共查询到20条相似文献,搜索用时 15 毫秒
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Elena Babiychuk Mayuree Fuangthong Marc Van Montagu Dirk Inz Sergei Kushnir 《Proceedings of the National Academy of Sciences of the United States of America》1997,94(23):12722-12727
Large quantities of DNA sequence information about plant genes are rapidly accumulating in public databases, but to progress from DNA sequence to biological function a mutant allele for each of the genes ideally should be available. Here we describe a gene trap construct that allowed us to disrupt transcribed genes with a high efficiency in Arabidopsis thaliana. In the T-DNA vector used, the expression of a bacterial reporter gene coding for neomycin phosphotransferase II (nptII) depends on the in vivo generation of a translation fusion upon the T-DNA integration into the Arabidopsis genome. Analysis of 20 selected transgenic lines showed that 12 lines are T-DNA insertion mutants. The disrupted genes analyzed encoded ribosomal proteins (three lines), aspartate tRNA synthase, DNA ligase, basic-domain leucine zipper DNA binding protein, ATP-binding cassette transporter, and five proteins of unknown function. Four tagged genes were new for Arabidopsis. The results presented here suggest that gene trapping, using nptII as a reporter gene, can be as high as 80% and opens novel perspectives for systematic gene tagging in A. thaliana. 相似文献
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Y Arai T Kubo K Kobayashi T Ikeda K Takahashi M Takigawa J Imanishi Y Hirasawa 《The Journal of rheumatology》1999,26(8):1769-1774
OBJECTIVE: To investigate whether the expressions of delivered Escherichia coli beta-galactosidase (LacZ) gene and transforming growth factor-beta1 (TGF-beta1) gene are regulated by the stress response of human chondrocyte-like cells (HCS-2/8) when heat shock protein 70B (HSP70B) promoter is inserted into the adenovirus vector. METHODS: Two adenovirus vectors that contain either LacZ gene or TGF-beta1 gene regulated by HSP70B promoter were constructed. One of the adenovirus vectors was added to the culture of HCS-2/8 and gene transduced cells were produced. We applied heat stress (43 degrees C) to the transduced cells for 2 h and examined whether the expression of transduced LacZ and TGF-beta1 genes is affected by the stress, using 5 bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal) staining, measurement of beta-galactosidase activity, Northern blotting, and ELISA. RESULTS: The percentage of X-gal positive stained cells in LacZ gene-delivered cells with heat stress was significantly higher than in controls (no heat stress). With heat stress, beta-galactosidase activity increased significantly, and the band of exogenous TGF-beta1 mRNA became more apparent and the expression was maintained during the 24 h monitoring period. TGF-beta1 level in culture supernatant of TGF-beta1 gene-delivered cells with heat stress (5477.3+/-321.1 pg/ml) was significantly higher than in the controls (853.2+/-29.2 pg/ml). CONCLUSION: HSP70B promoter could regulate the expression of delivered genes according to the intensity of heat stress. 相似文献
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Inducible gene expression and protein translocation using nontoxic ligands identified by a mammalian three-hybrid screen 下载免费PDF全文
Stephen D. Liberles Steven T. Diver David J. Austin Stuart L. Schreiber 《Proceedings of the National Academy of Sciences of the United States of America》1997,94(15):7825-7830
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ChristineT.N. Pham DebraM. MacIvor BruceA. Hug JonathanW. Heusel TimothyJ. Ley 《Proceedings of the National Academy of Sciences of the United States of America》1996,93(23):13090-13095
