作者: James L. Manley
DOI: 10.1016/0092-8674(83)90440-3
关键词: DNA 、 Lysis 、 Biochemistry 、 RNA 、 Polyadenylation 、 Messenger RNA 、 In vivo 、 In vitro 、 Biology 、 Polymerase 、 General Biochemistry, Genetics and Molecular Biology
摘要: Abstract Conditions have been developed which allow for the efficient, accurate, and specific polyadenylation of exogenously added mRNA precursors in a wholecell lysate derived from HeLa cells. Precursors are prepared by vitro transcription using linear DNA templates whole-cell lysate, purified, to another lysate. Under optimal conditions (which quite precise with respect several variables), 70% or more precursor molecules become polyadenylated, length poly(A) segment is controlled much as it vivo, giving rise 150–300-nucleotide long stretches poly(A). suboptimal conditions, both fraction RNA that becomes polyadenylated also reduced. The reaction remarkably specific: Only vitro-synthesized pre-mRNAs contain 3′ end located at slightly downstream corresponding vivo site can be efficiently vitro. These results suggest polymerase requires one protein (or RNA) factors order bring about accurate vitro, complex must interact recognition signal catalyze subsequent polyadenylation.