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At least 793 records · Page 44Linked to original sources
PubMed1995

RNA viruses as vectors for the expression of heterologous proteins.

RNA viruses comprise a wide variety of infectious agents, some of which are the cause of disease in humans, animals, and plants. Recombinant DNA technology is now making it feasible to modify these genomes and engineer them to express heterologous proteins. Several different schemes are being employed that depend on the genome organization of the virus and on the strategy of replication of the particular virus. Several different examples are illustrated and potential uses as well as possible problems are discussed. In the future reverse genetics may convert some of these viruses from agents of disease to agents of cure.

Animals↗
PubMed1989

Unraveling the mysteries of Duchenne and Becker muscular dystrophy.

Through a process that has come to be known as reverse genetics, the gene and gene product involved in Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) have been identified. The DMD/BMD gene is over 2 million base pairs in size and over 50% of DMD/BMD patients harbor submicroscopic deletions for portions of the gene. The gene product, named dystrophin, is 400 Kd in size. Dystrophin is present in skeletal, cardiac, and smooth muscles, as well as brain. The protein is absent or altered in DMD/BMD patient muscle. The normal function of dystrophin and the reasons why its alteration results in the DMD/BMD phenotypes are presently unknown. The discoveries to date, however, provide a starting point for investigating the fundamental pathogenetic mechanisms involved in DMD/BMD.

Animals↗
PubMed2003

Recently discovered functions of glucosylceramides in plants and fungi.

Glycosphingolipids are ubiquitous membrane lipids of eukaryotic organisms and a few bacteria. Whereas inositol-containing glycosphingolipids are restricted to plants and fungi, galactosylceramide occurs only in fungi and animals. In contrast, glucosylceramide is the unique glycosphingolipid which plants, fungi and animals have in common. However, there are specific differences in the structure of the ceramide backbone of glucosylceramides from these organisms. A comparison of the structural features and the biosynthesis of glucosylceramides from plants, fungi and animals will contribute to our understanding of their functions, which so far have been analysed mainly in animals. The availability of nearly all genes involved in the biosynthesis of glucosylceramides enables the specific manipulation of glycosphingolipid metabolism by techniques of forward and reverse genetics. Application of this approach to unicellular organisms like yeasts, multicellular filamentous fungi, as well as to complex organisms like plants will reveal common and different glucosylceramide functions in these organisms. These glycolipids play a role both in intracellular processes and in cell-to-cell interactions. These interactions may occur between cells of a multicellular organism or between cells of different species, as in host-pathogen interactions.

Animals↗
PubMed1999

Efficient gene delivery into adult cardiomyocytes by recombinant Sindbis virus.

Somatic gene therapy as a potential strategy for the treatment of myocardial diseases relies on an efficient gene transfer into cardiac muscle cells. The difficulty of delivering genes into adult cardiomyocytes exists not only in vivo but also in primary culture systems. Therefore, possibilities for ex vivo gene transfer and the in vitro study of physiological processes by reverse genetics are limited. We investigated the potential of an alphavirus-based vector system to transduce adult rat cardiomyocytes (ARC) in culture using a replication-deficient Sindbis virus (SIN) encoding beta-galactosidase (SIN-LacZ). Transduction efficiency depended on the virus concentration used, with expression of the reporter gene being detectable in up to 80% of cultured ARC as early as 24 h after infection. We observed a remarkably lower cytotoxicity of this viral vector in ARC than in other cells such as fibroblasts and neonatal cardiomyocytes. Additionally, no perceptible changes in the morphology of the nuclei or cytoskeleton were found in ARC 48 h after infection with SIN-LacZ. We conclude that SIN vectors are useful for gene delivery into adult cardiomyocytes and believe that improved versions of this viral system may be useful for cardiovascular gene therapy in the future.

Animals↗
PubMed2003

RNA interference: from an ancient mechanism to a state of the art therapeutic application?

