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PubMed2003

Avermectin: biochemical and molecular basis of its biosynthesis and regulation.

Avermectin and its analogues, produced by Streptomyces avermitilis, are major commercial antiparasitic agents in the field of animal health, agriculture, and human infections. They are 16-membered pentacyclic lactone compounds derived from polyketide and linked to a disaccharide of the methylated deoxysugar l-oleandrose. Labeling studies, analyses of the biosynthetically blocked mutants, and the identification of the avermectin gene cluster allows characterization of most of the biosynthetic pathway. Recent completion of S. avermitilis genome sequencing is also expected to help in revealing the precise biosynthetic sequence and the complicated regulatory mechanism for avermectin biosynthesis, which has been long-awaited to be elucidated. The well characterized avermectin biosynthetic pathway and availability of S. avermitilis genome information in combination with the recent development of combinatorial biosynthesis should allow us to redesign more potent avermectin analogues and to engineer S. avermitilis as a more efficient host for the production of important commercial analogues.

Antiparasitic Agents↗

PubMed2004

Functional prediction of a T-DNA tagged gene of Arabidopsis thaliana by in silico analysis.

We have employed a gene-knockout approach using T-DNA tagging and in vivo gene fusion in Arabidopsis thaliana for identification and isolation of specific plant genes. Screening of about 3,000 T-DNA tagged lines resulted in identification of a mutant line (no. 197) exhibiting a significant delay in flowering. From this line a 600-bp plant DNA fragment downstream of the left T-DNA junction was cloned by inverse PCR. BLAST searching in the A. thaliana genomic database indicated a putative gene, frf (flowering regulating factor), with unknown function downstream of the T-DNA insert. Bioinformatic tools were used to predict possible protein structure and function. The protein structure predicted by fold recognition indicates that frf is a transcriptional regulator, a ligand-binding receptor responsive to steroids and hormones. Analyzing the predicted results and the phenotype of the T-DNA tagged plant we hypothesized that FRF might be involved in hormone response in A. thaliana. For verification of this hypothesis we exposed the plants of line no. 197 to gibberellic acid (GA3), a potential growth regulator in higher plants. This treatment resulted in an earlier onset of flowering, almost similar to that in wild type control plants.

Amino Acid Sequence↗

PubMed2003

Huntington's disease: a decade beyond gene discovery.

Huntington's disease is a dominantly inherited neurodegenerative disease that causes a progressive movement disorder, cognitive decline, and varying degrees of psychiatric dysfunction. The identification of the mutant gene in 1993 paved the way for a decade of basic research. The resultant advances in our understanding of the pathogenesis of the disorder are moving us toward rational therapies to slow the progression and delay the onset of the illness.

Animals↗

PubMed1981

The morphology of the cervical giant fiber neuron of Drosophila.

The morphology of the cervical giant fiber (CGF) neuron of Drosophila melanogaster was studied by intracellular injection of Lucifer yellow dye. The CGF neuron is the command cell in a motor circuit causing visually driven escape behavior: a single action potential in a CGF axon produces patterned activity in jump and flight muscles. The present study identified the CGF cell body, a large soma located in the posterior part of the lower ipsilateral protocerebrum. The main process runs anteriorly from the cell body, extends three branches, and turns posteromedially while descending through the brain. The CGF axon courses through the cervical connective and ends within the mesothoracic neuromere of the thoracic ganglion. Thus, the CGF neuron is an interneuron, not a motoneuron as previously believed. We have been isolating mutants that affect CGF neuron-mediated behavior. Comparison of CGF neuron morphology in wildtype strains with that in these mutants will allow identification of genes that affect the development, structure, and connections of the CGF neuron.

Action Potentials↗

PubMed1976

A comparative study of different experimental protocols for mutagenesis assays with the 9-azaguanine resistance system in cultured Chinese hamster cells.

Both spontaneous and EMS-induced mutant frequencies were determined in cultured cells from V79 Chinese hamsters using three different experimental protocols. After optimal expression time was attained, mutation frequencies only remained constant when a protocol was used in whcih the cell density was maintained below critical values both before and during mutant selection. The identification of such a plateau allows, besides more reliable and reproducible estimates of mutation frequency, reduction in the size of experiments for quantitative evaluation of mutagenicity. Determination of mutation frequencies over a wide range of expression times becomes in fact unnecessary.

