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首页> 外文期刊>Protoplasma: An International Journal of Cell Biology >CELL CYCLE-DEPENDENT EXPRESSION OF THE CHO2 ANTIGEN, A MINUS-END DIRECTED KINESIN-LIKE MOTOR IN MAMMALIAN CELLS
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CELL CYCLE-DEPENDENT EXPRESSION OF THE CHO2 ANTIGEN, A MINUS-END DIRECTED KINESIN-LIKE MOTOR IN MAMMALIAN CELLS

机译：CHO2抗原的细胞周期依赖性表达，CHO2抗原是负端导向的类似哺乳动物细胞的驱动蛋白动子。

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A 69 kDa protein present in the interphase centrosome and mitotic spindle/pole was identified with the CHO2 monoclonal antibodies raised against mitotic spindles isolated from Chinese hamster ovary (CHO) cells. Isolation and characterization of antigen-specific cDNAs and recombinant proteins demonstrated that the protein is a minus-end-directed microtubule motor with the motor domain located at the C terminus. Affinity-purified polyclonal antibodies prepared to bacterially expressed fusion proteins revealed the presence of the antigen in interphase nuclei, and the degree of nude ar immunostaining intensity varied among cells at different cell cycle stages. In order to examine the change of antigen expression during the cell cycle, we prepared synchronized populations of CHO cells, double stained with CHO2 and PCNA (proliferating cell nuclear antigen) antibodies, and quantitated the amounts of nuclear fluorescence using the MetaMorpho image analysis software package. Cells right after cell division contained nuclei with the lowest level of CHO2 immunofluorescence. The immunofluorescence intensity progressively increases through G(1) to S, reaching a maximum level by the end of G(2). The antibody uniformly stained the entire nuclear region, and the total amount of fluorescence detected in G(2) cells was greater than three rimes that of G(1) cells. Cell cycle dependent accumulation of the CHO2 antigen was further confirmed by immunoblot analysis of the protein included in whole cell extracts and nuclei isolated from synchronized CHO cells. Northern blot analysis showed that, although the CHO2-transcript accumulated during later stages of the cell cycle, its abundance declined through G(1) to S, and was lowest in cells at the early S phase. The difference in the expression pattern of the antigen protein and its transcript may suggest the presence of multiple mechanisms controlling the level of CHO2 antigen during the course of the cell cycle. [References: 31]
机译：利用从中国仓鼠卵巢（CHO）细胞分离的针对有丝分裂纺锤体的CHO2单克隆抗体，鉴定出存在于相间中心体和有丝分裂纺锤体/极中的69 kDa蛋白。抗原特异性cDNA和重组蛋白的分离和鉴定表明，该蛋白是负端定向的微管马达，其马达结构域位于C末端。针对细菌表达的融合蛋白制备的亲和纯化多克隆抗体揭示了抗原存在于相间核中，并且裸ar免疫染色强度的程度在不同细胞周期阶段的细胞之间有所不同。为了检查抗原在细胞周期中的表达变化，我们准备了同步化的CHO细胞群体，用CHO2和PCNA（增殖细胞核抗原）抗体双重染色，并使用MetaMorpho图像分析软件包定量了核荧光的量。 。细胞分裂后的细胞中含有的核具有最低水平的CHO2免疫荧光。免疫荧光强度通过G（1）逐渐增加到S，到G（2）结束时达到最大水平。该抗体均匀染色整个核区域，并且在G（2）细胞中检测到的荧光总量大于G（1）细胞的三个边缘。通过对包含在全细胞提取物中的蛋白质和从同步化CHO细胞分离的细胞核进行免疫印迹分析，进一步证实了CHO2抗原的细胞周期依赖性积累。 Northern印迹分析表明，尽管CHO2转录物在细胞周期的后期积累，但其丰度从G（1）下降到S，在S期早期最低。抗原蛋白及其转录物表达模式的差异可能表明在细胞周期过程中存在多种控制CHO2抗原水平的机制。 [参考：31]

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