Now that the sequencing of many genomes has been completed, the basic challenges are finding the genes and predicting their functions. Up until now, a large information gap has existed between the knowledge of genome sequence and our knowledge of protein function. The assessment of gene function may be performed using the tools of reverse genetics, including knock-out mice, antisense oligomers, aptamers, and ribozymes. These approaches have been superseded by RNA interference (RNAi), which exhibits much more potency for the investigation of protein function than the techniques listed above. As already known some years ago, RNAi is based on an ancient anti-viral defense mechanism in lower eukaryotes. It is induced by double-stranded RNA and its processing to 21-23 nt small interfering RNAs (siRNAs), which cause the degradation of homologous endogenous mRNA. The way RNAi works has still to be determined, but it already serves as a first-choice approach to generate loss-of-function phenotypes among a broad variety of eukaryotic species, such as nematodes, flies, plants, fungi and mammals. RNAi also represents an extremely powerful tool, becoming a therapeutic approach to curing infectious diseases originated by viral or parasitic invasion. In this review we present the current view of how RNAi works in different eukaryotic species and its high potential for functional genomics and in rational drug design.

Animals↗
PubMed1998

A gene hunting we will go!

Two general approaches toward the identification of new genes which may contribute to specific developmental, physiologic, or pathologic processes can be pursued: (1) a strategy of positional cloning or reverse genetics and linkage analysis in which affected individuals or families are studied to identify defective genes, and (2) an immediately focused approach which uses available partial information about a gene to screen cDNA libraries toward the isolation and characterization of the complete gene. The general principles that form the basis of how these two strategies aid the gene hunter in the pursuit of new genes is discussed. The modern day urologist should become familiar with these techniques and the promise they hold for the future diagnosis and treatment of urologic diseases.

Cloning, Molecular↗
PubMed2005

Influenza vaccines: recent advances in production technologies.

In spite of ongoing annual vaccination programs, the seasonal influenza epidemics remain a major cause of high morbidity and mortality. The currently used "inactivated" vaccines provide very short-term and highly specific humoral immunity due to the frequent antigenic variations in the influenza virion. These intra-muscularly administered vaccines also fail to induce protective mucosal immunity at the portal of viral entry and destruction of the virally infected cells by induction of cytotoxic T lymphocytes. Therefore, it is necessary to develop immunologically superior vaccines. This article highlights some of the recent developments in investigational influenza vaccines. The most notable recent developments of interest include the use of immunopotentiators, development of DNA vaccines, use of reverse genetics, and the feasibility of mammalian cell-based production processes. Presently, due to their safety and efficacy, the cold-adapted "live attenuated" vaccines are seen as viable alternatives to the "inactivated vaccines". The DNA vaccines are gaining importance due to the induction of broad-spectrum immunity. In addition, recent advances in recombinant technologies have shown the possibility of constructing pre-made libraries of vaccine strains, so that adequately preparations can be made for epidemics and pandemics.

Adjuvants, Immunologic↗
PubMed2003

Cloning and characterization of katA, encoding the major monofunctional catalase from Xanthomonas campestris pv. phaseoli and characterization of the encoded catalase KatA.

The first cloning and characterization of the gene katA, encoding the major catalase (KatA), from Xanthomonas is reported. A reverse genetic approach using a synthesized katA-specific DNA probe to screen a X. campestris pv. phaseoli genomic library was employed. A positively hybridizing clone designated pKat29 that contained a full-length katA was isolated. Analysis of the nucleotide sequence revealed an open reading frame of 1,521 bp encoding a 507-amino acid protein with a theoretical molecular mass of 56 kDa. The deduced amino acid sequence of KatA revealed 84% and 78% identity to CatF of Pseudomonas syringae and KatB of P. aeruginosa, respectively. Phylogenetic analysis places Xanthomonas katA in the clade I group of bacterial catalases. Unexpectedly, expression of katA in a heterologous Escherichia coli host resulted in a temperature-sensitive expression. The KatA enzyme was purified from an overproducing mutant of X. campestris and was characterized. It has apparent K(m) and V(max) values of 75 m M [H(2)O(2)] and 2.55 x 10(5) micromol H(2)O(2) micromol heme(-1) s(-1), respectively. The enzyme is highly sensitive to 3-amino-1,2,4-triazole and NaN(3), has a narrower optimal pH range than other catalases, and is more sensitive to heat inactivation.