Azaguanine↗

PubMed1989

Vaccinia virus gene D12L encodes the small subunit of the viral mRNA capping enzyme.

Vaccinia virus gene D12L, which lies between nucleotides 14,350 and 13,487 in the HindIII D fragment, is transcribed at early times in infection and is capable of encoding a protein 287 amino acids in length with a predicted molecular mass of 33,331. A polyclonal antiserum was raised in rabbits to a fusion protein containing 279 amino acids of the D12L protein, and this serum was used to investigate both the time of synthesis and the function of the D12L protein. A combination of Western blot analysis and immunoprecipitation from pulse-labeled and pulse-chased cell extracts demonstrated that the synthesis of a 31-kDa protein begins early in infection, that it reaches a plateau by about 4 hr, and that it is stable in the infected cell. The D12L protein was localized by Western blot analysis of detergent-solubilized virions to the sodium deoxycholate soluble fraction which suggested that it may be a virion core-associated enzyme. Due to the similarity in apparent molecular weight between the D12L protein and the small subunit of the vaccinia mRNA capping complex the anti-D12L antiserum was employed in Western blot analysis of fractions generated during the purification of the virion mRNA capping enzyme. The 31-kDa D12L protein copurified with the virus capping enzyme through chromatography on heparin-agarose and phosphocellulose and also cosedimented with the capping enzyme through a glycerol density gradient. In addition, the anti-D12L antiserum coprecipitated the large subunit of the capping enzyme, confirming that gene D12L encodes the small subunit of the viral mRNA capping enzyme. An insertion mutation which destroys the gene D12L coding sequence was constructed in a plasmid containing a portion of both genes D11L and D12L and this plasmid was used to rescue a ts mutation, in a single step, in the adjacent gene D11L. Southern blot analysis of the re-plaque-purified virus permitted the identification of the mutant virus only when the mutant was propagated in the presence of wild-type helper virus. We concluded from these data that gene D12L is essential for virus propagation in tissue culture.

Base Sequence↗

PubMed1981

The duplicated human alpha-globin genes: their relative expression as measured by RNA analysis.

The human alpha-globin genes are duplicated and encode identical polypeptides. Recently we detected in cloned genomic DNAs characteristic sequence differences between the 3' untranslated regions of the 5' (alpha 2) and 3' (alpha 1) genes, not previously recognized by direct analysis of mRNA and cDNA transcripts. Based on these untranslated region differences, we have now used S1 nuclease mapping of RNA to detect and quantitate the two predicted alpha-mRNA species. With this assay we have examined the relative expression of the alpha-globin genes during normal development and in alpha-thalassemia syndromes. In normal adult reticulocytes, alpha 2 RNA is slightly more abundant than the alpha 1 species (ratio 60:40). This relative abundance of the alpha RNAs was consistently observed in fetal blood and liver RNA samples from 10 weeks of gestation to birth. In both deletion and nondeletion forms of alpha thalassemia, only the alpha 1 RNA and establish the normal pattern of relative alpha-gene expression during development independent of protein variants. RNA analysis also permits for the first time identification of the mutant genes in nondeletion forms of thalassemia.

Chromosome Deletion↗

PubMed1989

Mapping replication units in animal cells.

A general approach for assaying the in vivo direction of replication for any DNA segment has been developed. This technique allows the scanning of genomic regions to detect bidirectional tail-to-tail replication, indicating the presence of a functional origin. By this criterion we identified the approximate positions of two origin sites downstream of the Chinese hamster DHFR gene. Further mapping revealed areas of head-to-head replication, signifying locations of replication termination and thus defining the landmarks of a complete animal cell replicon. Genetic proof for the existence of the DHFR origin was obtained by showing that this region serves as a bidirectional DNA synthesis initiation point following its integration into other sites in the genome by transfection. To show the general applicability of this methodology, we studied the APRT domain. Replication mapping together with the use of deletion mutants allowed the identification of an origin at a far-upstream locus.

