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91.
Adeno-associated virus type 2-mediated transfer of ecotropic retrovirus receptor cDNA allows ecotropic retroviral transduction of established and primary human cells. 总被引:1,自引:0,他引:1 下载免费PDF全文
K Qing T Bachelot P Mukherjee X S Wang L Peng M C Yoder P Leboulch A Srivastava 《Journal of virology》1997,71(7):5663-5667
The cellular receptors that mediate binding and internalization of retroviruses have recently been identified. The concentration and accessibility of these receptors are critical determinants in accomplishing successful gene transfer with retrovirus-based vectors. Murine retroviruses containing ecotropic glycoproteins do not infect human cells since human cells do not express the receptor that binds the ecotropic glycoproteins. To enable human cells to become permissive for ecotropic retrovirus-mediated gene transfer, we have developed a recombinant adeno-associated virus type 2 (AAV) vector containing ecotropic retroviral receptor (ecoR) cDNA under the control of the Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter (vRSVp-ecoR). Established human cell lines, such as HeLa and KB, known to be nonpermissive for murine ecotropic retroviruses, became permissive for infection by a retroviral vector containing a bacterial gene for resistance to neomycin (RV-Neo(r)), with a transduction efficiency of up to 47%, following transduction with vRSVp-ecoR, as determined by the development of colonies that were resistant to the drug G418, a neomycin analog. No G418-resistant colonies were present in cultures infected with either vRSVp-ecoR or RV-Neo(r) alone. Southern and Northern blot analyses revealed stable integration and long-term expression, respectively, of the transduced murine ecoR gene in clonal isolates of HeLa and KB cells. Similarly, ecotropic retrovirus-mediated Neo(r) transduction of primary human CD34+ hematopoietic progenitor cells from normal bone marrow was also documented, but only following infection with vRSVp-ecoR. The retroviral transduction efficiency was approximately 7% without prestimulation and approximately 14% with prestimulation of CD34+ cells with cytokines, as determined by hematopoietic clonogenic assays. No G418-resistant progenitor cell colonies were present in cultures infected with either vRSVp-ecoR or RV-Neo(r) alone. These results suggest that sequential transduction of primary human cells with two different viral vectors may overcome limitations encountered with a single vector. Thus, the combined use of AAV- and retrovirus-based vectors may have important clinical implications for ex vivo and in vivo human gene therapy. 相似文献
92.
Evolution and probable transmission of intersubtype recombinant human immunodeficiency virus type 1 in a Zambian couple. 总被引:5,自引:3,他引:2 下载免费PDF全文
93.
Hidekazu Nishimura Kanetsu Sugawara Peng Gao Yasushi Muraki Seiji Hongo Fumio Kitame Kiyoto Nakamura 《Microbiology and immunology》1995,39(9):737-740
The HMV-II cells infected with influenza C virus were labeled with inorganic [32P]phosphate to identify phosphorylated proteins. Analysis by radioimmunoprecipitation with antiviral serum or monoclonal antibodies revealed that three major structural proteins of the virus, hemagglutinin-esterase (HE), nucleoprotein (NP), and matrix protein (M1) are all phosphorylated in both infected cells and virions. It was also observed that, in the presence of trypsin (10 μg/ml), the unphosphorylated form of the HE glycoprotein was cleaved efficiently whereas the phosphorylated form was not, raising the possibility that phosphorylation of HE may influence its susceptibility to degradation by proteolytic enzymes. 相似文献
94.
95.
Stabilization of double-stranded oligonucleotides using backbone-linked disulfide bridges. 总被引:1,自引:1,他引:0 下载免费PDF全文
A convenient, practical route to the synthesis of disulfide-bridged oligonucleotides has been developed. Aliphatic linkers with terminal thiol groups have been attached to the phosphodiester backbones of partially or fully complementary oligonucleotide sequences and oxidized to yield covalently closed oligonucleotides with disulfide bridges. This procedure has been used to prepare a duplex with disulfide bridges at both ends and stem-loop sequences with single disulfide bridges. Oxidation of a self-complementary duplex possessing terminal thiol groups produced both hairpin and duplex structures with disulfide bridges, the relative proportions of each being dependent upon the reaction conditions. These bridged hairpin and duplex structures were shown to be interconvertible by reduction and re-oxidation. The melting profiles of disulfide-bridged oligonucleotides were compared with the same sequences without bridges and with sequences possessing triethylene glycol bridges, and in all cases the introduction of disulfide bridges resulted in a considerable increase in thermal stability. EcoRI endonuclease was capable of cleaving a disulfide-bridged duplex possessing a recognition site for this enzyme, thus supporting a lack of distortion of the recognition site. The disulfide bridges could be cleaved using a large excess of DTT to regenerate the corresponding sulfhydryl compounds. A study of the serum stabilities of disulfide-bridged oligonucleotides showed that the bridged duplexes were much more stable than their unmodified counterparts, whereas the rate of degradation of the stem-loop structures was more dependent upon the size of the loop than the presence or absence of the disulfide bridge. In summary, we have described a novel methodology, employing commercially available reagents, for the stabilization of oligonucleotide duplexes or stem-loop structures by disulfide bridge formation. 相似文献
96.
97.
Rhizobium etli strain TAL182 and R. leguminosarum bv phaseoli strain 8002, both of which produce melanin pigment, were tested for their nodulation competitiveness on beans by paired inoculation with two strains which do not produce melanin: R. tropici strain CIAT899 and Rhizobium sp. strain TAL1145. An assay was developed to distinguish nodules formed by the melanin-producing and non-producing strains. Strain TAL182 had discrete competitive superiority over CIAT899 and TAL1145 for nodulation of beans. Nodulation competitiveness was not correlated with the ability to produce melanin pigment or the host range of the Rhizobium strains tested.The authors are with the Department of Plant Molecular Physiology, University of Hawaii, 3050 Maile Way, Gillmore 402, Honolulu, HI 96822, USA 相似文献
98.
本文报道了采自贵州沿河土地坳下奥陶统的牙形石,计有25属39种,根据牙形石组合特片分析,认为其与黔北其它地区下奥陶统牙形石组合面貌基本一致,属北大西洋牙形石动物地理区。 相似文献
99.
中国生态环境区划初探 总被引:10,自引:1,他引:9
中国生态环境区划初探高密来(中国人民大学区域经济研究所北京100872)AnApproachtotheRegionalizationofEcologicalEnvironmentinChina.¥GaoMilai(InstituteofRegiona... 相似文献
100.