Catalase↗
PubMed2000

Cloning, sequencing, and function of sanF: A gene involved in nikkomycin biosynthesis of Streptomyces ansochromogenes.

A 111-bp DNA fragment related to nikkomycin biosynthesis of Streptomyces ansochromogenes 7100 was obtained with the method of reverse genetics. Then, a 2.2-kb DNA fragment was cloned from the DNA library of S. ansochromogenes 7100 by using the 111-bp fragment as a probe. Sequence analysis showed that the fragment contains one complete open reading frame (ORF) that encodes a 219-amino acid (aa) protein, and this gene was designated sanF (GenBank Accession No. AF223971). The function of the sanF gene was studied by a strategy of gene disruption, and the resulting sanF mutants lost the ability to synthesize biologically active nikkomycin, indicating that sanF is essential for nikkomycin biosynthesis.

Amino Acid Sequence↗
PubMed1999

Characterization of the mesB gene and expression of bacteriocins by Leuconostoc mesenteroides Y105.

Leuconostoc mesenteroides Y105, previously described for production of mesentericin Y105, an anti-Listeria bacteriocin, was shown to secrete a second bacteriocin. The latter was purified, and its molecular mass of 3446 Da, obtained by mass spectrometric analysis, indicates that this bacteriocin should be identical to mesenterocin 52B [Revol-Junelles et al., Lett Appl Microbiol 23:120, 1996]. This second bacteriocin produced by L. mesenteroides Y105 was named mesentericin B105. Its structural gene, mesB, was then localized by a reverse genetic approach, cloned, and sequenced. MesB was found on the pHY30 plasmid, next to mesY gene clusters. Curing experiments led to isolation of two L. mesenteroides Y105 derivatives, named L. mesenteroides Y29 and Y30. The latter had lost pHY30 plasmid, encoding bacteriocin determinants, therefore explaining its phenotype (MesY-, MesB-). On the contrary, Y29 derivative still harbors the pHY30 but did not produce any bacteriocin. Thus, its phenotype could likely result from a point mutation within a gene, probably encoding a protein involved in production of both mesentericin Y105 and mesentericin B105.

Amino Acid Sequence↗
PubMed2005

Unexplained complexity of the mitochondrial genome and transcriptome in kinetoplastid flagellates.

Kinetoplastids are flagellated protozoans, whose members include the pathogens Trypanosoma brucei, T. cruzi and Leishmania species, that are considered among the earliest diverging eukaryotes with a mitochondrion. This organelle has become famous because of its many unusual properties, which are unique to the order Kinetoplastida, including an extensive kinetoplast DNA network and U-insertion/deletion type RNA editing of its mitochondrial transcripts. In the last decade, considerable progress has been made in elucidating the complex machinery of RNA editing. Moreover, our understanding of the structure and replication of kinetoplast DNA has also dramatically improved. Much less however, is known, about the developmental regulation of RNA editing, its integration with other RNA maturation processes, stability of mitochondrial mRNAs, or evolution of the editing process itself. Yet the profusion of genomic data recently made available by sequencing consortia, in combination with methods of reverse genetics, hold promise in understanding the complexity of this exciting organelle, knowledge of which may enable us to fight these often medically important protozoans.

Animals↗
PubMed2005

RNA silencing in Chlamydomonas: mechanisms and tools.