Recent studies have suggested that the retention of selectable marker cassettes (like PGK–Neo, in which a hybrid gene consisting of the phosphoglycerate kinase I promoter drives the neomycin phosphotransferase gene) in targeted loci can cause unexpected phenotypes in “knockout” mice due to disruption of expression of neighboring genes within a locus. We have studied targeted mutations in two multigene clusters, the granzyme B locus and the β-like globin gene cluster. The insertion of PGK–Neo into the granzyme B gene, the most 5′ gene in the granzyme B gene cluster, severely reduced the normal expression of multiple genes within the locus, even at distances greater than 100 kb from the mutation. Similarly, the insertion of a PGK–Neo cassette into the β-globin locus control region (LCR) abrogates the expression of multiple globin genes downstream from the cassette. In contrast, a targeted mutation of the promyelocyte-specific cathepsin G gene (which lies just 3′ to the granzyme genes in the same cluster) had minimal effects on upstream granzyme gene expression. Although the mechanism of these long distance effects are unknown, the expression of PGK–Neo can be “captured” by the regulatory domain into which it is inserted. These results suggest that the PGK–Neo cassette can interact productively with locus control regions and thereby disrupt normal interactions between local and long-distance regulatory regions within a tissue-specific domain. 相似文献
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Inducible expression of a cloned heat shock fusion gene in sea urchin embryos. 总被引:9,自引:1,他引:9 下载免费PDF全文
A P McMahon T J Novak R J Britten E H Davidson 《Proceedings of the National Academy of Sciences of the United States of America》1984,81(23):7490-7494
A fusion gene construct, in which the coding sequence for bacterial chloramphenicol acetyltransferase (CAT; acetyl-CoA: chloramphenicol 3-O-acetyltransferase, EC 2.3.1.28) was placed under the control of the regulatory region of the Drosophila gene encoding the 70-kilodalton heat shock protein [Di Nocera, P.P. & Dawid, I.B. (1983) Proc. Natl. Acad. Sci. USA 80, 7095-7098], was microinjected into the cytoplasm of unfertilized sea urchin eggs. Pluteus-stage embryos developing from the injected eggs were exposed to high temperature conditions that we found would elicit an endogenous sea urchin heat shock response. These embryos express the gene for CAT and, after heat treatment, display 8-10 times more CAT enzyme activity than do extracts from control embryos cultured at normal temperatures. The injected DNA is present in high molecular weight concatenates and, during development, is amplified about 100-fold. Amplified sequences are responsible for all or most of the induced CAT enzyme activity. 相似文献
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Discrimination of a single base change in a ribozyme using the gene for dihydrofolate reductase as a selective marker in Escherichia coli 下载免费PDF全文
Satoshi Fujita Tetsuhiko Koguma Jun Ohkawa Kazuyuki Mori Takeo Kohda Hideo Kise Satoshi Nishikawa Masahiro Iwakura Kazunari Taira 《Proceedings of the National Academy of Sciences of the United States of America》1997,94(2):391-396
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Identification of a novel negative retinoic acid responsive element in the promoter of the human matrix Gla protein gene 下载免费PDF全文
Jutta Kirfel Manuela Kelter Leonor M. Cancela Paul A. Price Roland Schüle 《Proceedings of the National Academy of Sciences of the United States of America》1997,94(6):2227-2232
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In vivo gene delivery to the liver using reconstituted chylomicron remnants as a novel nonviral vector 下载免费PDF全文
Toshifumi Hara Yadi Tan Leaf Huang 《Proceedings of the National Academy of Sciences of the United States of America》1997,94(26):14547-14552
Lipoproteins are emulsion particles that consist of lipids and apolipoproteins. Their natural function is to transport lipids and/or cholesterol to different tissues. We have taken advantage of the hydrophobic interior of these natural emulsions to solubilize DNA. Negatively charged DNA was first complexed with cationic lipids containing a quaternary amine head group. The resulting hydrophobic complex was extracted by chloroform and then incorporated into reconstituted chylomicron remnant particles (≈100 nm in diameter) with an efficiency ≈65%. When injected into the portal vein of mice, there were ≈5 ng of a transgene product (luciferase) produced per mg of liver protein per 100 μg injected DNA. This level of transgene expression was ≈100-fold higher than that of mice injected with naked DNA. However, such a high expression was not found after tail vein injection. Histochemical examination revealed that a large number of parenchymal cells and other types of cells in the liver expressed the transgene. Gene expression in the liver increased with increasing injected dose, and was nearly saturated with 50 μg DNA. At this dose, the expression was kept at high level in the liver for 2 days and then gradually reduced and almost disappeared by 7 days. However, by additional injection at day 7, gene expression in the liver was completely restored. By injection of plasmid DNA encoding human α1-antitrypsin, significant concentrations of hAAT were detected in the serum of injected animals. This is the first nonviral vector that resembles a natural lipoprotein carrier. 相似文献
