions in which genetic events accumulate during malignant progression in human gliomas. Current studies are examining the functional role of genes implicated in glioma malignancy and investigating the mechanisms of their dysregulation in the generation of the malignant phenotype.

Animals↗
PubMed2001

Evolving concepts in epithelial magnesium transport.

Magnesium is an important, predominantly intracellular cation that is required for a wide variety of cellular processes. The mammalian kidney plays a key role in whole-body magnesium homeostasis, but the molecular and cellular mechanisms that underlie renal epithelial magnesium reabsorption are poorly understood. Traditional physiologic approaches have been severely hampered by the lack of a useful radioisotope of magnesium that can be used for tracer flux studies. The present review discusses physiologic insights gained from recent reverse-genetic studies that have identified a plethora of genes involved in inherited renal magnesium wasting syndromes.

Animals↗
PubMed2006

Generation of gene knockouts and mutant models in the laboratory rat by ENU-driven target-selected mutagenesis.

OBJECTIVE: The rat is one of the most important model organisms for biomedical and pharmacological research. However, the generation of novel models for studying specific aspects of human diseases largely depends on selection for specific traits using existing rat strains, thereby solely depending on naturally occurring variation. This study aims to provide the tools to manipulate the rat genome in a more directed way. METHODS: We developed robust, automated, and scaleable reverse genetic methodology based on ENU (N-ethyl-N-nitrosourea)-driven target-selected mutagenesis. Optimal mutagenesis conditions have been determined in three different rat strains and a universal, rapid, and cost-effective dideoxy resequencing-based screening setup was established for mutation discovery. The effectiveness of the approach is illustrated by the identification of 120 induced mutations in a set of genes of interest, including six that result in unique rat knockout models due to the introduction of premature stop codons. In addition, 56 mutations were found that change amino acids, including critical residues in transmembrane domains of receptors and channels. CONCLUSIONS: The approach described here allows for the systematic generation of knockout and protein function altering alleles in the rat. The resulting induced rat models will be powerful tools for studying many aspects of a wide variety of human diseases.

Alkylating Agents↗
PubMed2004

Vaccination for pandemic influenza: a six point agenda for interpandemic years.

BACKGROUND: The demand for influenza vaccine is driven by recognition of its health and economic benefits. Vaccine reduces all cause mortality in the elderly by 30 to 50% and prevents > or =30% of hospital admissions for influenza-related respiratory disease, heart disease and stroke. However, because most influenza vaccine (85%) is produced in only eight countries, adequate production and equitable distribution of vaccine throughout the world will pose a serious challenge when the next influenza pandemic appears. METHODS: This article reviews a six point agenda for pandemic vaccination that should be undertaken during interpandemic years. The agenda includes preparing vaccine seed strains using reverse genetics, determining the characteristics of a pandemic vaccine and vaccination schedule, considering global registration of pandemic vaccines, increasing vaccination in interpandemic years, documenting the epidemiology of vaccine use and addressing political issues that will affect the global supply of pandemic vaccines. CONCLUSIONS: Planning for pandemic vaccination must begin during the interpandemic period to ensure a vaccine supply that will be adequate to meet demand in all countries. This will require the skills not only of experts in virology, epidemiology and public health but also those in politics, economics and law. The task will be complex, but its promised benefits will be immense.

Disease Outbreaks↗
PubMed2004

Human metapneumovirus as a major cause of human respiratory tract disease.

BACKGROUND: Human metapneumovirus (hMPV) is a newly identified paramyxovirus that appears to be one of the most significant and common viral infections in humans. The virus, first isolated in 2001, is a clear cause of lower respiratory tract disease in both the very young and the frail elderly. The virus causes acute wheezing in children or, less commonly, croup or pneumonia. METHODS/RESULTS: Molecular epidemiology studies have shown that field strains exhibit sufficient sequence diversity to designate 2 subgroups of circulating viruses. Small animal and nonhuman primate models of infection have been described, which will allow studies of pathogenesis and immunity. Recombinant viruses have already been generated by several groups using reverse genetics, which facilitates the study of the biology of the virus and the generation of live attenuated vaccine candidates. CONCLUSIONS: Ongoing research promises to elucidate the molecular basis for pathogenesis and immunity of human metapneumovirus infections and to pave the way for rapid vaccine development.