Adenine Phosphoribosyltransferase↗

PubMed1994

Construction and application of streptomycete promoter probe vectors which employ the Streptomyces glaucescens tyrosinase-encoding gene as reporter.

We have constructed multicopy and integrative streptomycete promoter probe vectors that employ the promoterless tyrosinase (melC) operon of Streptomyces glaucescens as the chromogenic transcriptional reporter. Each vector contains the reporter cassette, RC3, which comprises part of the melC operon flanked by transcription terminators; RC3 may be easily inserted into any vector. We demonstrate the use of the pIJ101-based mel vector, pMT3010, for the isolation of mutations which alter expression of the S. coelicolor glycerol operon. The S. glaucescens mel reporter system is well-suited for the visual, non-selective identification of regulatory mutants and can be used efficiently for screening several thousand clones at high colony density.

Base Sequence↗

PubMed2006

Regulation of Stat1 protein expression by phenylalanine 172 in the coiled-coil domain.

Stat1 plays an essential role in signal transduction and gene expression of various cytokines including interferons (IFNs). Although the mechanism of cytokine-induced activation of Stat1 and transcriptional regulation of Stat1 gene expression have been established, post-transcriptional regulation of Stat1 protein expression is not fully understood. Here, we report identification of a mutant of Stat1 that has decreased expression levels by using inducible translocation trap (ITT), a reporter gene-based detection system of nuclear translocation. The substitution of serine for phenylalanine 172 (F172S) in the coiled-coil domain causes marked decrease in Stat1 protein expression in various cell lines without decreasing its mRNA levels. Our results suggest that the decrease is caused by translational/post-translational mechanisms independent of proteasome machinery. These results suggest a novel potential mechanism of determination of specificity of Stat proteins and showed that the ITT system is a powerful technique to identify mutants of nuclear translocating signal transducers.

Amino Acid Sequence↗

PubMed2005

Mitochondrial encephalomyopathies: an update.

A genetic classification of the mitochondrial encephalomyopathies includes disorders due to defects of mitochondrial DNA (mtDNA) and disorders due to defects of nuclear DNA (nDNA). Recent progress in mtDNA-related diseases includes: (i) new pathogenic mutations in protein-coding genes, especially those encoding subunits of complex I (ND genes); (ii) the pathogenic nature of homoplasmic mutations, whose expression is regulated by environmental and genetic factors; (iii) increasing interest in the functional and pathophysiological role of haplotypes. Advances in mendelian mitochondrial diseases include: (i) new mutations in genes for complex I subunits; (ii) identification of new mutant ancillary proteins associated with complex IV and complex V deficiencies; (iii) better molecular understanding of disorders due to faulty intergenomic communication, which are associated with multiple mtDNA deletions, mtDNA depletion, or defects of mtDNA translation; (iv) the pathogenic role of alterations of the inner mitochondrial membrane phospholipid components, especially cardiolipin; (v) the emerging importance of defects in mitochondrial motility, fission, or fusion.

DNA, Mitochondrial↗

PubMed2006

Protein aggregate myopathies.

Protein aggregate myopathies (PAMs) based on the morphologic phenomenon of aggregation of proteins within muscle fibers may occur in children (selenoproteinopathies, actinopathies, and myosinopathies) or adults (certain myofibrillar myopathies and myosinopathies). They may be mutation related, which includes virtually all childhood forms but certain other forms as well, or sporadic, which are largely seen in adults. Their classification as myofibrillar or desmin-related myopathies, actinopathies, or myosinopathies is based on the identification of respective mutant proteins, most of them components of the sarcomeres. Recognition of PAM requires muscle biopsy and an extensive immunohistochemical and electron microscopic workup of the biopsied muscle tissue after which molecular analysis of morphologically ascertained proteins should ensue to permit recognition of individual entities and genetic counseling of patients and families. Because pathogenetic principles in PAMs are still incompletely known, causative therapy, at this time, is not available.

Animals↗

PubMed2003

Importance of plant sterols in pattern formation and hormone signalling.

The recent identification of sterol mutants in plants has shown that these molecules play essential roles in development. Although several such mutants are dwarfed, predominantly because of the reduced accumulation of brassinosteroids, others show distinctive phenotypes. We put forward the view that sterols also have roles in mediating brassinosteroid-independent signalling, in the trafficking of membrane vesicles that transport key regulatory proteins and in correct signalling protein conformation and function in membranes.

