GENETIC COMPLEMENTATION IN HETEROKARYONS OF HUMAN FIBROBLASTS DEFECTIVE IN COBALAMIN METABOLISM
GENETIC COMPLEMENTATION IN HETEROKARYONS OF HUMAN FIBROBLASTS DEFECTIVE IN COBALAMIN METABOLISM
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DOI：
10.1073/pnas.72.8.3181
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发表时间：
1975-01-01
影响因子：
11.1
通讯作者：
ROSENBERG, LE
中科院分区：
文献类型：
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作者：
GRAVEL, RA;MAHONEY, MJ;ROSENBERG, LE
由于甲基丙二酰辅酶 A 变位酶（甲基丙二酰辅酶 A 辅酶 A 羰基变位酶；EC 5.4.99.2）活性缺乏而导致的遗传性甲基丙二酸血症是由影响钴胺素（Cbl：维生素 B12）代谢的至少三类生化上不同的缺陷（cbl A、cbl B 和 cbl C 突变体）和第四类产生有缺陷的变位酶脱辅酶引起的。我们使用仙台病毒介导的细胞融合制备的异核体，获得了支持这种生化异质性的遗传证据。来自 Cbl 代谢缺陷患者的 9 个成纤维细胞系（4 个 cbl A、3 个 cbl B 和 2 个 cbl C）、两个来自变位酶脱辅基酶缺陷患者的两个和两个来自对照的成对组合融合，并使用放射自显影程序检测功能性变位酶全酶，该程序检测原位成纤维细胞单层中三氯乙酸可沉淀材料中[14C]丙酸盐的掺入。与对照细胞相比，每个突变体的放射性可以忽略不计。当不同突变体在没有病毒的情况下混合或通过自融合产生同核体时，活性也可以忽略不计。通过将四个突变类别中的每一个的成员与任何其他类别的代表融合而产生的异核体恢复了掺入[14C]丙酸盐的能力，其水平与对照细胞相当。然而，同一类成员之间产生的异核体在所有情况下都无法互补。我们得出的结论是，Cbl 代谢缺陷的突变体（cbl A、cbl B、cbl C）包含三个互补组，第四组对应于变位酶脱辅基酶缺陷，并且所有四类突变都是隐性遗传的。
Inherited methylmalonicacidemia due to deficiency of methylmalonyl-CoA mutase (methylmalonyl-CoA CoA-carbonylmutase; EC 5.4.99.2) activity results from at least three classes of biochemically distinct defects affecting cobalamin (Cbl: vitamin B12) metabolism (cbl A, cbl B, and cbl C mutants) and a fourth class producing a defective mutase apoenzyme. We have obtained genetic evidence in support of this biochemical heterogeneity, using heterokaryons prepared by Sendai-virus-mediated cell fusion. Nine fibroblast lines from patients with defective Cbl metabolism (4 cbl A, 3 cbl B, and 2 cbl C), two from patients with defective mutase apoenzyme, and two from controls were fused in pairwise combinations and tested for functional mutase holoenzyme using a radioautographic procedure which detects [14C]propionate incorporation into trichloroacetic-acid-precipitable material in fibroblast monolayers in situ. Each of the mutants incorporates negligible radioactivity compared to control cells. Activity is also negligible when different mutants are mixed without virus or when homokaryons are produced by self-fusion. Heterokaryons produced by fusing members of each of the four mutant classes with representatives of any other class recover the ability to incorporate [14C]propionate to levels comparable to those of control cells. However, heterokaryons produced between members of the same class fail to complement in all cases. We conclude that the mutants with defective Cbl metabolism (cbl A, cbl B, cbl C) comprise three complementation groups, that a fourth group corresponds to mutase apoenzyme deficiency, and that all four classes of mutations are recessively inherited.