Animals↗
PubMed2005

Human metapneumovirus: a ubiquitous and long-standing respiratory pathogen.

BACKGROUND: Human metapneumovirus (hMPV) is a recently described human pathogen first identified in respiratory specimens of young children suffering from respiratory syndromes ranging from mild to severe. METHODS AND RESULTS: Virological studies have reported the presence of hMPV infections in many countries from all continents. Seroprevalence studies have indicated that the virus has been circulating in humans for more than 50 years and that it infects virtually all children by the ages of 5-10 years. In young children, hMPV has been mainly associated with bronchiolitis but also with pneumonitis, otitis media and acute exacerbation of asthma. The contribution of hMPV in respiratory syndromes of adults has been studied considerably less; initial studies have indicated a role for this pathogen in flu-like syndromes and in significant percentages of chronic obstructive pulmonary disease exacerbations and cases of community-acquired pneumonia during the winter-spring period. Both primate and rodent experimental models have been used to characterize the pathogenesis of this respiratory virus. In some of these models, intranasal hMPV inoculation has elicited not only important viral replication but also significant pulmonary inflammation and clinical disease. Recently a few groups have developed reverse genetic systems for hMPV, allowing a better understanding of viral pathogenesis and generation of attenuated viral strains for immunization. CONCLUSIONS: Recent studies on hMPV have provided a better understanding of the epidemiology and pathogenesis associated with this viral infection, and have enhanced the prospect of developing efficient therapeutic agents and vaccine candidates.

Animals↗
PubMed2005

Newer respiratory virus infections: human metapneumovirus, avian influenza virus, and human coronaviruses.

PURPOSE OF REVIEW: Recently, several previously unrecognized respiratory viral pathogens have been identified and several influenza A virus subtypes, previously known to infect poultry and wild birds, were transmitted to humans. Here we review the recent literature on these respiratory viruses. RECENT FINDINGS: Human metapneumovirus has now been detected worldwide, causing severe respiratory tract illnesses primarily in very young, elderly and immunocompromised individuals. Animal models and reverse genetic techniques were designed for human metapneumovirus, and the first vaccine candidates have been developed. Considerable genetic and antigenic diversity was observed for human metapneumovirus, but the implication of this diversity for vaccine development and virus epidemiology requires further study. Two previously unrecognized human coronaviruses were discovered in 2004 in The Netherlands and Hong Kong. Their clinical impact and epidemiology are largely unknown and warrant further investigation. Several influenza A virus subtypes were transmitted from birds to humans, and these viruses continue to constitute a pandemic threat. The clinical symptoms associated with these zoonotic transmissions range from mild respiratory illnesses and conjunctivitis to pneumonia and acute respiratory distress syndrome, sometimes resulting in death. More basic research into virus ecology and evolution and development of effective vaccines and antiviral strategies are required to limit the impact of influenza A virus zoonoses and the threat of an influenza pandemic. SUMMARY: Previously unknown and emerging respiratory viruses are an important threat to human health. Development of virus diagnostic tests, antiviral strategies, and vaccines for each of these pathogens is crucial to limit their impact.

Communicable Diseases, Emerging↗
PubMed1984

The expression of human interferon alpha genes.

