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61.
Sessions A Burke E Presting G Aux G McElver J Patton D Dietrich B Ho P Bacwaden J Ko C Clarke JD Cotton D Bullis D Snell J Miguel T Hutchison D Kimmerly B Mitzel T Katagiri F Glazebrook J Law M Goff SA 《The Plant cell》2002,14(12):2985-2994
A collection of Arabidopsis lines with T-DNA insertions in known sites was generated to increase the efficiency of functional genomics. A high-throughput modified thermal asymmetric interlaced (TAIL)-PCR protocol was developed and used to amplify DNA fragments flanking the T-DNA left borders from approximately 100000 transformed lines. A total of 85108 TAIL-PCR products from 52964 T-DNA lines were sequenced and compared with the Arabidopsis genome to determine the positions of T-DNAs in each line. Predicted T-DNA insertion sites, when mapped, showed a bias against predicted coding sequences. Predicted insertion mutations in genes of interest can be identified using Arabidopsis Gene Index name searches or by BLAST (Basic Local Alignment Search Tool) search. Insertions can be confirmed by simple PCR assays on individual lines. Predicted insertions were confirmed in 257 of 340 lines tested (76%). This resource has been named SAIL (Syngenta Arabidopsis Insertion Library) and is available to the scientific community at www.tmri.org. 相似文献
62.
Lamin A/C binding protein LAP2alpha is required for nuclear anchorage of retinoblastoma protein
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Markiewicz E Dechat T Foisner R Quinlan RA Hutchison CJ 《Molecular biology of the cell》2002,13(12):4401-4413
The phosphorylation-dependent anchorage of retinoblastoma protein Rb in the nucleus is essential for its function. We show that its pocket C domain is both necessary and sufficient for nuclear anchorage by transiently expressing green fluorescent protein (GFP) chimeras of Rb fragments in tissue culture cells and by extracting the cells with hypotonic solutions. Solid phase binding assays using glutathione S-transferase-fusion of Rb pockets A, B, and C revealed a direct association of lamin C exclusively to pocket C. Lamina-associated polypeptide (LAP) 2alpha, a binding partner of lamins A/C, bound strongly to pocket C and weakly to pocket B. When LAP2alpha was immunoprecipitated from soluble nuclear fractions, lamins A/C and hypophosphorylated Rb were coprecipitated efficiently. Similarly, immunoprecipitation of expressed GFP-Rb fragments by using anti-GFP antibodies coprecipitated LAP2alpha, provided that pocket C was present in the GFP chimeras. On redistribution of endogenous lamin A/C and LAP2alpha into nuclear aggregates by overexpressing dominant negative lamin mutants in tissue culture cells, Rb was also sequestered into these aggregates. In primary skin fibroblasts, LAP2alpha is expressed in a growth-dependent manner. Anchorage of hypophosphorylated Rb in the nucleus was weakened significantly in the absence of LAP2alpha. Together, these data suggest that hypophosphorylated Rb is anchored in the nucleus by the interaction of pocket C with LAP2alpha-lamin A/C complexes. 相似文献
63.
P.J. Gregory D. J. Hutchison D. B. Read P. M. Jenneson W. B. Gilboy E. J. Morton 《Plant and Soil》2003,255(1):351-359
X-ray micro-tomography is a well-established technique for non-invasive imaging and evaluation of heterogeneous materials. An inexpensive X-ray micro-tomography system has been designed and built for the specific purposes of examining root growth and root/soil interactions. The system uses a silver target X-ray source with a focal spot diameter of 80 m, an X-ray image intensifier with a sampling aperture of about 100 m, and a sample with a diameter of 25 mm. Pre-germinated wheat and rape seeds were grown for up to 8–10 days in plastic containers in a sandy loam soil sieved to < 250 m, and imaged with the X-ray system at regular intervals. The quality of 3 D image obtained was good allowing the development and growth of both root axes and some first-order laterals to be observed. The satisfactory discrimination between soil and roots enabled measurements of root diameter (wheat values were 0.48–1.22 mm) in individual tomographic slices and, by tracking from slice to slice, root lengths were also measured. The measurements obtained were generally within 10% of those obtained from destructive samples measured manually and with a flat-bed scanner. Further developments of the system will allow more detailed examination of the root:soil interface. 相似文献
64.
