Isolation of alpha-fetoprotein messenger RNA from mouse yolk sac.
Isolation of alpha-fetoprotein messenger RNA from mouse yolk sac.
复制标题
从小鼠卵黄囊中分离甲胎蛋白信使 RNA。
DOI：
10.1016/s0021-9258(18)50625-6
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发表时间：
1979
期刊：
影响因子：
--
通讯作者：
T. Tamaoki
中科院分区：
文献类型：
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作者：
K. Miura;S. Law;S. Nishi;T. Tamaoki
纯化的AFPmRNA在酸/尿素/琼脂糖凝胶上于21 S处迁移为单一条带。分子量估计为9 × 10 - 6，对应于约2700个核苷酸，比编码AFP多肽所需的长约1000个核苷酸。在麦胚提取物中，AFP mRNA指导蛋白质的合成，其中98%与特异性抗AFP反应。在十二烷基硫酸钠存在和不存在的情况下，通过聚丙烯酰胺凝胶电泳进一步确定翻译产物为AFP。AFPcDNA-AFPmRNA杂交体的高T_（92 C）值表明了转录的保真度。用此cDNA，在AFP mRNA纯化的各个阶段的AFP mRNA序列进行了测定。甲胎蛋白mRNA的物理纯化与麦胚提取物中甲胎蛋白mRNA生物活性的富集完全一致。杂交测定也证实了先前报道的非多聚腺苷酸化的AFP mRNA的存在（Tamaoki，T.，Miura，K.，林，T.，和Banks，P.（1976）在Onco-DeueZ-opmental Gene Expression（Fishman，WH和Sell，S.，编），pp. 115-122，Academic Press，纽约）。用该cDNA探针检测的成年小鼠肝脏AFP mRNA序列的含量远低于胎鼠肝脏。这一点，再加上以前的结果，无细胞分析AFP mRNA活性在胎儿和成人肝脏，表明转录或转录后加工是主要的网站控制AFP合成的发展。小鼠卵黄囊AFP cDNA与大鼠卵黄囊多核糖体RNA广泛反应，形成稳定的杂交体。小鼠甲胎蛋白cDNA也与
The purified AFP mRNA migrated as a single band at 21 S on an acid/urea/agarose gel. The molecular weight was estimated to be 9 x lo”, corresponding to about 2700 nucleotides, about 1000 nucleotides longer than required for coding for the AFP polypeptide. In wheat germ extracts, the AFP mRNA directed the synthesis of proteins, 98% of which reacted with specific anti-AFP. Identity of the translation product as AFP was further established by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate.DNA complementary to the AFP mRNA (AFP cDNA) was synthesized using avian myeloblastosis virus reverse transcriptase. The fidelity of transcription was indicated by high T,,,(92 C) of AFP cDNA-AFP mRNA hybrids. Using this cDNA, AFP mRNA sequences at various stages of AFP mRNA purification were assayed. Physical purification of AFP mRNA was in good agreement with enrichment of biological activity of AFP mRNA assayed in wheat germ extracts. Hybridization assays also confirmed the existence of nonpolyadenylated AFP mRNA reported earlier (Tamaoki, T., Miura, K., Lin, T., and Banks, P.(1976) in Onco-DeueZ-opmental Gene Expression(Fishman, WH, and Sell, S., eds) pp. 115-122, Academic Press, New York). The amount of AFP mRNA sequences in the adult mouse liver assayed by the cDNA probe was much lower than that of the fetal mouse liver. This, coupled with the previous results of cell-free assays of AFP mRNA activity in fetal and adult liver, indicates that transcription or post-transcriptional processing is the major site of control of AFP synthesis in development. The mouse yolk sac AFP cDNA reacted with polysomal RNA from rat yolk sac extensively forming stable hybrids. The mouse AFP cDNA also cross-reacted