We have determined the levels of mRNAs for IFN-beta, IFN-gamma and various alpha-IFNs (IFN-alpha 1, -alpha 2, -alpha 4, -alpha 5, -alpha 6, -alpha 7, -alpha 8 and -alpha 14) in normal and leukaemic human blood leucocytes and several cell lines induced in different fashions. The ratio of alpha to beta IFN transcripts varied greatly, depending on the cell type. The levels of the individual IFN-alpha RNAs were very different: IFN-alpha 1, -alpha 2 and -alpha 4 RNAs constituted the major fraction of the IFN-alpha transcripts measured, while IFN-alpha 6, -alpha 7, -alpha 8 and -alpha 14 were minor components in normal, induced leucocytes. Moreover, there was a striking difference in the proportion of individual IFN-alpha mRNA species in different cell types, in particular between normal and leukaemic cells; for example all cases of myeloblastic leukaemia examined showed a high expression of IFN-alpha 14. Use of different induction protocols did not significantly affect the proportion of IFN mRNAs. Analysis of the human IFN-alpha 1 gene by reversed genetics led to the identification of a segment of 5' flanking sequence between positions 117 and 68 upstream of the cap site which is required for inducibility by virus.

Cell Line↗
PubMed1993

Role of a raf proto-oncogene during Caenorhabditis elegans vulval development.

During Caenorhabditis elegans vulval induction, multipotent precursors respond to an inductive signal by generating vulval cells as opposed to non-specialized epidermal cells. Both classical and 'reverse' genetic approaches have revealed that a cascade of nematode homologues of mammalian proto-oncogenes is necessary for induction of the vulva. The inductive signal is a growth factor encoded by the lin-3 gene and its candidate receptor is a tyrosine kinase encoded by the let-23 gene. let-23 acts via a Ras protein encoded by the let-60 gene. A nematode homologue of mammalian raf family protein serine/threonine kinases has been cloned and found to be encoded by the lin-45 gene. Dominant negative lin-45 raf mutants prevent vulval induction. A recessive lin-45 raf mutation prevents the excessive vulval differentiation caused by activated ras, indicating that raf might act downstream of ras during vulval induction.

Animals↗
PubMed2001

RNA-binding proteins and circadian rhythms in Arabidopsis thaliana.

An Arabidopsis transcript preferentially expressed at the end of the daily light period codes for the RNA-binding protein AtGRP7. A reverse genetic approach in Arabidopsis thaliana has revealed its role in the generation of circadian rhythmicity: AtGRP7 is part of a negative feedback loop through which it influences the oscillations of its own transcript. Biochemical and genetic experiments indicate a mechanism for this autoregulatory circuit: Atgrp7 gene transcription is rhythmically activated by the circadian clock during the day. The AtGPR7 protein accumulates with a certain delay and represses further accumulation of its transcript, presumably at the post-transcriptional level. In this respect, the AtGRP7 feedback loop differs from known circadian oscillators in the fruitfly Drosophila and mammals based on oscillating clock proteins that repress transcription of their own genes with a 24 h rhythm. It is proposed that the AtGRP7 feedback loop may act within an output pathway from the Arabidopsis clock.

Arabidopsis↗
PubMed1996

Cell-free cloning and biolistic inoculation of an infectious cDNA of potato virus Y.

Potato virus Y (PVY) full-length cDNA has been found to be refractory to cloning in Escherichia coli cells. A full-length 9.7 kb PVY cDNA was obtained by reverse transcription polymerase chain reaction (RT-PCR) from the RNA of PVY (tuber necrotic strain, PVYNTN). Double-stranded DNA fragments were used as primers (ds megaprimers), to include signals for transcription in vivo (a cauliflower mosaic virus 35S RNA promoter and a nopaline synthase terminator) in the final PCR product. Biolistic bombardment with a helium particle gun was used to inoculate the amplified product to detached tobacco leaves. Inoculation of tobacco plants with ground inoculated leaves followed by northern blot, ELISA and immuno-electron microscopy demonstrated that the DNA was highly infectious with up to 90% of bombarded leaves containing the virus. This methodology will allow the use of reverse genetics in the study of PVY-plant interactions and will also be useful for obtaining infectious cDNA from other viruses with large RNA genomes.

Cloning, Molecular↗
PubMed1997

Mutational analysis of the RNA-fork model of the influenza A virus vRNA promoter in vivo.

