Molecular cloning of a cDNA complementary to a UDP-glucose pyrophosphorylase mRNA of dictyostelium discoideum.
Molecular cloning of a cDNA complementary to a UDP-glucose pyrophosphorylase mRNA of dictyostelium discoideum.
复制标题
与盘基网柄菌的 UDP-葡萄糖焦磷酸化酶 mRNA 互补的 cDNA 的分子克隆。
DOI：
10.1016/0012-1606(85)90096-x
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发表时间：
1985
影响因子：
2.7
通讯作者：
Dottin,RP
中科院分区：
文献类型：
--
作者：
Fishel,BR;Ragheb,JA;Rajkovic,A;Haribabu,B;Schweinfest,CW;Dottin,RP
尿苷二磷酸葡萄糖焦磷酸化酶（UTP：-α-d-葡萄糖-1-磷酸尿苷转移酶，EC 2.7.7.9）是盘基网柄藻正常发育所必需的酶，其比活性在发育后期增加3 ~ 10倍。先前的实验已经表明，伴随着这种增加，酶的其他形式出现，并且在发育的任何阶段，两种尿苷二磷酸葡萄糖焦磷酸化酶（UDPGP）多肽从总细胞RNA的体外翻译产物中免疫沉淀（B）。F.菲舍尔河E. Manrow和R. P. Dottin，1982，Dev. 92，175 -187）。使用体外免疫沉淀法测定UDPGP mRNA，我们发现，增加翻译mRNA的量与积累的酶在发展过程中。cDNA文库是从mRNA群体中构建的，该mRNA群体通过在含有甲基汞的蔗糖梯度上进行大小分级富集UDPGP mRNA（C. W.施魏菲斯特河W. Kwiatkowski和R. P. Dottin，1982，Proc. Natl. Acad. Sci. USA79，4997-5000）。通过杂交选择和翻译筛选文库后，鉴定出与UDPGP mRNA互补的1.8Kb cDNA。只有编码高分子量体外翻译产物的mRNA被该cDNA杂交选择。在杂交抑制翻译实验中，该cDNA的编码链选择性地抑制两种体外翻译产物中的仅一种的翻译。因此，存在两种不同的UDPGP mRNA。
Uridine diphosphoglucose pyrophosphorylase (UTP: -α-d-glucose-1-phosphate uridyltransferase, EC 2.7.7.9) is an essential enzyme for normal development ofDictyostelium discoideumand its specific activity increases 3- to 10-fold by the later stages of development. Previous experiments have shown that additional forms of the enzyme appear concomitantly with this increase and that two uridine diphosphoglucose pyrophosphorylase (UDPGP) polypeptides are immunoprecipitated from thein vitrotranslation products of total cellular RNA at any stage of development (B. F. Fishel, R. E. Manrow and R. P. Dottin, 1982,Dev. Biol.92,175–187). Using anin vitrotranslation-immunoprecipitation assay of UDPGP mRNA, we show that an increase in the amount of translatable mRNA is correlated with the accumulation of enzyme during development. A cDNA bank was constructed from a mRNA population that had been enriched for UDPGP mRNA by size fractionation on sucrose gradients containing methylmercuric hydroxide (C. W. Schweinfest, R. W. Kwiatkowski, and R. P. Dottin, 1982,Proc. Natl. Acad. Sci. USA79,4997–5000). A 1.8-Kb cDNA complementary to a UDPGP mRNA was identified after screening the bank by hybridization selection and translation. Only the mRNA encoding the higher molecular weightin vitrotranslation product is hybrid selected by this cDNA. In hybrid-arrested translation experiments, the coding strand of this cDNA selectively inhibits the translation of only one of the twoin vitrotranslation products. Therefore, there are two distinct UDPGP mRNAs.