Grandchamp B, Romeo P H, Dubart A, Raich N, Rosa J, Nordmann Y, Goossens M
Proc Natl Acad Sci U S A. 1984 Aug;81(16):5036-40. doi: 10.1073/pnas.81.16.5036.
A cDNA clone containing sequences complementary to the mRNA coding for anemic rat spleen porphobilinogen deaminase (EC 4.3.1.8) has been isolated. A cDNA library was prepared from partially purified mRNA (1% purity). This library was then screened by colony hybridization, using a cDNA probe derived from porphobilinogen deaminase mRNA further enriched (10-20% purity) by gel electrophoresis in the presence of methylmercury hydroxide. Colonies hybridizing with the probe were analyzed by hybrid-selected translation using anemic rat spleen mRNA. Four recombinant plasmids containing porphobilinogen deaminase cDNA sequences were identified by specific immunoprecipitation of the translational product from hybrid-selected mRNA. Porphobilinogen deaminase mRNA was shown to contain 1800 bases by blot hybridization analysis. The cloned cDNA sequence consists of 1500 bases. Hybridization analysis of poly(A)+ RNA from uninduced and induced mouse erythroleukemic cells indicated that induction to erythroid differentiation by dimethyl sulfoxide results in a 10-fold increase of porphobilinogen deaminase mRNA. The rat cDNA clones hybridize to the corresponding sequences encoding human porphobilinogen deaminase. This property will be useful for isolation of human gene(s) and further characterization of the molecular lesion(s) responsible for acute intermittent porphyria.
已分离出一个cDNA克隆，其包含与编码贫血大鼠脾脏胆色素原脱氨酶（EC 4.3.1.8）的mRNA互补的序列。从部分纯化的mRNA（纯度为1%）制备了一个cDNA文库。然后使用在氢氧化甲基汞存在下通过凝胶电泳进一步富集（纯度为10 - 20%）的来自胆色素原脱氨酶mRNA的cDNA探针，通过菌落杂交对该文库进行筛选。与探针杂交的菌落通过使用贫血大鼠脾脏mRNA的杂交选择翻译进行分析。通过对杂交选择的mRNA的翻译产物进行特异性免疫沉淀，鉴定出四个含有胆色素原脱氨酶cDNA序列的重组质粒。通过印迹杂交分析表明胆色素原脱氨酶mRNA含有1800个碱基。克隆的cDNA序列由1500个碱基组成。对未诱导和诱导的小鼠红白血病细胞的聚腺苷酸加尾RNA（poly(A)+ RNA）进行杂交分析表明，二甲基亚砜诱导向红系分化会导致胆色素原脱氨酶mRNA增加10倍。大鼠cDNA克隆与编码人胆色素原脱氨酶的相应序列杂交。这一特性将有助于分离人类基因以及进一步表征导致急性间歇性卟啉症的分子病变。
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