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At least 775 records · Page 43Linked to original sources
PubMed2005

The virulence of the 1918 pandemic influenza virus: unraveling the enigma.

The 1918 influenza pandemic caused acute illness in 25-30% of the world's population and resulted in the death of up to 40 million people. Using lung tissue of 1918 influenza victims, the complete genomic sequence of the 1918 influenza virus is being deduced. Neither the 1918 hemagglutinin nor neuraminidase genes possess mutations known to increase tissue tropicity that account for virulence of other influenza virus strains, such as A/WSN/33 or the highly pathogenic avian influenza H5 or H7 viruses. Using reverse genetics approaches, influenza virus constructs containing the 1918 hemagglutinin and neuraminidase on an A/WSN/33 virus background were lethal in mice. The genotypic basis of this virulence has not yet been elucidated. The complete sequence of the non-structural (NS) gene segment of the 1918 virus was deduced and also tested to determine the validity of the hypothesis that enhanced virulence in 1918 could have been due to type I interferon inhibition by the NS1 protein. Results from these experiments suggest that in human cells the 1918 NS1 is a very effective interferon antagonist. Sequence analysis of the 1918 influenza virus is allowing us to test hypotheses as to the origin and virulence of this strain. This information should help elucidate how pandemic influenza virus strains emerge and what genetic features contribute to virulence in humans.

Animals↗
PubMed2005

Recombinant rhabdoviruses: vectors for vaccine development and gene therapy.

The establishment of methods to recover rhabdoviruses from cDNA, so-called reverse genetics systems, has made it possible to genetically engineer rhabdoviruses and to study all aspects of the virus life cycle by introducing defined mutations into the viral genomes. It has also opened the way to make use of the viruses in biomedical applications such as vaccination, gene therapy, or oncolytic virotherapy. The typical gene expression mode of rhabdoviruses, a high genetic stability, and the propensity to tolerate changes in the virus envelope have made rhabdoviruses attractive, targetable gene expression vectors. This chapter provides an overview on the possibilities to manipulate biological properties of the rhabdoviruses that may be important for further development of vaccine vectors and examples of recombinant rhabdoviruses expressing foreign genes and antigens.

Animals↗
PubMed2001

The cell biology of coronavirus infection.

The ability to obtain entire volume data on infected cells will allow us to define much more accurately the interactions of viral proteins with host cell structures such as ER, Golgi, and cytoskeletal elements. In addition, the demonstrated ability to express viral proteins fused to fluorescent markers in in live cells will allow us to follow specific proteins or complexes during the course of infection and to determine if exogenously expressed proteins are able to target to sites of active viral replication. This in turn will allow new approaches to the study of viral and cellular protein-protein interactions, as methods to study the biology and pathogenesis of MHV infection at a cellular level. Finally, the approaches described here will allow us to define protein complementation of defective viruses at a cellular level, rather than being dependent on population measurements of RNA, protein, or progeny virus. By combining these approaches with available biochemical and molecular biological approaches and the emerging reverse genetic and recombinant genetic approaches, rapid progress in understanding the details of coronavirus-cell interactions should be possible.

Animals↗
PubMed2007

Metabolomic profiling of natural volatiles: headspace trapping: GC-MS.

Plants are a fabulously rich source of naturally volatile metabolites, which are derived from a range of contrasting biochemical pathways (e.g., mono-, di-, and sesquiterpenoids, benzoates, alcohols, esters). Such volatiles may immediately be released from the plant or they may be stored, e.g., in glycosylated form for release later "on demand." Certain roles for these molecules have already been determined in that they can function as attractants (e.g., to pollinators, seed dispersers, and others) or as protectants (repellants, pathogen inhibitors, and so on). The flavor and fragrance of plant materials to humans and other animals are also, to a great extent, determined by natural volatiles. Other more sophisticated roles have also been elucidated where plant volatiles have been shown to be involved either as signal molecules to attract the predators of damaging herbivorous insects or potentially even as signal molecules warning other plants of imminent danger. As such, detailed knowledge of these components can be valuable in relation to breeding crop varieties for enhanced product quality or for achieving improved resistance to pathogens and insects. Furthermore, knowledge of the metabolites can result in a corresponding knowledge of the genes responsible for their synthesis and this can lead to dedicated strategies for their in vitro production through, e.g., reverse genetics in heterologous microbial expression systems in fermentors for the production of high-value fine chemicals. Various analytical techniques based on gas chromatography-mass spectrometry have been devised for the analysis of this complex group of metabolites. Two of these key methods are detailed in this chapter.