BACKGROUND: Post-traumatic psychopathology (PTP) is important to the orthopaedic surgeon as it may complicate the recovery from musculoskeletal injury. PTP is associated with a disturbance of the hypothalamic-pituitary-adrenal axis, and may lead to impaired healing. We have investigated the relationship between PTP and pro-inflammatory markers of the metabolic response to trauma. mATERIALS AND METHODS: A prospective cohort study of 82 patients with musculoskeletal injuries, correlating development of psychopathology (measured by general health questionnaire) and pro-inflammatory markers (CRP, IL-6, sIL-6r, TNF-alpha) two and six months after their injury. RESULTS: Psychological disturbance was found in 39% of patients at two months and 18% at six months. This disturbance was associated with significantly increased levels of IL-6 at two months and of sIL-6r and TNF-alpha at six months. CRP levels were not related to the development of PTP. CONCLUSIONS: The relationship between PTP and disturbances of pro-inflammatory markers needs further exploration, but may explain in part the impaired functional recovery when musculoskeletal trauma is complicated by psychological disturbance. 相似文献
65.
Endotoxic activity of lipopolysaccharides isolated from emergent potential cystic fibrosis pathogens
Hutchison ML Bonell EC Poxton IR Govan JR 《FEMS immunology and medical microbiology》2000,27(1):73-77
Improved antimicrobial therapies against the classical spectrum of pathogenic bacteria which colonise the lungs of cystic fibrosis (CF) patients has resulted in improved life expectancy and quality of life. Bacterial species that are resistant to a broad range of antibiotics including Stenotrophomonas maltophilia and Alcaligenes xylosoxidans have now emerged as potential new pathogens to fill the niche. At present, it is unclear from clinical data whether these microbes are commensal or pathogenic. In this study we have quantified the inflammatory potential of lipopolysaccharide (LPS) from eight species of Gram-negative organisms which have been cultured with increasing frequency from CF patients. Inflammatory responses induced by LPS from whole human blood and a human-derived monocyte cell line (THP-1) were assessed. Enzyme-linked immunosorbent assays were used to detect interleukin-6, interleukin-8, and tumour necrosis factor alpha (TNF). A bioassay was also used to assess TNF activity. With the exception of S. maltophilia, LPS extracted from all of the bacteria tested upregulated, by varying degrees, expression of each of the proinflammatory cytokines assayed. This study represents the first comprehensive report of the endotoxic potential of a new wave of microbes which are associated with CF. 相似文献
66.
67.
We present a field-based approach to detect and monitor insects with resistance to insecticidal toxins produced by transgenic plants. Our objective is to estimate the phenotypic frequency of resistance in a population by relating the densities of insects on genetically transformed plants to densities on nontransformed plants. We focus on European corn borer, Ostrinia nubilalis (Hübner), in sweet corn, Zea mays L., expressing Cry1Ab from Bacillus thuringiensis subsp. kurstaki Berliner to illustrate principles underlying the method. The probability of detecting one or more rare, resistant larvae depends on sample size, the density of larvae on nontransformed plants, and an assumed frequency of resistant phenotypes in a given population. Probability of detection increases with increases in sample size, background density, or the frequency of resistant individuals. Following binomial probability theory, if a frequency of 10(-4) is expected, 10(3)-10(4) samples must be collected from a B. thuringiensis (Bt) crop to have at least a 95% probability of locating one or more resistant larvae. In-field screens using transgenic crops have several advantages over traditional laboratory-based methods, including exposure to a large number of feral insects, discrimination of resistant individuals based on Bt dosages expressed in the field, incorporation of natural and Bt-induced mortality factors, simultaneous monitoring for more than one insect species, and ease of use. The approach is amenable to field survey crews working in research, extension, and within the seed corn industry. Estimates of the phenotypic frequency of resistance from the in-field screen can be useful for estimating initial frequency of resistant alleles. Bayesian statistical methods are outlined to estimate phenotype frequencies, allele frequencies, and associated confidence intervals from field data. Results of the approach are discussed relative to existing complementary methods currently available for O. nubilalis and corn earworm, Helicoverpa zea (Boddie). 相似文献
68.