The generation of a comprehensive EST library and the sequencing of its genome set the stage for reverse genetics approaches in Chlamydomonas reinhardtii. However, these also require tools for the specific downregulation of target gene expression. Consequently, a large number of diverse constructs were developed aimed at reducing target gene expression in Chlamydomonas via the stable expression of antisense or inverted repeat-containing RNA. Double-stranded RNA (dsRNA) generated by the annealing of antisense and sense RNAs or by hairpin formation of an inverted repeat, feeds into the RNA silencing pathway. In this pathway, dsRNA is cleaved into approximately 25-bp small interfering RNAs (siRNAs) by the endonuclease Dicer. One of the two complementary strands of a siRNA is then loaded onto an Argonaute-like protein present as core component within larger complexes. Guided by this single-stranded RNA, the Argonaute-like protein either detects homologous transcripts and cleaves these endonucleolytically, or initiates transcriptional gene silencing. This article summarizes current information derived mainly from the Chlamydomonas genome project on components that are assumed to be involved in RNA silencing mechanisms in Chlamydomonas. Furthermore, all approaches employed in Chlamydomonas to date to downregulate target gene expression by antisense or inverted repeat constructs are reviewed and discussed critically.

Amino Acid Sequence↗
PubMed1998

The manganese superoxide dismutase from the penicillin producer Penicillium chrysogenum.

The antioxidant enzyme superoxide dismutase has been studied in order to define mechanisms for the influence of oxygen on penicillin production. Manganese-containing SOD activity was purified from penicillin-producing cultures of the filamentous fungus Penicillium chrysogenum and reverse genetics was used to identify full-length cDNA and genomic clones. Sequence analysis revealed a 630-bp ORF containing three exons and two introns with fungal consensus splice-site junctions. The deduced amino-acid sequence (210 amino acids; 23.13 kDa) includes conserved residues required for enzymatic activity and metal binding, and shares significant similarity with Mn- and Fe-containing superoxide dismutases. The sod gene is present as a single copy in the genome of different P. chrysogenum strains and its expression level is not correlated with penicillin-G productivity.

Amino Acid Sequence↗
PubMed2003

A tool for understanding homologous recombination in plants.

Attempts for establishing an efficient gene targeting (GT) system in seed plants have hitherto not been successful. In contrast, GT based on homologous recombination is highly efficient in Physcomitrella, making this moss a novel tool in reverse genetics. However, why homologous and illegitimate recombination are differently regulated between Physcomitrella and seed plants is still enigmatic. Here we update the state of the art of GT in Physcomitrella and discuss approaches to unravel this enigma. Identification of molecular factors significantly enhancing GT and their subsequent transfer to crop plants will have a great impact on plant biotechnology by enabling precise genetic engineering. Physcomitrella appears to be the most useful model system in this context.

Bryopsida↗
PubMed2003

Post-transcriptional gene silencing in plants by RNA.

RNA silencing, which is termed post-transcriptional gene silencing in plants, is an RNA degradation process through sequence-specific nucleotide interactions induced by double-stranded RNA. In plants, RNA silencing not only serves as a component of the defense mechanism, but also participates in the regulation of endogenous gene expression in a variety of developmental processes. This review elaborates the current progress on the understanding of the molecular basis of RNA silencing including a mechanistic link between the regulation of microRNA and RNA silencing. The practical use of RNA silencing as a reverse genetics approach in plant functional genomics is also discussed.

Gene Silencing↗
PubMed2005

Decreased sucrose-6-phosphate phosphatase level in transgenic tobacco inhibits photosynthesis, alters carbohydrate partitioning, and reduces growth.