Arabidopsis↗
PubMed2004

Identification and characterization of viral antagonists of type I interferon in negative-strand RNA viruses.

Interferons are cytokines secreted in response to viral infections with potent antiviral activity, and they represent a critical component of the innate immune response against viruses. It has now become apparent that many viruses have evolved different mechanisms to counteract the interferon response, allowing their efficient replication and propagation in their hosts. This review discusses how the development of reverse genetics techniques and the increase in our knowledge of the interferon response have led to the discovery of interferon-antagonistic functions of different genes of viruses belonging to the negative-strand RNA virus group. In many cases, these viral genes encode accessory pro- teins that are not required for viral infectivity but are critical for optimal replication and for virulence in the host.

Animals↗
PubMed2004

Toward novel vaccines and therapies based on negative-strand RNA viruses.

The study of negative-strand RNA viruses has suggested new strategies to produce more attenuated viruses. Reverse genetics has allowed the implementation of the strategies, and new or improved monovalent vaccines are being developed. In addition, recombinant viruses expressing foreign proteins or epitopes have been produced with the aim of developing multivalent vaccines capable of stimulating humoral and cellular immune responses against more than one pathogen. Finally, recombinant viruses that selectively enter cells expressing tumor markers or the HIV envelope protein have been engineered and shown to lyse target cells. Preclinical and clinical trials of improved and multivalent vaccines and therapeutic (oncolytic) viruses are ongoing.

Animals↗
PubMed1993

Mitochondrial DNA polymorphism and feminizing sex factors dynamics in a natural population of Armadillidium vulgare (Crustacea, Isopoda).

Sex determination in Armadillidium vulgare may be under the control of two parasitic sex factors that reverse genetic males into functional neo-females. The first feminizing factor (F) is a Wolbachia and the other (f) is probably a sequence of the F bacterial DNA unstably integrated into the host genome. Both of these feminizing factors are mainly maternally transmitted. Here we investigate the mitochondrial DNA polymorphism of wild iso-female lineages harbouring either F or f. Among the four haplotypes present in the population, two were the f-harbouring lineages, while two were common to the F- and f-harbouring lineages. This result suggests that there has been an introgression of the f factor into lineages infected by F Wolbachia. Based on previous data, we propose two different ways to account for such introgression. Given the particular dynamics of feminizing factors (f-harbouring lineages increase in populations at the expense of F-harbouring lineages), such an introgression should prevent the replacement of F-linked mitochondrial types by f-linked mitochondrial types in wild populations.

Animals↗
PubMed1993

A nucleotide substitution in one of the beta-tubulin genes of Trichoderma viride confers resistance to the antimitotic drug methyl benzimidazole-2-yl-carbamate.

We characterized a Trichoderma viride strain that is resistant to the antimitotic drug methyl benzimidazole-2-yl-carbamate (MBC). This species has two beta-tubulin genes (tub1 and tub2) and by reverse genetics we showed that a mutation in the tub2 gene confers MBC resistance in this strain. Comparison of the tub2 sequence of the mutant strain with that of the wild type revealed that a single amino acid substitution of tyrosine for histidine at a position 6 is responsible for the MBC tolerance. Furthermore, we showed that this gene can be used as a homologous dominant selectable marker in T. viride transformation. Both tubulin genes were completely sequenced. They differ by 48 residues and the degree of identity between their deduced amino acid sequences is 86.3%.

Amino Acid Sequence↗
PubMed1992

DNA markers closely linked to nematode resistance genes in sugar beet (Beta vulgaris L.) mapped using chromosome additions and translocations originating from wild beets of the Procumbentes section.

