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The NF-kappa B binding sites in the human immunodeficiency virus type 1 long terminal repeat are not required for virus infectivity. 总被引:20,自引:30,他引:20 下载免费PDF全文
J Leonard C Parrott A J Buckler-White W Turner E K Ross M A Martin A B Rabson 《Journal of virology》1989,63(11):4919-4924
Mutations were introduced into the regulatory sequences in the long terminal repeat of an infectious molecular clone of the human immunodeficiency virus. Viruses in which the NF-kappa B binding sites were deleted or ones in which one or two Sp1 binding sites were mutated still replicated efficiently in human T lymphocytes. A deletion of the two NF-kappa B sites plus the three Sp1 sites or a mutation of the tat-responsive region rendered the virus replication incompetent. Thus, the NF-kappa B sequences are not required for human immunodeficiency virus infectivity; however, a tat-responsive region is essential. 相似文献
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Gallerano Daniela Devanaboyina Siva Charan Swoboda Ines Linhart Birgit Mittermann Irene Keller Walter Valenta Rudolf 《Amino acids》2011,40(3):981-989
HIV-1 virus infectivity factor (Vif) is one of the four accessory proteins that are characteristic of primate lentiviruses
and critically required for the infection of host cells. Vif plays a key role in replication and transmission of the virus
in non-permissive cells, such as primary T cells and macrophages. Using co-precipitation and co-fractionation techniques,
evidence has been provided that Vif interacts with a variety of host proteins, such as the cytidine deaminases APOBEC3G and
3F, the Cullin5/EloBC ubiquitin–ligase complex, Fyn and Hck tyrosine kinases, as well as with viral components, such as the
immature Gag precursor and viral RNA. We report on the expression, purification and molecular characterization of a folded
recombinant subtype C Vif. Vif was expressed in E. coli with a C-terminal hexahistidine tag and purified by nickel affinity chromatography. We obtained approximately 5 mg protein
per liter of bacterial culture, with a purity >95%. The expected molecular mass of 23.7 kDa was confirmed by mass spectrometry.
Although dynamic light scattering and small angle X-ray scattering measurements revealed the presence of high molecular weight
aggregates in the protein preparation, circular dichroism analysis showed that the protein contains mainly folded β-sheet
elements and exhibits remarkable thermal stability (T
m > 95°C). Recombinant Vif may be used as a tool to study its biological functions and tertiary structure, as well as for the
development of diagnostic, therapeutic and preventive strategies for HIV-1 infections. 相似文献
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Transcription signals and protein binding sites for sericin gene transcription in vitro 总被引:8,自引:0,他引:8
K Matsuno C C Hui S Takiya T Suzuki K Ueno Y Suzuki 《The Journal of biological chemistry》1989,264(31):18707-18713
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Introduction
Ultra-deep pyrosequencing (UDPS) has been used to detect minority variants within HIV-1 populations. Some aspects of the quality and reproducibility of UDPS have been previously evaluated, but comprehensive studies are still needed.Principal Finding
In this study the UDPS technology (FLX platform) was evaluated by analyzing a 120 base pair fragment of the HIV-1 pol gene from plasma samples from two patients and artificial mixtures of molecular clones. UDPS was performed using an optimized experimental protocol and an in-house data cleaning strategy. Nine samples and mixtures were analyzed and the average number of reads per sample was 19,404 (range 8,858–26,846). The two patient plasma samples were analyzed twice and quantification of viral variants was found to be highly repeatable for variants representing >0.27% of the virus population, whereas some variants representing 0.11–0.27% were detected in only one of the two UDPS runs. Bland-Altman analysis showed that a repeated measurement would have a 95% likelihood to lie approximately within ±0.5 log10 of the initial estimate. A similar level of agreement was observed for variant frequency estimates in forward vs. reverse sequencing direction, but here the agreement was higher for common variants than for rare variants. UDPS following PCR amplification with alternative primers indicated that some variants may be incorrectly quantified due to primer-related selective amplification. Finally, the in vitro recombination rate during PCR was evaluated using artificial mixtures of clones and was found to be low. The most abundant in vitro recombinant represented 0.25% of all UDPS reads.Conclusion
This study demonstrates that this UDPS protocol results in low experimental noise and high repeatability, which is relevant for future research and clinical use of the UDPS technology. The low rate of in vitro recombination suggests that this UDPS system can be used to study genetic variants and mutational linkage. 相似文献15.
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The CFTR gene exhibits a complex pattern of expression that shows temporal and spatial regulation though the control mechanisms have not been fully elucidated. We have mapped DNase I hypersensitive sites (DHS) flanking the CFTR gene to identify potential regulatory elements. We previously characterized DHS at -79.5 and -20.9 kb with respect to the CFTR translational start site, DHS 3' to the gene at 4574 + 5.4-7.4 and 4574 + 15.6 kb, and a regulatory element in the first intron of the gene at 185 + 10 kb. We generated a cosmid contig to provide probes to evaluate the whole of the CFTR gene for DHS and have now mapped novel sites in introns 2, 3, 10, 16, 17a, 18, 20, and 21. These DHS show different patterns of cell-specific expression. 相似文献