The genome of influenza A virus consists of eight negative-stranded RNA segments which have partially complementary non-coding terminal sequences. Previous transcription studies of the virion RNA promoter in vitro have shown that the 5' terminus forms an integral part of the promoter and an 'RNA-fork' model has been proposed for the initiation of transcription. According to this model part of the promoter is formed by an RNA-duplex which involves complementary residues 10 to 1 2 of the 3' end and residues 11' to 13' of the 5' end. With a reverse genetics system, based on the chloramphenicol acetyltransferase (CAT) gene, we have now tested this part of the promoter in vivo. Single mutations of the conserved residues at positions 11 and 12 of the 3' terminus and at positions 12' and 13' of the 5' terminus abolished promoter activity. The introduction of complementary mutations into both termini partially restored activity. On the other hand, mutations at positions 10 of the 3' terminus and 11' of the 5' terminus inhibited activity independently of whether a base-pair was formed or not. Thus, at these positions, the nature of the residues is apparently more important than their ability to form base-pairs. These results extend our previous virion 'RNA-fork' model and are consistent with in vitro findings that the 5' terminus is involved in the initiation of transcription.

Base Composition↗
PubMed1997

Complete nucleotide sequence of tobacco necrosis virus strain DH and genes required for RNA replication and virus movement.

The complete genome sequence of tobacco necrosis virus strain D (Hungarian isolate, TNV-DH) was determined. The genome (3762 nt) has an organization identical to that reported for TNV-D. Highly infectious synthetic transcripts from a full-length TNV-DH cDNA clone were prepared, the first infectious necrovirus transcript reported. This clone was used for reverse genetic studies to map the viral genes required for replication and movement. Protoplast inoculation with delta 22 and delta 82 mutants revealed that both the 22 kDa and 82 kDa gene products are required for RNA replication. Although the products of three small central genes (p7(1), p7a and p7b) were not essential for RNA replication in protoplasts, mutations in these ORFs prevented infection of plants. In contrast, viral RNA accumulation and cell-to-cell movement were observed in the inoculated, but not the systemically infected, leaves of Nicotiana benthamiana challenged with RNA lacking the intact coat protein (CP) gene. These results strongly suggest that p7(1), p7a, p7b and CP are involved in TNV-DH cell-to-cell and long-distance movement, respectively.

Base Sequence↗
PubMed1998

A host restriction-based selection system for influenza haemagglutinin transfectant viruses.

During the 1996 influenza epidemic in Vienna we obtained influenza A virus specimens (Vienna/47/96, Vienna/81/96) which grow efficiently in African green monkey kidney (Vero) cells but not in embryonated chicken eggs. Amplification of the specimens in Vero cells resulted in progeny that agglutinated human but not chicken erythrocytes. Reassortment analysis suggested that the haemagglutinin (HA) might be responsible for the host restriction. Vero cells were infected with the Vienna/47/96 virus and then transfected with reconstituted ribonucleoprotein complexes containing HA genes from egg-adapted strains. Subsequent selective passages in embryonated chicken eggs resulted in selection of transfectant viruses, growing in eggs and containing the transfected HAs. The results demonstrate that host restriction of the Vero-adapted Vienna/47/96 virus is due to its HA. Moreover, the experiments showed that the Vienna/47/96 strain can be used as helper virus for reverse genetics experiments.

Animals↗
PubMed1999

Tissue culture infectivity of different strains of infectious bursal disease virus is determined by distinct amino acids in VP2.

Two types of strains of serotype I of infectious bursal disease virus (IBDV) have been described, on the basis of their ability (IBDV-TC) or inability (IBDV-BU) to infect chicken embryonic cells in culture. However, both types infect B lymphocytes in the bursa of Fabricius of young chickens. To determine the molecular basis for tissue culture infectivity, virus recombinants with chimeric segments A were constructed from IBDV-TC and IBDV-BU by reverse genetics. The region responsible for the different phenotypes was located in VP2. Site-directed mutagenesis identified single amino acids that are responsible for the restriction in infectivity. However, the appropriate amino acid exchanges are strain-specific.