Genes conferring resistance to the beet cyst nematode (Heterodera schachtii Schm.) have been transferred to sugar beet (Beta vulgaris L.) from three wild species of the Procumbentes section using monosomic addition and translocation lines, because no meiotic recombination occurs between chromosomes of cultured and wild species. In the course of a project to isolate the nematode resistance genes by strategies of reverse genetics, probes were cloned from DNA of a fragmented B. procumbens chromosome carrying a resistance gene, which had been isolated by pulsed-field gel electrophoresis. One probe (pRK643) hybridized with a short dispersed repetitive DNA element, which was found only in wild beets, and thus may be used as a molecular marker for nematode resistance to progeneis of monosomic addition lines segregating resistant and susceptible individuals. Additional probes for the resistance gene region were obtained with a polymerase chain reaction (PCR)-based strategy using repetitive primers to amplify DNA located between repetitive elements. One of these probes established the existence of at least six different chromosomes from wild beet species, each conferring resistance independently of the others. A strict correlation between the length of the wild beet chromatin introduced in fragment addition and translocation lines and the repeat copy number has been used physically to map the region conferring resistance to a chromosome segment of 0.5-3 Mb.

Animals↗
PubMed1989

Chromosomal mapping and gene disruption of the ADHIII gene in aspergillus nidulans.

There are at least three alcohol dehydrogenases in Aspergillus nidulans. ADHIII has no obvious physiological function. We describe here the cloning of the ADHIII gene (alcC), its mapping on linkage group VII by "reverse genetics", and the properties of multicopy transformants tested for their ability to grow on a range of alcohols (butan-1-ol being the best substrate tested for growth). We were unable to detect any obvious alteration in phenotype of a strain carrying a disrupted copy of the ADHIII gene.

Alcohol Dehydrogenase↗
PubMed1993

Dopaminergic defect in hypertension.

Reverse genetics and the candidate gene approach have been utilized to identify the genetic defect(s) in hypertension. We have proposed the dopamine receptor gene as one candidate in the pathogenesis of hypertension. Because some forms of hypertension are sodium dependent or aggravated by sodium loading and because dopamine is important in aiding the organism to eliminate "excess" sodium, an abnormality in the renal dopaminergic system may be responsible for the sodium retention in hypertension. Both human and animal models of hypertension are associated with renal dopamine production and/or post first messenger defects. The Dahl salt-sensitive rat, which has a decreased ability to generate renal dopamine, and the spontaneously hypertensive rat (SHR), which has no such limitation, have a defective coupling of a D1 receptor to a G protein/adenylyl cyclase complex. This coupling defect is: (1) genetic, since it precedes the onset of hypertension and co-segregates with the hypertensive phenotype, (2) receptor specific, since it is not shared by other humoral agents, and (3) organ and nephron segment selective, since it occurs in proximal tubules but not in cortical collecting ducts or the brain striatum. A consequence of the defective dopamine receptor/adenylyl cyclase coupling in the SHR is a decreased ability of D1 agonists to inhibit Na+/H+ exchange activity. A resistance to the natriuretic effect of dopamine and D1 agonists in the SHR is due mainly to decreased cyclic AMP production, although with maturation a post cyclic AMP defect is acquired. Radioligand binding studies suggest a "loss" of the high-affinity D1 binding site in the SHR.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Construction of an infectious DNA clone of the Y1 strain of canine parvovirus and characterization of the virus derived from the clone.

We have cloned genome fragments of canine parvovirus strain Y1 from replicative-form DNA and double-stranded DNA synthesized from virion DNA in vitro, and constructed a recombinant plasmid containing a full-length Y1 genome (pCPVY 1). When this recombinant plasmid was transfected into cell cultures, an infectious virus could be recovered. To characterize this pCPVY 1-derived virus, its biological properties were compared with those of the parental strain. No difference was observed between them in antigen expression, viral DNA replication, hemagglutination ability, and virus multiplication, indicating that the virus derived from the infectious plasmid inherited the biological properties of the authentic Y1 strain. Therefore, this recombinant plasmid appears to be useful for reverse genetics of canine parvovirus.

Animals↗
PubMed1991

Transgenic regulation in laboratory animals.

This chapter is an attempt to summarize some commonly accepted and some more subjective opinions about the regulation of transgene expression in laboratory animals. After a short historical introduction, I present some general notions regarding gene structure/function. The spotlight shifts then to the description of the most popular techniques for gene transfer, including the targeted gene replacement. The different approaches are briefly discussed in terms of intrinsic advantages and limitations regarding gene expression patterns. Furthermore, the role of enhancers, promoters and other cis-acting elements such as silencers and dominant control regions as well as their involvement in the chromatin on-off state are discussed on the basis of a specific example studied in our laboratory. The review concludes by presenting recent results and the new perspectives opening in the field of 'surrogate' (also called 'reversed') genetics. Some problems which remain to be solved both at the technical as well as at the social-ethical level are also briefly presented.

