Bresnick E, Brosseau M, Levin W, Reik L, Ryan D E, Thomas P E
Proc Natl Acad Sci U S A. 1981 Jul;78(7):4083-7. doi: 10.1073/pnas.78.7.4083.
Poly(A)+-RNA obtained from the livers of 3-methylcholanthrene (3MC)-treated rats was translated into cytochrome P-450c in a cell-free reticulocyte system. In this translational system, no precursor cytochrome P-450c was observed. The mRNA responsible for the synthesis of this cytochrome was isolated by immunoprecipitation of liver polyribosomes obtained at 15 hr after 3MC treatment, and a cDNA was constructed by the reverse transcriptase reaction. The cDNA was further purified by hybridizing at a high R0t (product of RNA concentration and incubation time) to poly(A)+-RNA isolated from control rat liver, and the nonhybridized, single-stranded cDNA was isolated by hydroxylapatite chromatography. This cDNAp-450c was employed in hybridization reactions with poly(A)+-RNA isolated from the livers of rats treated with 3MC for various times. These studies indicated a maximal induction of mRNAp-450c at about 15 hr after 3MC injection, although levels of this mRNA were significantly increased by 7 hr. The mRNAp-450c concentration had diminished by 24 hr but remained higher than control levels for at least 48 hr. These studies establish an effect of 3MC upon the accumulation of mRNAp-450c in rat liver.
从经3-甲基胆蒽（3MC）处理的大鼠肝脏中获得的聚腺苷酸（Poly(A)+）RNA，在无细胞网织红细胞系统中被翻译为细胞色素P-450c。在这个翻译系统中，未观察到细胞色素P-450c的前体。通过对3MC处理后15小时获得的肝脏多核糖体进行免疫沉淀，分离出负责合成这种细胞色素的mRNA，并通过逆转录酶反应构建了cDNA。通过在高R0t（RNA浓度与孵育时间的乘积）下与从对照大鼠肝脏中分离的聚腺苷酸（Poly(A)+）RNA杂交，进一步纯化该cDNA，然后通过羟基磷灰石柱色谱法分离出未杂交的单链cDNA。该cDNAp-450c用于与从经不同时间3MC处理的大鼠肝脏中分离的聚腺苷酸（Poly(A)+）RNA进行杂交反应。这些研究表明，在3MC注射后约15小时，mRNAp-450c诱导达到最大值，尽管该mRNA水平在7小时时就已显著增加。mRNAp-450c浓度在24小时时有所下降，但至少在48小时内仍高于对照水平。这些研究证实了3MC对大鼠肝脏中mRNAp-450c积累的影响。