Head and/or CaaX domain deletions of lamin proteins disrupt preformed lamin A and C but not lamin B structure in mammalian cells
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The nuclear lamina is an important determinant of nuclear architecture. Mutations in A-type but not B-type lamins cause a range of human genetic disorders, including muscular dystrophy. Dominant mutations in nuclear lamin proteins have been shown to disrupt a preformed lamina structure in Xenopus egg extracts. Here, a series of deletion mutations in lamins A and B1 were evaluated for their ability to disrupt lamina structure in Chinese hamster ovary cells. Deletions of either the lamin A "head" domain or the C-terminal CaaX domain formed intranuclear aggregates and resulted in the disruption of endogenous lamins A/C but not lamins B1/B2. By contrast, "head-less" lamin B1 localized to the nuclear rim with no detectable effect on endogenous lamins, whereas lamin B1 CaaX domain deletions formed intranuclear aggregates, disrupting endogenous lamins A/C but not lamins B1/B2. Filter binding assays revealed that a head/CaaX domain lamin B1 mutant interacted much more strongly with lamins A/C than with lamins B1/B2. Regulated induction of this mutant in stable cell lines resulted in the rapid elimination of all detectable lamin A protein, whereas lamin C was trapped in a soluble form within the intranuclear aggregates. In contrast to results in Xenopus egg extracts, dominant negative lamin B1 (but not lamin A) mutants trapped replication proteins involved in both the initiation and elongation phases of replication but did not effect cellular growth rates or the assembly of active replication centers. We conclude that elimination of the CaaX domain in lamin B1 and elimination of either the CaaX or head domain in lamin A constitute dominant mutations that can disrupt A-type but not B-type lamins, highlighting important differences in the way that A- and B-type lamins are integrated into the lamina. 相似文献
69.
Differential effects of chilling-induced photooxidation on the redox regulation of photosynthetic enzymes 总被引:10,自引:0,他引:10
Photosynthesis in plant species that are evolutionarily adapted for growth in warm climates is highly sensitive to illumination under cool conditions. Although it is well documented that illumination of these sensitive species under cool conditions results in the photosynthetic production of reactive oxygen molecules, the underlying mechanism for the inhibition of photosynthesis remains uncertain. Determinations of chloroplast fructose-1,6-bisphosphatase and sedoheptulose-1,7-bisphosphatase activity showed that the light-dependent, reductive activation of these key carbon reduction cycle enzymes was substantially inhibited in tomato (Lycopersicon esculentum) following illumination at 4 degrees C. However, other chloroplast enzymes also dependent on thioredoxin-mediated reductive activation were largely unaffected. We performed equilibrium redox titrations to investigate the thermodynamics of the thiol/disulfide exchange between thioredoxin f and the regulatory sulfhydryl groups of fructose-1,6-bisphosphatase, sedoheptulose-1,7-bisphosphatase, phosphoribulokinase, NADP-glyceraldehyde phosphate dehydrogenase, and the chloroplast ATPsynthase. We determined that the redox midpoint potentials for the regulatory sulfhydryl groups of the various enzymes spanned a broad range ( approximately 50 mV at pH 7. 9). The electron-sharing equilibria among thioredoxin f and its target enzymes largely explained the differential effects of photooxidation induced at low temperature on thioredoxin-mediated activation of chloroplast enzymes in tomato. These results not only provide a plausible mechanism for the low-temperature-induced inhibition of photosynthesis in this important group of plants, but also provide a quantitative basis to evaluate the influence of thioredoxin/target enzyme electron-sharing equilibria on the differential activation and deactivation kinetics of thioredoxin-regulated chloroplast enzymes. 相似文献
70.
Songbirds have a complex neural network for learning and production of song, namely the neural song system. Several nuclei of the song system contain androgen receptors (AR), and the neostriatal nucleus HVc also contains alpha type estrogen receptors (ER). Many songbird species show seasonal changes in both song and the neural song system that are correlated with seasonal variations in the circulating levels of gonadal steroids. However, there is increasing evidence that the sensitivity of the song system to gonadal steroids also changes seasonally. This could involve changes in the expression and activity of steroid receptors and steroid-metabolizing enzymes, such as the estrogen-synthesizing enzyme aromatase (AROM). The seasonal regulation of brain AR, ER, and AROM has not been studied before in the same individual songbirds. In this work, we compared plasma levels of androgens and estrogens, the expression level of AR-, ER-, and AROM-mRNA in the telencephalon, and brain AROM activity in male canaries between autumn (November) and spring (April) periods of high singing activity. Plasma levels of androgens and estrogens were higher in April than in November. The expression level of ER in HVc was higher in November than in April. In contrast, the expression level of AROM in the caudomedial neostriatum was higher in April than in November. However, we found no seasonal differences in the level of expression of AR and the volume of HVc as delimited by AR expression. Thus, AR expression in HVc was not correlated with circulating androgen levels. This study shows that both steroid-dependent and -independent seasonal factors regulate the action of gonadal hormones on the song system. In addition, we report a new site of AROM expression in the songbird brain, the nucleus interfacialis. 相似文献