ISOLATION OF A SOMATIC-CELL MUTANT DEFECTIVE IN PHOSPHATIDYLSERINE BIOSYNTHESIS
ISOLATION OF A SOMATIC-CELL MUTANT DEFECTIVE IN PHOSPHATIDYLSERINE BIOSYNTHESIS
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DOI：
10.1073/pnas.82.7.1926
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发表时间：
1985-01-01
影响因子：
11.1
通讯作者：
AKAMATSU, Y
中科院分区：
文献类型：
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作者：
KUGE, O;NISHIJIMA, M;AKAMATSU, Y
用原位酶法测定固定在聚酯布上的细胞集落中磷脂与胆碱的碱交换反应，分离出中国仓鼠卵巢（CHO）细胞中磷脂与胆碱碱交换反应缺陷的突变克隆。在33 ℃下生长的一种突变体（指定为64）的提取物中胆碱交换反应的比活性。C和40度。C分别为亲本（CHO-K1）细胞的13%和6%。突变体中的胆碱交换活性在细胞提取物中比在亲本中更热不稳定，这表明胆碱交换酶的结构基因中的突变可能在该突变体中被诱导。在补充有脂蛋白缺乏血清的培养基中，突变体64在33 ℃几乎正常生长。C，但在40 °只分两次。C，然后停止生长。用[32 P]Pi标记完整细胞表明，突变体64在40 ° C的磷脂酰丝氨酸的生物合成中也有显著缺陷。C，但正常为33.0度。C.突变体64的大多数耐温回复突变体在40 ℃下表现出几乎正常的合成磷脂酰丝氨酸的能力。C，也表现出类似的亲本细胞的胆碱交换活性。向补充有新生小牛血清的培养基中加入磷脂酰丝氨酸，其中突变体64在40 ℃下比亲本细胞生长更慢。C，使突变体的生长速率恢复到亲本水平。胆碱交换酶可能作为形成磷脂酰丝氨酸的主要途径起作用，突变体64的温度敏感性生长是由于在40 ℃时磷脂酰丝氨酸生物合成的缺陷。C.
Mutant clones of Chinese hamster ovary (CHO) cells defective in the base-exchange reaction of phospholipids with choline were isolated by using an in situ enzymatic assay for the reaction in cell colonies immobilized on polyester cloth. The specific activities of the choline-exchange reaction in extracts of one of the mutants (designated 64) grown at 33.degree. C and 40.degree. C were 13% and 6% of those in parental (CHO-K1) cells, respectively. The choline-exchange activity in the mutant was more thermolabile in cell extracts than that in the parent, suggesting that a mutation in the structural gene for the choline-exchange enzyme might be induced in this mutant. In culture medium supplemented with lipoprotein-deficient serum, mutant 64 grew almost normally at 33.degree. C but divided only twice at 40.degree. C and then stopped growing. Labeling of intact cells with [32P]Pi showed that mutant 64 was also strikingly defective in the biosynthesis of phosphatidylserine at 40.degree. C but was normal at 33.degree. C. Most temperature-resistant revertants of mutant 64 exhibited nearly normal ability to synthesize phosphatidylserine at 40.degree. C and also showed choline-exchange activity similar to that in parental cells. The addition of phosphatidylserine to medium supplemented with newborn calf serum, in which mutant 64 grew more slowly than parental cells at 40.degree. C, restored the growth rate of the mutant to the parental level. The choline-exchange enzyme may function as the major route for the formation of phosphatidylserine and that the temperature-sensitive growth of mutant 64 is due to a defect in phosphatidylserine biosynthesis at 40.degree. C.