噬菌体ε15吸附装置中结构蛋白的遗传分析
Genetic analysis of structural proteins in the adsorption apparatus of bacteriophage epsilon 15.
作者信息
Guichard Jared A, Middleton Paula C, McConnell Michael R
机构信息
Jared A Guichard, Paula C Middleton, Michael R McConnell, Department of Biology, Point Loma Nazarene University, San Diego, CA 92106, United States.
出版信息
World J Virol. 2013 Nov 12;2(4):152-9. doi: 10.5501/wjv.v2.i4.152.
AIM
To probe the organizational structure of the adsorption apparatus of bacteriophage epsilon 15 (E15) using genetic and biochemical methodology
METHODS
Hydroxylamine was used to create nonsense mutants of bacteriophage E15. The mutants were then screened for defects in their adsorption apparatus proteins, initially by measuring the concentrations of free tail spike proteins in lysates of cells that had been infected by the phage mutants under non-permissive growth conditions. Phage strains whose infected cell lysates contained above-average levels of free tail spike protein under non-permissive growth conditions were assumed to contain nonsense mutations in genes coding for adsorption apparatus proteins. These mutants were characterized by classical genetic mapping methods as well as automated sequencing of several of their genes. Finally, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography were used to examine the protein compositions of the radioactive particles produced when the various mutants were grown on a non-permissive host cell in the presence of (35)S-methionine and co-purified along with E15wt phage on CsCl block gradients.
RESULTS
Our results are consistent with gp4 forming the portal ring structure of E15. In addition, they show that proteins gp15 and gp17 likely comprise the central tube portion of the E15 adsorption apparatus, with gp17 being more distally positioned than gp15 and dependent upon both gp15 and gp16 for its attachment. Finally, our data indicates that tail spike proteins comprised of gp20 can assemble onto nascent virions that contain gp7, gp10, gp4 and packaged DNA, but which lack both gp15 and gp17, thereby forming particles that are of sufficient stability to survive CsCl buoyant density centrifugation.
CONCLUSION
The portal ring (gp4) of E15 is bound to tail spikes (gp20) and the tail tube (gp15 and gp17); gp17's attachment requires both gp15 and gp16.
目的
运用遗传学和生物化学方法探究噬菌体ε15（E15）吸附装置的组织结构
方法
使用羟胺构建噬菌体E15的无义突变体。随后，最初通过测量在非允许生长条件下被噬菌体突变体感染的细胞裂解物中游离尾刺蛋白的浓度，筛选这些突变体在其吸附装置蛋白方面的缺陷。在非允许生长条件下，其感染细胞裂解物中游离尾刺蛋白水平高于平均水平的噬菌体菌株被假定在编码吸附装置蛋白的基因中含有无义突变。这些突变体通过经典遗传图谱绘制方法以及对其几个基因的自动测序进行表征。最后，使用十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳和放射自显影来检查当各种突变体在存在（35）S - 甲硫氨酸的非允许宿主细胞上生长并与E15wt噬菌体一起在CsCl阻滞梯度上共纯化时产生的放射性颗粒的蛋白质组成。
结果
我们的结果与gp4形成E15的门户环结构一致。此外，它们表明蛋白gp15和gp17可能构成E15吸附装置的中心管部分，其中gp17比gp15更位于远端，并且其附着依赖于gp15和gp16两者。最后，我们的数据表明由gp20组成的尾刺蛋白可以组装到含有gp7、gp10、gp4和包装DNA，但缺乏gp15和gp17的新生病毒粒子上，从而形成具有足够稳定性以在CsCl浮力密度离心中存活的颗粒。
结论
E15的门户环（gp4）与尾刺（gp20）和尾管（gp15和gp17）结合；gp17的附着需要gp15和gp16两者。
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