Determination of Intracellular Trehalose and Glycogen in Saccharomyces cerevisiae

Schulze, Ulrik; Larsen, Malene Erup

doi:10.1006/abio.1995.1325

Cited by 56 publications

(38 citation statements)

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“…Aliquots (200 l) of the supernatant were placed into two tubes; one was used as background, and the other was digested by trehalase (0.05 units/ml, 37°C overnight). Glucose concentration was measured using Infinity TM glucose reagent (Sigma) (17).…”

Section: Methodsmentioning

confidence: 99%

Role of Trehalose Phosphate Synthase in Anoxia Tolerance and Development in Drosophila melanogaster

Chen¹,

Ma²,

Behar³

et al. 2002

Journal of Biological Chemistry

162

136

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Recent studies have shown that trehalose plays a protective role in yeast in a variety of stresses, including heat, freezing and thawing, dehydration, hyperosmotic shock, and oxidant injury. Because (a) heat shock and anoxia share mechanisms that allow organisms to survive, (b) Drosophila melanogaster is tolerant to anoxia, and (c) trehalose is present in flies and is metabolically active, we asked whether trehalose can protect against anoxic stress. Here we report on a new role of trehalose in anoxia resistance in Drosophila. We first cloned the gene trehalose-6-phosphate synthase (tps1), which synthesizes trehalose, and examined the effect of tps1 overexpression as well as mutation on the resistance of Drosophila to anoxia. Upon induction of tps1, trehalose increased, and this was associated with increased tolerance to anoxia. Furthermore, in vitro experiments showed that trehalose reduced protein aggregation caused by anoxia. Homozygous tps1 mutant (P-element insertion into the third intron of the gene) leads to lethality at an early larval stage, and excision of the Pelement rescues totally the phenotype. We conclude that trehalose contributes to anoxia tolerance in flies; this protection is likely to be due to a reduction of protein aggregation.

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Section: Methodsmentioning

confidence: 99%

Role of Trehalose Phosphate Synthase in Anoxia Tolerance and Development in Drosophila melanogaster

Chen¹,

Ma²,

Behar³

et al. 2002

Journal of Biological Chemistry

162

136

View full text Add to dashboard Cite

show abstract

“…As visualized by transmission electron microscopy, the bulges and vesicles contain densely packed material which appears to be storage material. Its nature is unknown, but trehalose (Schulze et al, 1995), glycogen (Preiss, 1984), polyhydroxybutyrate (Steinbiichel & Schlegel, 1991), or a predominant type of lipid can be excluded as possibilities. In addition to glycogen, triacylglycerol has been found recently within Streptomyces species, including S. lividans (Olukoshi & Packter, 1994).…”

Section: Discussionmentioning

confidence: 99%

“…To the supernatant 1 mg pronase ml-l was added, and after 60 min cell debris was removed by low-speed centrifugation (2500 g for 10 min), and granules were sedimented at 50000 g. Tests for glycogen and trehalose. Previously described enzymic methods were used to test for the presence of glycogen and trehalose (Schulze et al, 1995).…”

Section: Methodsmentioning

confidence: 99%

Identification of genes in Rhizobium leguminosarum bv. trifolii whose products are homologues to a family of ATP-binding proteins

1997

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The wild-type Streptomyces lividans 66 genome contains a 4 3 kb amplifiable DNA unit (AUD), and its four ORFs encode proteins that could not be identified by sequence comparison with databases. One of the gene products (encoded by orf-2) was purified and determined to be a novel 23 kDa protein. This protein is synthesized by the wild-type strain, absent in a variant lacking the AUD and overproduced in a variant in which the AUD is amplified (ADS). Immunological studies and analyses by confocal laser microscopy showed that the 23 kDa protein is associated with the substrate hyphae of the wild-type and the ADS-containing variant. Examination by microscopy revealed that the strain carrying the ADS forms bulges within the substrate hyphae and apical vesicles. These bulges have high levels of associated 23 kDa protein and contain storage-I i ke material.

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“…Glucose concentrations were measured using Infinity TM glucose reagent (Sigma). 48 The mass of trehalose per wet weight of first instar larvae was determined for 4 replicates of 100 larvae treated with either gus-dsRNA (controls) or tps1-dsRNA.…”

Section: Methodsmentioning

confidence: 99%

Biolistics for high-throughput transformation and RNA interference inDrosophila melanogaster

Yuen

Read²,

Brubacher³

et al. 2008

Fly

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With twelve Drosophila genomes now sequenced, there is a growing need to develop higher-throughput methods for identifying the functions of the many newly identified genes. Genetic transformation and RNA interference are two technologies that have been used extensively to facilitate gene-function studies in Drosophila melanogaster, to introduce genes or block the expression of endogenous genes, respectively. Both of these technologies typically require the delivery of nucleic acids into developing insect embryos, and virtually all studies to date have relied on microinjection as the DNA delivery method of choice. In this study, we describe the use of biolistics as a higher-throughput method of nucleic acid delivery. By bombarding dechorionated D. melanogaster embryos with 1 μm gold beads coated with P-element or piggyBac transformation vectors, we observed transformation frequencies (3-4%) that are comparable to those achieved using microinjection methods, but in only a fraction of the time required for the DNA delivery. Biolistic delivery of double-stranded RNA (dsRNA) specific to a β-glucuronidase (gus) transgene resulted in a significant (71%) reduction in gus transcripts in embryos and the RNA interference (RNAi) persisted through two successive larval molts, albeit at reduced levels. DsRNAs specific to four essential genes were delivered to embryos and resulted in arrested development and phenotypes that closely match that of null mutations. These results suggest that biolistic delivery of dsRNA into embryos could be adapted for high throughput RNAi screens of early Drosophila developmental genes.

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Determination of Intracellular Trehalose and Glycogen in Saccharomyces cerevisiae

Cited by 56 publications

References 0 publications

Role of Trehalose Phosphate Synthase in Anoxia Tolerance and Development in Drosophila melanogaster

Role of Trehalose Phosphate Synthase in Anoxia Tolerance and Development in Drosophila melanogaster

Identification of genes in Rhizobium leguminosarum bv. trifolii whose products are homologues to a family of ATP-binding proteins

Biolistics for high-throughput transformation and RNA interference inDrosophila melanogaster

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