Biological Transport↗

PubMed2000

Molecular genetics of Oguchi disease, fundus albipunctatus, and other forms of stationary night blindness: LVII Edward Jackson Memorial Lecture.

PURPOSE: To compare the clinical findings of the various forms of stationary night blindness caused by mutations in identified genes encoding proteins of photoreceptors or the retinal pigment epithelium. METHODS: Review of the visual acuities, visual fields, fundi, dark-adaptation curves, and electroretinograms from patients with stationary night blindness caused by mutations in the genes RHO, GNAT1, PDE6B, RHOK, SAG, RDH5, and CACNA1F, respectively encoding rhodopsin, the alpha subunit of rod transducin, the beta subunit of rod cGMP-phosphodiesterase, rhodopsin kinase, arrestin, 11-cis retinol dehydrogenase, and a retinal L-type calcium channel. RESULTS: In the evaluated forms of stationary night blindness, the time course of dark adaptation and the characteristics of the electroretinogram indicate that rod photoreceptors are present and that they function, although abnormally. In night blindness resulting from defects in rhodopsin, the alpha subunit of rod transducin, or the beta subunit of rod cGMP phosphodiesterase, rod photoreceptors respond only to light intensities far brighter than normal, and the sensitivity of rods to light is similar to that of normal individuals who are not dark adapted. In fundus albipunctatus and in Oguchi disease, the rod photoreceptors can achieve normal sensitivity to dim light but only after 2 or more hours of dark adaptation, compared with approximately 0.5 hours for normal individuals. In each of these forms of stationary night blindness, the poor rod sensitivity and the time course of dark adaptation correlate with the known or presumed physiologic abnormalities caused by the identified gene defects. Patients with some forms of stationary night blindness, such as fundus albipunctatus and Oguchi disease, may develop degeneration of the retina leading to severe loss of vision in later life. CONCLUSIONS: The identification of the mutant genes causing forms of stationary night blindness refines the classification of these diseases and enhances our understanding of the underlying physiologic defects. Ophthalmologists must be aware that although these diseases are traditionally categorized as "stationary," some of them lead to reduced visual acuity or constricted visual fields, especially in older patients. Efforts to develop therapies for these diseases should concentrate on these more severe forms.

Amino Acid Sequence↗

PubMed1999

Advances in the molecular genetics of corneal dystrophies.

PURPOSE: To improve our understanding of the role of specific genes on corneal transparency through a review of linkage to specific chromosomal loci and the identification of the mutant genes dealing with the corneal dystrophies. METHOD: Relevant recent literature on the corneal dystrophies is reviewed. RESULTS: Molecular genetic studies of the corneal dystrophies suggest that genes on at least 10 human chromosomes are involved in the maintenance of corneal transparency (chromosomes 1, 5, 9, 10, 12, 16, 17, 20, 21, and X). Within the 10 chromosomes to which corneal dystrophies have been mapped, specific genetic mutations in seven genes (GSN, BIGH3, KRT3, See also pp. 687-691. KRT12, MSS1, GLA, and ARSC1) have been identified in 15 corneal dystrophies. Some corneal dystrophies that are considered distinct clinicopathologic entities are actually caused by different mutations in the same gene. For example, lattice dystrophy types I and IIIA, granular corneal dystrophy types I, II (Avellino dystrophy), and III (Reis-Bucklers dystrophy), and Thiel-Behnke corneal dystrophy are the result of mutations in BIGH3. Mutations in three genes (GSN, BIGH3, MSS1) are associated with amyloid deposition in the cornea. A gene for keratoconus has been mapped to chromosome 21, which is noteworthy because of the established association of keratoconus in Down syndrome (trisomy 21). CONCLUSION: Recent genetic studies on the corneal dystrophies provide insight into some of these disorders at a basic molecular level. Some corneal dystrophies that were previously believed to be distinct clinicopathologic entities are closely related at the molecular level with the different phenotypes resulting from distinct mutations in the same gene. This new knowledge is leading to a revised classification of the corneal dystrophies and to the development of animal models of corneal dystrophies. The latter will lead to a better understanding of the pathogenesis of the disorders and hence to novel therapeutic approaches to those dystrophies that cause significant visual impairment. Research of this nature is only in its infancy.

