QUANTITATION OF TYPE-II PROCOLLAGEN MESSENGER-RNA LEVELS DURING CHICK LIMB CARTILAGE DIFFERENTIATION
QUANTITATION OF TYPE-II PROCOLLAGEN MESSENGER-RNA LEVELS DURING CHICK LIMB CARTILAGE DIFFERENTIATION
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DOI：
10.1016/0012-1606(85)90018-1
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发表时间：
1985-01-01
影响因子：
2.7
通讯作者：
UPHOLT, WB
中科院分区：
文献类型：
--
作者：
KRAVIS, D;UPHOLT, WB
一个单链DNA探针互补鸡II型前胶原mRNA被用来定量水平的mRNA存在于鸡肢间充质软骨分化过程中。将从克隆在M13 mp 9中的c[互补]DNA模板制备的过量标记探针与增加量的总RNA杂交至完全，并通过保护免于S1核酸酶消化进行测定。使用含有编码链的M13 mp 9模板作为标准品，测定II型前胶原RNA绝对水平的估计值。与探针互补的RNA从第24阶段肢的每个二倍体基因组20个拷贝增加到约2000拷贝/二倍体基因组，在培养物中已分化为软骨的第24期肢体间充质中。来自第31阶段肢体的软骨中的水平相似。来自17天胚胎的胸骨软骨含有. apprx。10，000个拷贝/二倍体基因组，表明该基因的表达水平在肢体生长软骨中与胸骨软骨相比是不同的。在第20-24阶段的肢体中观察到与探针互补的低但可检测水平的RNA。由于在这些早期肢体中II型前胶原RNA的大部分与多聚核糖体相关，因此II型前胶原基因在表型分化之前和II型胶原的免疫学可检测水平的积累之前似乎以低水平表达。
A single-stranded DNA probe complementary to chicken type II procollagen mRNA was used to quantitate levels of that mRNA present in chicken limb mesenchyme during cartilage differentiation. Excess labeled probe prepared from a c[complementary]DNA template cloned in M13mp9 was hybridized to completion to increasing amounts of total RNA and assayed by protection from S1 nuclease digestion. Estimates of the absolute levels of type II procollagen RNA were determined using the M13mp9 template containing the coding strand as a standard. RNA complementary to the probe increased from 20 copies per diploid genome in stage 24 limb to .apprx. 2000 copies/diploid genome in stage 24 limb mesenchyme which had differentiated to cartilage in culture. Similar levels were in cartilage from stage 31 limb. Sternal cartilage from 17-day embryos contained .apprx. 10,000 copies per diploid genome suggesting that the level of expression of this gene is different in limb growth cartilage compared with sternal cartilage. Low but detectable levels of RNA complementary to the probe were observed in limb at stages 20-24. Since a large fraction of the type II procollagen RNA in these early limbs is assocated with polysomes, the type II procollagen gene appears to be expressed at a low level prior to phenotypic differentiation and prior to the accumulation of immunologically detectable levels of type II collagen.