Animals↗
PubMed1995

Dual role of cAMP during Dictyostelium development.

cAMP plays an essential role during Dictyostelium development both outside and inside the cell. Membrane-bound receptors and adenylyl cyclase are responsible for sensing and producing extracellular cAMP, whereas a phosphodiesterase is responsible for maintaining a low basal level. The molecular events underlying this type of hormone like signalling, which are now beginning to be deciphered, will be presented, in the light of cAMP analogue studies. The importance of intracellular cAMP for cell differentiation has been demonstrated by the central role of the cAMP dependent protein kinase. Mutants as well as strains obtained by reverse genetics will be reviewed which lead to our current understanding of the role of intracelluar cAMP in the differentiation of both stalk and spore cells.

Adenylyl Cyclases↗
PubMed1996

The role of gap junction membrane channels in development.

In most developmental systems, gap junction-mediated cell-cell communication (GJC) can be detected from very early stages of embryogenesis. This usually results in the entire embryo becoming linked as a syncytium. However, as development progresses, GJC becomes restricted at discrete boundaries, leading to the subdivision of the embryo into communication compartment domains. Analysis of gap junction gene expression suggests that this functional subdivision of GJC may be mediated by the differential expression of the connexin gene family. The temporal-spatial pattern of connexin gene expression during mouse embryogenesis is highly suggestive of a role for gap junctions in inductive interactions, being regionally restricted in distinct developmentally significant domains. Using reverse genetic approaches to manipulate connexin gene function, direct evidence has been obtained for the connexin 43 (Cx43) gap junction gene playing a role in mammalian development. The challenges in the future are the identification of the target cell populations and the cell signaling processes in which Cx43-mediated cell-cell interactions are critically required in mammalian development. Our preliminary observations suggest that neural crest cells may be one such cell population.

Animals↗
PubMed1996

chs-4, a class IV chitin synthase gene from Neurospora crassa.

In Saccharomyces cerevisiae, most of the cellular chitin is produced by chitin synthase III, which requires the product encoded by the CSD2/CAL1/DIT101/KT12 gene. We have identified, isolated and structurally characterized as CSD2/CAL1/DIT101/KT12 homologue in the filamentous ascomycete Neurospora crassa and have used a "reverse genetics" approach to determine its role in vivo. The yeast gene was used as a heterologous probe for the isolation of a N. crassa gene(designated chs-4) encoding a polypeptide belonging to a class of chitin synthases which we have designated class IV. The predicted polypeptide encoded by this gene is highly similar to those of S. cerevisiae and Candida albicans. N. crassa strains in which chs-4 had been inactivated by the Repeat-Induced point mutation (RIP) process grew and developed in a normal manner under standard growth conditions. However, when grown in the presence of sorbose (a carbon source which induces morphological changes accompanied by elevated chitin content), chitin levels in the chs-4RIP strain were significantly lower than those observed in the wild type. We suggest that CHS4 may serve as an auxiliary enzyme in N. crassa and that, in contrast to yeasts, it is possible that filamentous fungi may have more than one class IV chitin synthase.

Acetylglucosamine↗
PubMed1995

The binding motifs for Ac transposase are absolutely required for excision of Ds1 in maize.

A reverse genetic system for studying excision of the transposable element Ds1 in maize plants has been established previously. In this system, the Ds1 element, as part of the genome of maize streak virus (MSV), is introduced into maize plants via agroinfection. In the presence of the Ac element, excision of Ds1 from the MSV genome results in the appearance of viral symptoms on the maize plants. Here, we used this system to study DNA sequences required in cis for excision of Ds1. The Ds1 element contains the Ac transposase binding motif AAACGG in only one of its subterminal regions (defined here as the 5' subterminal region). We showed that mutation of these motifs abolished completely the excision capacity of Ds1. This is the first direct demonstration that the transposase binding motifs are essential for excision. Mutagenesis with oligonucleotide insertions in the other (3') subterminal region resulted in elements with either a reduced or an increased excision efficiency, indicating that this subterminal region also has an important function.

Base Sequence↗