The aim of this work was to examine the role of sucrose-6-phosphate phosphatase (SPP; EC 3.1.3.24) in photosynthetic carbon partitioning. SPP catalyzes the final step in the pathway of sucrose synthesis; however, until now the importance of this enzyme in plants has not been studied by reversed-genetics approaches. With the intention of conducting such a study, transgenic tobacco plants with reduced SPP levels were produced using an RNA interference (RNAi) strategy. Transformants with less than 10% of wild-type SPP activity displayed a range of phenotypes, including those that showed inhibition of photosynthesis, chlorosis, and reduced growth rates. These plants had strongly reduced levels of sucrose and hexoses but contained 3-5 times more starch than the control specimens. The leaves were unable to export transient starch during extended periods of darkness and as consequence showed a starch- and maltose-excess phenotype. This indicates that no alternative mechanism for carbon export was activated. Inhibition of SPP resulted in an approximately 1,000-fold higher accumulation of sucrose-6-phosphate (Suc6P) compared to wild-type leaves, whereas the content of hexose-phosphates was reduced. Although the massive accumulation of Suc6P in the cytosol of transgenic leaves was assumed to impair phosphate-recycling into the chloroplast, no obvious signs of phosphate-limitation of photosynthesis became apparent. 3-Phosphoglycerate (3-PGA) levels dropped slightly and the ATP/ADP ratio was not reduced in the transgenic lines under investigation. It is proposed that in SPP-deficient plants, long-term compensatory responses give rise to the observed acceleration of starch synthesis, increase in total cellular Pi content, decrease in protein content, and related reduction in photosynthetic activity.

Carbohydrate Metabolism↗
PubMed2005

High-efficiency transformation of the diploid strawberry (Fragaria vesca) for functional genomics.

Fragaria vesca L., a diploid (2n = 2x = 14) relative of the commercial octoploid strawberry, is an attractive model for functional genomics research in Rosaceae. Its small genome size, short reproductive cycle, and facile vegetative and seed propagation make F. vesca a promising candidate for forward and reverse genetics experiments. However, the lack of a high-efficiency transformation protocol required for systematic production of thousands of T-DNA insertional mutant lines and high-throughput gene validation is a major bottleneck. We describe a new transformation procedure that uses leaf explants from newly unfolded trifoliate leaves obtained from stock plants 6-7 weeks after seed germination, co-cultivation with Agrobacterium strain GV3101, and stringent selection on MS medium containing 4 mg l(-1) hygromycin. Using this protocol we achieved 100% transformation efficiency for 6 of 14 F. vesca accessions tested. Accession PI 551572 was determined to be the best candidate for a model in F. vesca functional genomics research, as it showed the greatest propensity for callus formation, transformation, shoot regeneration, ex vitro establishment, and plant growth, requiring only 14-15 weeks to complete its life cycle in different seasons in the greenhouse.

Cinnamates↗
PubMed2002

Antisense repression of cytosolic phosphoglucomutase in potato (Solanum tuberosum) results in severe growth retardation, reduction in tuber number and altered carbon metabolism.

The aim of this work was to investigate the role of cytosolic phosphoglucomutase (PGM; EC 5.4.2.2) in the regulation of carbohydrate metabolism. Many in vitro studies have indicated that PGM plays a central role in carbohydrate metabolism; however, until now the importance of this enzyme in plants has not been subject to reverse-genetics investigations. With this intention we cloned the cytosolic isoform of potato PGM (StcPGM) and expressed this in the antisense orientation under the control of the CaMV 35 S promoter in potato plants. We confirmed that these plants contained reduced total PGM activity and that loss in activity was due specifically to a reduction in cytosolic PGM activity. These plants were characterised by a severe phenotype: stunted aerial growth combined with limited root growth and a reduced tuber yield. Analysis of the metabolism of these lines revealed that leaves of these plants were inhibited in sucrose synthesis whereas the tubers exhibited decreased levels of sucrose and starch as well as decreased levels of glycolytic intermediates but possessed unaltered levels of adenylates. Furthermore, a broader metabolite screen utilising GC-MS profiling revealed that these lines contained altered levels of several intermediates of the TCA cycle and of amino acids. In summary, we conclude that cytosolic PGM plays a crucial role in the sucrose synthetic pathway within the leaf and in starch accumulation within the tuber, and as such is important in the maintenance of sink-source relationships.

Amino Acids↗