Chromosome Mapping↗

PubMed2002

Longevity determination genes in Drosophila melanogaster.

Identification of longevity mutants is crucial for genetic approach to dissect the molecular mechanism of aging and longevity determination. In Drosophila melanogaster, several mutations have been shown to extend the longevity: methuselah encoding a putative G-protein coupled receptor, Indy encoding a sodium dicarboxylate cotransporter, chico encoding insulin receptor substrate, and InR encoding the insulin-like receptor. Extended longevity phenotypes were also observed in transgenic flies overexpressing antioxidant enzymes, Cu/Zn superoxide dismutase and Catalase, Cu/Zn SOD only, or a molecular chaperone, hsp70. Pleiotropism of mutations is a limitation associated with conventional mutagenesis for efficient detection of longevity determination genes. Using a conditional misexpression system, we identified Drosophila POSH (DPOSH), a scaffold protein containing RING finger and four SH3 domains, whose ubiquitous overexpression in adult stage extends the longevity. Neural-specific overexpression of DPOSH is sufficient to extend the longevity, whereas overexpression in non-neural tissues during development induces apoptosis through activation of JNK/SAPK pathway.

Animals↗

PubMed2003

The interplay between Salmonella typhimurium and its macrophage host--what can it teach us about innate immunity?

Salmonella enterica sv. Typhimurium (S. typhimurium) is a genetically tractable, facultative intracellular pathogen, whose capacity to cause systemic disease in mice depends upon its ability to survive and replicate within macrophages. The identification of Salmonella mutants that lack this activity, has provided a tool with which to dissect the mechanisms used by Salmonella to establish a permissive niche, and identify host activities which it must overcome in order to achieve this. Salmonella actively maintains itself within an intracellular vacuole, thereby shielding itself from an antibacterial activity of host macrophage cytosol. Salmonella controls the maturation of its vacuole, segregating itself from the macrophage degradative pathway. Like several other pathogens, Salmonella reduces the effectiveness of bacteriocidal and bacteriostatic free radicals generated by macrophages, by synthesising enzymes and products that counteract them. Recent evidence indicates that Salmonella also avoids free radical-dependent macrophage antimicrobial mechanisms by more novel means. Here, we review recent studies of the interplay between pathogen and host, with particular emphasis on those areas that suggest new facets to the cell biology of macrophages, and their innate immune functions.

Animals↗

PubMed2003

Glutamate-115 renders specificity of human 11beta-hydroxysteroid dehydrogenase type 2 for the cofactor NAD+.

The renal 11beta-hydroxysteroid dehydrogenase (11beta-HSD) type 2 catalyzes the NAD(+)-dependent oxidation of the C11-alcohol on cortisol and corticosterone to yield inactive 11-ketosteroids. The lack of purified active enzyme complicates structure-function analyses of 11beta-HSD2. Here, we constructed a 3D-structural model of 11beta-HSD2, based on known 3D-structures of other short-chain dehydrogenases/reductases (SDR), and functionally analyzed 11beta-HSD2 mutants predicted to be involved in cofactor binding. Our 3D-model explains the preference for NAD(+) over NADP(+) by the coulombic repulsion between the adenosine ribose 2'-phosphate on NADP(+) and the carboxylate on Glu(115) and to steric hindrance with the side chain on Glu(115). Indeed, replacement of Glu(115) with serine or threonine, lacking repulsive charge and unfavorable steric interactions, showed only 3-fold preference for NAD(+), compared to 40-fold for wild-type 11beta-HSD2. Mutation of both Asp(91) and Glu(115) to serine raised NADP(+)-dependent activity to that with NAD(+), but caused reduced enzymatic activity. The 3D-model predicted that this is due to a loss of stabilizing interactions of Asp(91) with Cys(90), Glu(115), Asn(117) and Gly(120). Thus, predictions using the 3D-model combined with analysis of mutants allowed the identification of residues critical for NAD(+)-dependent activity of 11beta-HSD2.

11-beta-Hydroxysteroid Dehydrogenase Type 2↗