Abstract
To map transcriptional events associated with mineralization in developing long bones, we have established protocols for preparing RNA from regions of chick epiphyseal cartilage. Using these RNA preparations, we have probed for appearance of mRNA coding for type I, II, and X collagen, as well as osteonectin and calmodulin. Type II collagen mRNA was found in proliferating cartilage and, in lower amounts, in hypertrophic/calcifying cartilage. Type X mRNA was absent from proliferating cartilage and present in hypertrophic/calcifying cartilage at steady state levels slightly lower than that of type II mRNA. Type I mRNA was the major collagen mRNA species in endochondral bone; however, significant amounts of type X mRNA were also found. Examination of type X/type II ratios suggest that the cells producing type X mRNA in bone are different from those in the hypertrophic/calcifying cartilage region. Osteonectin mRNA was present in endochondral bone; however, significant amounts were also detected in precalcified cartilage. Indeed, the level of osteonectin mRNA was significantly higher in the resting/proliferating region than in the hypertrophic/calcifying region of the cartilage. No correlation was observed between calmodulin mRNA and the development of mineralization; levels of this message were slightly lower in endochondral bone, embryonic sterna, and calvaria than they were in chick liver and considerably lower than the calmodulin mRNA levels in chick brain.
Highlights
To map transcriptional events associated with min- Of the considerable number of proteinspresentin the eralization in developing long bones, we have estab- calcifying epiphysis, only a few have been directly linked with lished protocols for preparing RNAfrom regions of the mineralization process [4, 5]
A double extraction procedure for preparing RNA has permitted us to carry out a quantitative analysis of mRNA changes associated with cartilage maturation and bone formation in chick tibia
Optical density/ng of tissue RNA and optical density/pg of plasmid DNA were calculated for each filter, and the results for HTC-CC and endochondral bone were expressed per pgof DNA, permitting comparison of values from different filters
Summary
To map transcriptional events associated with min- Of the considerable number of proteinspresentin the eralization in developing long bones, we have estab- calcifying epiphysis, only a few have been directly linked with lished protocols for preparing RNAfrom regions of the mineralization process [4, 5]. We have addressed tion was observed between calmodulinmRNA and the this analyticalproblem and report here procedures for isolatdevelopment of mineralization;levels of this message ing mRNA from chick epiphyseal cartilage and endochondral were slightly lower in endochondral bone, embryonic bone.
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