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Methods were developed for the isolation of plastids from mature green and ripening tomatoes (Lycopersicon esculentum Mill.) and purification by sucrose or Percoll density-gradient centrifugation. Assessment of the purity of preparations involved phase-contrast and electron microscopy, assays for marker enzymes and RNA extraction and analysis. Proteins were extracted from isolated plastids at different ripening stages and separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The profiles obtained from chloroplasts and chromoplasts showed many qualitative and quantitative differences. Labelling of proteins with [35S]methionine in vivo showed that there was active protein synthesis throughout ripening, but there was a change in the plastid proteins made as ripening proceeded. The cellular location of synthesis of specific proteins has yet to be established.Abbreviations CS
citrate synthase
- EDTA
ethylenediaminetetraacetic acid,-acetate
- GAPDH
NADP+-glyceraldehyde-3-phosphate dehydrogenase
- rRNA
ribosomal RNA
- SDS
sodium dodecyl sulphate
- SDS-PAGE
SDS-polyacrylamide gel electrophoresis
- Tris
2-amino-2(hydroxymethyl)-1,3-propanediol 相似文献
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Studies on the maintenance and expression of cloned DNA fragments in the nuclear genome of the green alga Chlamydomonas Reinhardtii 总被引:3,自引:0,他引:3
Anil Day Robert Debuchy Jeanette van Dillewijn Saul Purton Jean-David Rochaix 《Physiologia plantarum》1990,78(2):254-260
Using a biolistic device built here and based on the principle of the device described by Klein et al. (1987). we have reproducibly obtained transformants of Chlamydomonas reinhardtii . The reproducibility of the method has allowed us to examine the maintenance and expression of cloned DNA fragments introduced into C. Reinhardtii . 相似文献
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A chimeric gene composed of the coding sequence of theble gene fromStreptoalloteichus hindustanus fused to the 5 and 3 untranslated regions of theChlamydomonas reinhardtii nuclear geneRBCS2 has been constructed. Introduction of this chimeric gene into the nuclear genome ofC. reinhardtii by co-transformation with theARG7 marker yields Arg+ transformants of which approximately 80% possess theble gene. Of these co-transformants, approximately 3% display a phleomycin-resistant (PmR) phenotype. Western blot analysis using antibodies against theble gene product confirms the presence of the protein in the PmR transformants and genetic analysis demonstrates the co-segregation of theble gene with the phenotype in progeny arising from the mating of a PmR transformant to wild-type strains. Direct selection of PmR transformants was achieved by allowing an 18-h period for recovery and growth of transformed cells prior to selection. This work represents the first demonstration of stable expression and inheritance of a foreign gene in the nuclear genome ofC. reinhardtii and provides a useful dominant marker for nuclear transformation. 相似文献
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Helen E. O'connor David R. Stevens Stuart V. Ruffle Jonathan H. A. Nugent Saul Purton 《Plant Molecular Biology Reporter》1993,11(3):207-211
The isolation of chloroplast DNA fromChlamydomonas reinhardtii requires the efficient separation of this AT-rich genome from the GC-rich nuclear genome by density-gradient centrifugation.
We describe a simple and efficient method for separating these DNA fractions by using a sodium iodide gradient in combination
with the DNA-binding dye, bisbenzimide. The yield of chloroplast DNA is close to the theoretical maximum and the DNA is suitable
for restriction enzyme analysis and cloning. This method is applicable to the isolation of AT-rich plastid genomes from other
organisms and may be appropriate as a general method for separating species of DNA that differ in their AT/GC ratios.
An erratum to this article is available at . 相似文献
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Beverly J. Hallahan Saul Purton Angela Ivison Derek Wright Michael C. W. Evans 《Photosynthesis research》1995,46(1-2):257-264
The psaA and psaB genes of the chloroplast genome in oxygenic photosynthetic organisms code for the major peptides of the Photosystem 1 reaction center. A heterodimer of the two polypeptides PsaA and PsaB is thought to bind the reaction center chlorophyll, P700, and the early electron acceptors A0, A1 and Fe-SX. Fe-SX is a 4Fe4S center requiring 4 cysteine residues as ligands from the protein. As PsaA and PsaB have only three and two conserved cysteine residues respectively, it has been proposed by several groups that Fe-SX is an unusual inter-peptide center liganded by two cysteines from each peptide. This hypothesis has been tested by site directed mutagenesis of PsaA residue C575 and the adjacent D576. The C575D mutant does not assemble Photosystem 1. The C575H mutant contains a photoxidisable chlorophyll with EPR properties of P700, but no other Photosystem 1 function has been detected. The D576L mutant assembles a modified Photosystem 1 in which the EPR properties of the Fe-SA/B centers are altered. The results confirm the importance of the conserved cysteine motif region in Photosystem 1 structure.Dedicated to the memory of Daniel I. Arnon. 相似文献
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The Gaussian density molecular model has been adapted for dissipative particle dynamics. The model, when combined with a soft potential, is shown to be a very flexible mesoscale model exhibiting a wide range of phase behaviour. The soft potential allows relatively large time steps to be used and hence a more rapid equilibration. In addition, the model can be used to study both uniaxial and biaxial systems. We have undertaken a number of pilot studies and have demonstrated that the Gaussian model is able to identify nematic–isotropic phase transitions in liquid crystals and the formation of ordered discotic phases. 相似文献
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The swimming behaviour of the green flagellated protist Chlamydomonas reinhardtii is influenced by several different external stimuli including light and chemical attractants. Common components are involved in both the photo- and chemo-sensory transduction pathways, although the nature and organisation of these pathways are poorly understood. To learn more about the mechanism of chemotaxis in Chlamydomonas, we have generated nonchemotactic strains by insertional mutagenesis. The arginine-requiring strain arg7-8 was transformed with DNA carrying the wild-type ARG7 gene. Of the 8630 arginine-independenttransformants obtained, five are defective in their chemotaxis towards various sugars. Two of the mutants (CTX2 and CTX3) are blocked only in their response to xylose. Mutant CTX1 is blocked in its response to xylose, maltose and mannitol, but displays normal taxis to sucrose. Mutants CTX4 and CTX5 lack chemotactic responses to all sugars tested. CTX1, CTX4 and CTX5 represent novel chemotactic phenotypes not previously obtained using ultra-violet or chemical mutagenesis. Genetic analysis confirms that each mutation maps to a single nuclear locus that is unlinked to the mating-type locus. Further analysis of CTX4 indicates that the mutant allele is tagged by the transforming ARG7 DNA. CTX4 appears to be defective in a component specific for chemotactic signal transduction since it exhibits wild-type photobehavioural responses (phototaxis and photoshock) as well as the wild-type responses of EGTA-induced trans-flagellum inactivation and acid-induced deflagellation. Insertional mutagenesis has thus permitted the generation of novel chemotactic mutants that will be of value in the molecular dissection of the signalling machinery. 相似文献
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