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Altered gene expression in the host brain caused by a trematode parasite: Neuropeptide genes are preferentially affected during parasitosis 下载免费PDF全文
Robert M. Hoek Ronald E. van Kesteren August B. Smit Marijke de Jong-Brink Wijnand P. M. Geraerts 《Proceedings of the National Academy of Sciences of the United States of America》1997,94(25):14072-14076
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Inheritance, gene expression, and lignin characterization in a mutant pine deficient in cinnamyl alcohol dehydrogenase 下载免费PDF全文
John J. MacKay David M. OMalley Timothy Presnell Fitzgerald L. Booker Malcolm M. Campbell Ross W. Whetten Ronald R. Sederoff 《Proceedings of the National Academy of Sciences of the United States of America》1997,94(15):8255-8260
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S. Randal Voss H. Bradley Shaffer 《Proceedings of the National Academy of Sciences of the United States of America》1997,94(25):14185-14189
Although adaptive evolution is thought to depend primarily on mutations of small effect, major gene effects may underlie many of the important differences observed among species in nature. The Mexican axolotl (Ambystoma mexicanum) has a derived mode of development that is characterized by metamorphic failure (paedomorphosis), an adaptation for an entirely aquatic life cycle. By using an interspecific crossing design and genetic linkage analysis, a major quantitative trait locus for expression of metamorphosis was identified in a local map of amplified fragment length polymorphisms. These data are consistent with a major gene hypothesis for the evolution of paedomorphosis in A. mexicanum. 相似文献
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Coordinate regulation of lipogenic gene expression by androgens: Evidence for a cascade mechanism involving sterol regulatory element binding proteins 下载免费PDF全文
Johannes V. Swinnen William Ulrix Walter Heyns Guido Verhoeven 《Proceedings of the National Academy of Sciences of the United States of America》1997,94(24):12975-12980
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Abnormal skeletal patterning in embryos lacking a single Cbp allele: A partial similarity with Rubinstein–Taybi syndrome 下载免费PDF全文
Yasunori Tanaka Ichiro Naruse Toshio Maekawa Hiroshi Masuya Toshihiko Shiroishi Shunsuke Ishii 《Proceedings of the National Academy of Sciences of the United States of America》1997,94(19):10215-10220
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Inactivation of the survival motor neuron gene, a candidate gene for human spinal muscular atrophy, leads to massive cell death in early mouse embryos 下载免费PDF全文
Bertold Schrank Rudolf Gtz Jennifer M. Gunnersen Janice M. Ure Klaus V. Toyka Austin G. Smith Michael Sendtner 《Proceedings of the National Academy of Sciences of the United States of America》1997,94(18):9920-9925
Proximal spinal muscular atrophy is an autosomal recessive human disease of spinal motor neurons leading to muscular weakness with onset predominantly in infancy and childhood. With an estimated heterozygote frequency of 1/40 it is the most common monogenic disorder lethal to infants; milder forms represent the second most common pediatric neuromuscular disorder. Two candidate genes—survival motor neuron (SMN) and neuronal apoptosis inhibitory protein have been identified on chromosome 5q13 by positional cloning. However, the functional impact of these genes and the mechanism leading to a degeneration of motor neurons remain to be defined. To analyze the role of the SMN gene product in vivo we generated SMN-deficient mice. In contrast to the human genome, which contains two copies, the mouse genome contains only one SMN gene. Mice with homozygous SMN disruption display massive cell death during early embryonic development, indicating that the SMN gene product is necessary for cellular survival and function. 相似文献