Amino Acid Sequence↗
PubMed2000

Unidirectional RNA polymerase I-polymerase II transcription system for the generation of influenza A virus from eight plasmids.

Recently, we developed a system for the generation of influenza A virus by cotransfecting only eight plasmids from which negative-sense vRNA and positive-sense mRNA are expressed (Hoffmann et al., Proceedings of the National Academy of Sciences, USA 97, 6108-6113, 2000). Here we report the establishment of a different transcription system for the expression of virus-like RNAs, allowing the intracellular synthesis of noncapped positive-sense cRNA and 5'-capped mRNA from one template. Cotransfection of eight RNA pol I-pol II tandem promoter plasmids containing the cDNA of A/WSN/33 (H1N1) resulted in the generation of infectious influenza A virus, albeit with a lower yield than the bidirectional system. Our approach of producing either vRNA and mRNA or cRNA and mRNA intracellularly from a minimum set of plasmids should be useful for the establishment or optimization of reverse genetics systems for other RNA viruses.

Animals↗
PubMed2000

Utilizing fowlpox virus recombinants to generate defective RNAs of the coronavirus infectious bronchitis virus.

Coronavirus defective RNAs (D-RNAs) have been used as RNA vectors for the expression of heterologous genes and as vehicles for reverse genetics by modifying coronavirus genomes by targetted recombination. D-RNAs based on the avian coronavirus infectious bronchitis virus (IBV) D-RNA CD-61 have been rescued (replicated and packaged into virions) in a helper virus-dependent manner following electroporation of in vitro-generated T7 transcripts into IBV-infected cells. In order to increase the efficiency of rescue of IBV D-RNAs, cDNAs based on CD-61, under the control of a T7 promoter, were integrated into the fowlpox virus (FPV) genome. The 3'-UTR of the D-RNAs was flanked by a hepatitis delta antigenomic ribozyme and T7 terminator sequence to generate suitable 3' ends for rescue by helper IBV. Cells were co-infected simultaneously with IBV, the recombinant FPV (rFPV) containing the D-RNA sequence and a second rFPV expressing T7 RNA polymerase for the initial expression of the D-RNA transcript, subsequently rescued by helper IBV. Rescue of rFPV-derived CD-61 occurred earlier and with higher efficiency than demonstrated previously for electroporation of in vitro T7-generated RNA transcripts in avian cells. Rescue of CD-61 was also demonstrated for the first time in mammalian cells. The rescue of rFPV-derived CD-61 by M41 helper IBV resulted in leader switching, in which the Beaudette-type leader sequence on CD-61 was replaced with the M41 leader sequence, confirming that helper IBV virus replicated the rFPV-derived D-RNA. An rFPV-derived D-RNA containing the luciferase gene under the control of an IBV transcription-associated sequence was also rescued and expressed luciferase on serial passage.

Animals↗
PubMed2000

Chimeras in noncoding regions between serotypes I and II of segment A of infectious bursal disease virus are viable and show pathogenic phenotype in chickens.

Two serotypes, I and II, have been identified for infectious bursal disease virus (IBDV), a member of the family BIRNAVIRIDAE: Here, the generation by reverse genetics of IBDV chimeras in segment A of the bisegmented genome is reported. The 5- and 3'-noncoding regions (NCRs) of a serotype II strain were exchanged with the NCRs of a full-length cDNA clone of segment A of a serotype I strain. Isolated chimeric viruses were characterized in cell culture and susceptible chickens. The results show that IBDV chimeras in segment A were able to replicate in cell culture and that VP1 encoded by a serotype I segment B is functionally active with serotype I NCRs as well as with serotype II NCRs. Chimeric viruses infected susceptible chickens and caused mild depletion of bursal cells. Thus, the noncoding regions of segment A are not responsible for the different pathotypes of IBDV serotypes I and II.

Animals↗