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Transfer functions of the Streptococcus faecalis plasmid pAD1: organization of plasmid DNA encoding response to sex pheromone. 总被引:29,自引:19,他引:10 下载免费PDF全文
The conjugative plasmid pAD1 (59.6 kilobases) of Streptococcus faecalis shows a 10,000-fold increase in transfer frequency following induction by the sex pheromone cAD1. Mutagenesis of the plasmid with transposon Tn917 was undertaken to determine the region(s) of pAD1 required for the mating response. The relevant genetic material was found to be distributed over a 31.2-kilobase contiguous region of the plasmid. Although insertions in two previously identified regions (traA and traB) exhibited increased transfer frequencies, insertions in five new regions (D, E, F, G, and H) decreased the ability of pAD1 to transfer. Insertions in region H allowed the cells to form visible mating aggregates, but the plasmid transfer frequency was decreased to levels below detection during a 1-h broth mating. Mutants with mutations in region G were able to form aggregates; however, insertions in regions D, E, and F prevented aggregate formation. Insertions in region C decreased the sensitivity of the cell to exogenous cAD1 and exhibited increased activity of the pheromone inhibitor iAD1. Surface protein profiles produced by a number of these mutants were examined, and in some cases were found to be different from those of the wild type. A map showing the various regions is presented, and related aspects of the regulation of the pAD1 mating response are discussed. 相似文献
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Leader protein of foot-and-mouth disease virus is required for cleavage of the p220 component of the cap-binding protein complex. 总被引:40,自引:24,他引:16 下载免费PDF全文
M A Devaney V N Vakharia R E Lloyd E Ehrenfeld M J Grubman 《Journal of virology》1988,62(11):4407-4409
Suppression of host protein synthesis in cells infected by poliovirus and certain other picornaviruses involves inactivation of the cap-binding protein complex. Inactivation of this complex has been correlated with the proteolytic cleavage of p220, a component of the cap-binding protein complex. Since picornaviral RNA is not capped, it continues to be translated as the cap-binding protein complex is inactivated. The cleavage of p220 can be induced to occur in vitro, catalyzed by extracts from infected cells or by reticulocyte lysates translating viral RNA. Expression of polioviral protease 2A is sufficient to induce p220 cleavage, and the presence in 2A of an 18-amino-acid sequence representing a putative cysteine protease active site correlates with the ability of different picornaviruses to induce p220 cleavage. Foot-and-mouth disease virus (FMDV) infection induces complete cleavage of p220, yet the FMDV genome codes for a 2A protein of only 16 amino acids, which does not include the putative cysteine protease active site. Using cDNA plasmids encoding various regions of the FMDV genome, we have determined that the leader protein is required to initiate p220 cleavage. This is the first report of a function for the leader protein, other than that of autocatalytic cleavage from the FMDV polyprotein. 相似文献
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A series of three-nucleotide insertions was engineered into the P2 and P3 coding regions of the T7 expression plasmid pT7(tau)-PV1, which encodes a full-length copy of poliovirus type 1 (Mahoney) cDNA. When RNA derived in vitro from these mutated templates was used to transfect HeLa cells, viable virus mutants were recovered. One mutant, Sel-3D-18, which contained a single amino acid insertion in the 3Dpol coding region, was temperature sensitive for growth at 39 degrees C and showed defects in both RNA synthesis and P1 protein processing at the nonpermissive temperature. The RNA replication defect in Se1-3D-18 was identified at the level of RNA chain elongation. A highly specific and sensitive method was developed for analyzing the ability of mutant RNA templates to replicate in the presence or absence of helper functions provided in trans. This approach was used to demonstrate that RNA synthesis in Se1-3D-18 can be rescued by helper functions provided in trans. 相似文献
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Accelerated transbilayer movement of phosphatidylcholine in sickled erythrocytes. A reversible process 总被引:9,自引:0,他引:9
P F Franck D T Chiu J A Op den Kamp B Lubin L L van Deenen B Roelofsen 《The Journal of biological chemistry》1983,258(13):8436-8442
The transbilayer mobility of phosphatidylcholine (PC) molecules in the membrane of homozygous reversible sickle cells (RSCs) was studied using a PC-specific exchange protein from beef liver. In deoxygenated RSCs, all of the PC present in the membrane of the intact cell is rapidly available for exchange, mediated by this protein. Since a substantial amount of the PC is present in the inner membrane leaflet of these cells, this observation implies that the PC molecules in their membranes do experience rapid transbilayer movements. To determine the actual rate of transbilayer movement of the PC, radioactive PC was introduced into the outer monolayer of oxygenated RSCs using the PC-specific exchange protein. Subsequently, the cells were incubated at 37 degrees C under oxy- and deoxygenating conditions to enable the PC to equilibrate within the bilayer. At various time intervals, samples were taken and treated with phospholipase A2, which selectively degrades the PC in the outer monolayer. Analysis of the specific radioactivities of the lyso-PC thus produced, as well as of the residual PC, enabled us to follow the fate of the radioactive PC previously introduced into the outer membrane layer. The half-time value for transbilayer equilibration of the PC in deoxygenated RSCs was determined to be 3.5 h, which is about four times lower than that for oxygenated RSCs. This increased transbilayer mobility of PC, observed in deoxygenated RSCs, is immediately restored to the normal low rate upon reoxygenation of the cells, indicating a complete reversibility of this phenomenon. 相似文献
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The existence of a soluble complex formed by polyuridylic acid (poly (U)) and 3′,5′-cyclic AMP (cAMP) is demonstrated by u.v. extinction vs. temperature curves, optical rotation, equilibrium dialysis, and reaction calorimetry. The complex hasthe stoichiometry of 2 poly (U)-cAMP and its formation is accompanied by an enthalpy change of ?13.0 kcal/mole of base triplet. The introuction of an empirical factor α in the equations given by Damle2 and Crothers2 leads to the evolution of a ΔH value of ?13.4 keal/mole. The parameter α is considered as a correction factor for the concentration dependence of the binding process. There is no relation between α and the reduction of monomer activity due to self-association of monomers. The study of the binding process at several temperatures showed that the cooperativity parameter, σ, is independent of temperature and its value of 6.5 × 10?3 is in good agreement with σ = 5 × 10?3 for the poly (U)·poly(A) system.3 相似文献