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Establishment of a transient expression system for Dictyostelium discoideum. 总被引:26,自引:4,他引:22 下载免费PDF全文
We have established a rapid and sensitive transient expression system for Dictyostelium discoideum. We constructed a gene fusion containing the promoter from the Dictyostelium Actin 15 gene fused to the firefly luciferase gene. The enzymatic activity of this gene fusion, expressed at very high levels in stable transformants, was measured to determine optimum conditions for transient expression using electroporation to introduce the DNA into cells. With these conditions, we show that a luciferase gene fusion driven by a prestalk, cell-type specific promoter from the pst-cathepsin gene expresses luciferase at the appropriate developmental stage. In addition, we present results suggesting that the system will be useful for expressing genes in non-axenic cell lines. Finally, we observe that electroporation is more efficient for obtaining stable transformations than the standard calcium phosphate procedure using extrachromosomally replicating shuttle vectors but less efficient for vectors that integrate into the Dictyostelium chromosomes. 相似文献
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Ute Mathis Frank Schaeffel Howard C. Howland 《Journal of comparative physiology. A, Neuroethology, sensory, neural, and behavioral physiology》1988,163(2):201-213
Aspects of visual optics were investigated in the American toad (Bufo americanus). The development of the refractive state of the eye during metamorphosis was followed with IR photoretinoscopy. Frozen sections documented the changes in optical parameters before and after metamorphosis. There is a difference in light sensitivity between juvenile and adult toads. Binocular accommodation in adult toads was observed. 1. IR photoretinoscopic measurements showed that the refractive state of the eye changed very rapidly during metamorphosis, about 10 D/h while the animal entered the terrestrial habitat. 2. Frozen sections showed that the almost spherical lens in a tadpole eye had flattened in a just metamorphosed toad's eye while at the same time the distance of the lens to the retina had decreased. However, the morphological measurements were not sufficiently sensitive to record the relatively small changes in ocular dimensions that were responsible for the rapid changes in refractive state during metamorphosis. 3. Schematic eyes, with homogeneous and non homogeneous lenses, were constructed for tadpoles, juvenile toads, and adult toads. 4. Nonparaxial raytracing studies in schematic eyes suggested that the lenses of animals of the three developmental stages tadpole, juvenile toad, and adult are not homogeneous but have a refractive index gradient. The raytracing studies indicated that the refractive index gradient is different for the different developmental stages, being highest in the tadpole lens. 5. The observations of toads during feeding behavior at different light levels showed an increased light sensitivity in the adult nocturnal toads in contrast to the juvenile animals, which are diurnal. The increased light sensitivity could partly be explained with an increase in aperture and an increase in red rod outer segments. To fully explain the higher light sensitivity in adult toads, changes in neuronal parameters had to be assumed. 6. Retinoscopic measurements of the resting refractive state in the adult toad showed a hyperopic defocus of about +8 D. By subtracting the measurement artefact for retinoscopy, the true resting focus was found to be nearly emmetropic. 7. The amount of natural accommodation in adult toads during normal feeding behavior was investigated with IR photoretinoscopy. Binocular accommodation of about 8 D was observed. 相似文献
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In past attempts, the experimental germination of the seeds of Euonymus americanus L. in vitro has had little success. However, treatment of seeds with ruminal fluid containing viable microflora has been successful in stimulating germination. In the presence of the cellulolytic ruminal bacterium, Clostridium cellobioparum ATCC 15832, seeds of E. americanus were stimulated to germinate. Subsequent studies were designed to determine whether the bacterium synthesized a cellulolytic enzyme responsible for initiating germination. The cell-free endocellulase from C. cellobioparum induced germination of the seeds. To support the hypothesis that the endocellulase from C. cellobioparum was responsible for triggering germination, a 1,4-beta-d-glucan glucanohydrolase (EC 3.2.1.4) from Penicillum funiculosum was used to treat the seeds. In addition, no germination was obtained from seeds treated with a commercial exocellulase enzyme. Also, Ruminococcus flavefaciens FD-1 was found to initiate germination of E. americanus seeds. Thus, cellulase activity is indicated in the degradation of the testa of the seed, allowing imbibition and germination. 相似文献
