Noncoordinate regulation of the mRNAs encoding cytochromes P-450BNF/MC-B and P-450ISF/BNF-G.
Noncoordinate regulation of the mRNAs encoding cytochromes P-450BNF/MC-B and P-450ISF/BNF-G.
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DOI：
10.1016/0003-9861(86)90116-5
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发表时间：
1986
影响因子：
3.9
通讯作者：
J. Fagan;J. Pastewka;S. Chalberg;E. Gozukara;F. Guengerich;H. Gelboin
中科院分区：
文献类型：
--
作者：
J. Fagan;J. Pastewka;S. Chalberg;E. Gozukara;F. Guengerich;H. Gelboin
编码主要的多环芳烃诱导的大鼠细胞染色质sp -450的mrna，P-450BNF/ mc - band - 450isf /BNF-G，使用三类重组质粒进行了表征：(a)仅与(P-450BNF/MC-BmRNA互补，(b)仅与(p - 450isf /BNF-GmRNA互补，(c)两个mrna都互补。利用6种单克隆和多克隆抗体，通过杂交选择翻译和免疫沉淀鉴定了这些类别，随后对其进行测序以确认其身份和特异性。这些发现表明，编码这两个op -450的mrna具有独特的区域，以及同源的区域。杂交选择翻译还表明，p - 450bnf / mc - band - 450isf / bnf - gmrna的主要体外翻译产物分别为55和52 kDa，并且具有独特和共同的结构特征，可以从免疫学上区分。通过Northern杂交，发现p - 450bnf /MC-BmRNA长2900个碱基，而p - 450isf /BNF-GmRNA长2100个碱基。p - 450bnf /MC-BmRNA检测到3500和5200个碱基的前体，而p - 450isf /BNF-GmRNA检测到3100个碱基的前体。β-萘黄酮、异黄樟醇和3-甲基胆蒽可诱导这两种mrna，而苯巴比妥不能诱导。在未经治疗的大鼠中，p - 450bnf /MC-BmRNA一直以非常低的水平存在，而p - 450isf /BNF-GmRNA以不同的量存在，这表明后者的mRNA可以由饮食或其他环境因素诱导。采用斑点杂交技术检测p - 450bnf / mc - band - 450isf / bnf - gmrna的诱导动力学。P-450BNF/MC-BmRNA增加迅速，在3-甲基胆酸处理后4 h达到一半最大值，而p - 450isf /BNF-GmRNA增加较慢，在12 h达到一半最大值。两种mrna的水平在24 h达到峰值，但随后以不同的速度下降；在接下来的24小时内，P-450BNF/MC-BmRNA下降了约20%，而ep - 450isf /BNF-GmRNA下降了50 - 70%。这些诱导动力学的差异和p - 450bnf / mc - band - 450isf / bnf - gmrna的明显稳定性，以及在未治疗的大鼠中观察到的它们的水平差异，表明这两种mrna虽然是由相同的化合物诱导的，但它们并没有协调调节。以芳烃羟化酶活性测量的功能性p - 450bnf /MC-B的诱导滞后于p - 450bnf /MC-BmRNA的诱导10 h，在14 h达到一半最大值。在24 h达到最大值后，芳烃羟化酶活性保持在这个升高的水平至少48 h，尽管在这段时间内p - 450bnf /MC-BmRNA的水平开始下降，这表明芳烃羟化酶比编码它的mRNA更稳定。
The mRNAs encoding the major polycyclic aromatic hydrocarbon-induced cytochromesP-450 from rat,P-450BNF/MC-BandP-450ISF/BNF-G, were characterized using three classes of recombinant plasmids: those complementary to (a) onlyP-450BNF/MC-BmRNA, (b) onlyP-450ISF/BNF-GmRNA, and (c) both mRNAs. These classes were identified by hybridization-selected translation and immunoprecipitation using six monoclonal and polyclonal antibodies and were later sequenced to confirm their identity and specificity. These findings indicated that the mRNAs encoding these twoP-450s have regions that are unique, as well as regions that are homologous. Hybridization-selected translation also showed that the primaryin vitrotranslation products of theP-450BNF/MC-BandP-450ISF/BNF-GmRNAs are 55 and 52 kDa, respectively, and have both unique and common structural characteristics that can be distinguished immunologically. By Northern hybridization, theP-450BNF/MC-BmRNA was found to be 2900 bases long, while theP-450ISF/BNF-GmRNA was 2100 bases long. Precursors of 3500 and 5200 bases were detected forP-450BNF/MC-BmRNA, while a 3100-base precursor was detected forP-450ISF/BNF-GmRNA. These two mRNAs were induced by β-naphthoflavone, isosafrole, and 3-methylcholanthrene, but not by phenobarbital. In untreated rats, theP-450BNF/MC-BmRNA was consistently present at very low levels while theP-450ISF/BNF-GmRNA was present in variable amounts, suggesting that the latter mRNA can be induced by dietary or other environmental factors. The kinetics of induction of theP-450BNF/MC-BandP-450ISF/BNF-GmRNAs were measured by dot blot hybridization.P-450BNF/MC-BmRNA increased rapidly, reaching half-maximum by 4 h after treatment with 3-methylcholantrene, while theP-450ISF/BNF-GmRNA increased more slowly, reaching half-maximum after 12 h. The levels of both mRNAs peaked at 24 h, but decreased thereafter at different rates;P-450BNF/MC-BmRNA dropped by about 20% during the next 24 h, whileP-450ISF/BNF-GmRNA dropped by 50 to 70%. These differences in the kinetics of induction and the apparent stabilities of theP-450BNF/MC-BandP-450ISF/BNF-GmRNAs, in conjunction with the observed differences in their levels in untreated rats, suggested that these two mRNAs were not coordinately regulated even though they were induced by the same compounds. The induction of functionalP-450BNF/MC-B, measured as aryl hydrocarbon hydroxylase activity, lagged 10 h behind the induction ofP-450BNF/MC-BmRNA, reaching half-maximum at 14 h. After reaching a maximum at 24 h, aryl hydrocarbon hydroxylase activity remained at this elevated level for at least 48 h, even though theP-450BNF/MC-BmRNA level began to drop during this time period, which suggests that the aryl hydrocarbon hydroxylase enzyme is more stable than the mRNA that encodes it.