Kravis D, Upholt W B
Dev Biol. 1985 Mar;108(1):164-72. doi: 10.1016/0012-1606(85)90018-1.
A single-stranded DNA probe complementary to chicken type II procollagen mRNA has been used to quantitate levels of that mRNA present in chicken limb mesenchyme during cartilage differentiation. Excess labeled probe prepared from a cDNA 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 approximately 2000 copies per diploid genome in stage 24 limb mesenchyme which had differentiated to cartilage in culture. Similar levels were found in cartilage from stage 31 limb. Sternal cartilage from 17-day embryos contained approximately 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 associated 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.
一种与鸡II型前胶原mRNA互补的单链DNA探针已被用于定量软骨分化过程中鸡肢体间充质中该mRNA的水平。从克隆于M13mp9的cDNA模板制备的过量标记探针与不断增加量的总RNA杂交至完全，并通过S1核酸酶消化保护进行测定。使用含有编码链的M13mp9模板作为标准来确定II型前胶原RNA的绝对水平估计值。与探针互补的RNA在第24阶段肢体中从每个二倍体基因组20拷贝增加到在培养中已分化为软骨的第24阶段肢体间充质中每个二倍体基因组约2000拷贝。在第31阶段肢体的软骨中发现了类似的水平。17天胚胎的胸骨软骨每个二倍体基因组含有约10,000拷贝，这表明该基因在肢体生长软骨中的表达水平与胸骨软骨不同。在第20 - 24阶段的肢体中观察到与探针互补的RNA水平较低但可检测到。由于这些早期肢体中大部分II型前胶原RNA与多核糖体相关，II型前胶原基因似乎在表型分化之前以及免疫可检测水平的II型胶原积累之前就以低水平表达。