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Howard Gest 《Photosynthesis research》1988,19(3):287-308
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Use of glycosyltransferases to restore pertussis toxin receptor activity to asialoagalactofetuin 总被引:10,自引:0,他引:10
Fetuin derivatives with enzymatically altered oligosaccharide units were tested for their ability to inhibit pertussis toxin-mediated agglutination of goose erythrocytes and the binding of 125I-labeled fetuin to pertussis toxin-coated polystyrene tubes. Fetuin oligosaccharides were sequentially degraded by treatment with: neuraminidase (asialofetuin) followed by beta-galactosidase (asialoagalactofetuin) and, lastly, with beta-N-acetylhexosaminidase (asialoagalacto-a[N-acetylglucosamino]fetuin). Asialofetuin retained only 19 and 53% of the inhibitory activity of native fetuin in the hemagglutination and 125I-fetuin binding assays, respectively. Asialoagalactofetuin showed no further reduction of inhibition in the hemagglutination system and, instead, resulted in partial recovery of inhibition in the 125I-fetuin-pertussis toxin binding assay. Asialoagalacto-a[N-acetylhexosamino]fetuin showed a further decrease in ability to inhibit pertussis toxin binding in both assays. The inhibitory activity of asialoagalactofetuin could be restored to that of native fetuin by adding back D-galactose with UDP-Gal:D-glucosyl-1,4-beta-galactosyltransferase, followed by the addition of terminal sialic acid residues with CMP-N-acetylneuraminic acid:beta-D-galactosyl-1,4-N-acetyl-beta-D-glucosamine-alpha-2,6-N- acetylneuraminyltransferase. The data suggested that a requirement for pertussis toxin binding to fetuin may be the presence of acetamido-containing sugar groups in the nonreducing terminal position of fetuin's oligosaccharides. 相似文献
49.
Regulation of the CD2 alternate pathway of T cell activation by CD3. Evidence for heterologous desensitization 总被引:2,自引:0,他引:2
W Holter O Majdic H Stockinger B H Howard W Knapp 《Journal of immunology (Baltimore, Md. : 1950)》1988,140(4):1043-1046
Normal resting T cells were stimulated through the alternate CD2 pathway. A CD3 mAb VIT3 completely blocked their proliferative response. The time interval for 50% inhibition lasted for 24 h after the onset of CD2 stimulation. Mitogen-activated cloned long term cultured T cells could also be stimulated via CD2. This proliferative response was again inhibitable by VIT3, indicating that CD3 regulates the CD2 pathway not only in resting cells, but also in lymphocytes actively involved in an Ir. T cells were further loaded with Quin2 and their free cytoplasmic Ca2+ levels were monitored in response to CD3 and CD2 stimulation. Antibodies directed against both surface R triggered a rapid elevation of Ca2+ levels. Both responses were abrogated when the cells had been treated overnight with VIT3. The free cytoplasmic Ca2+ levels of VIT3-pretreated cells, however, were not higher than those of control cells. These results point to a functional interaction between CD3 and CD2 possibly at the level of signal transducing proteins. Finally, cholera toxin was found to inhibit the Ca2+ response in Jurkat T cells. Both the CD3 and CD2 stimulation were sensitive to cholera toxin, indicating that a GTP-binding protein may be involved in signal transduction for both surface structures. 相似文献
50.
Andrew W. Thomas Jay Lewington Steve Hope Andrew W. Topping Andrew J. Weightman J. Howard Slater 《Archives of microbiology》1992,158(3):176-182
Favourable mutations involving the two dehalogenases (DehI and DehII) of Pseudomonas putida PP3 and derivative strains containing the cloned gene for DehI (dehI) occurred in response to specific environmental conditions, namely: starvation conditions; the presence of dehalogenase substrates (halogenated alkanoic acids — HAAs) which were toxic to P. putida; and/or the presence of a potential growth substrate. Fluctuation tests showed that these mutations were environmentally directed by the presence of HAAs. the mutations were associated with complex DNA rearrangements involving the movement of dehI located on a transposon DEH. Some mutations resulted in switching off the expression of either one or both of the dehalogenases, events which were effective in protecting P. putida from toxic compounds in its growth environment. Other mutations partially restored P. putida's dehalogenating capability under conditions where toxic substrates were absent. Restoration of the capability to untilize HAAs was favoured when normal growth substrates were present in the environment. 相似文献