Expression of a human cartilage procollagen gene (COL2A1) in mouse 3T3 cells.
Expression in a recombinant system has been difficult to obtain for any of the major fibrillar collagens that require processing by eight or more post-translational enzymes. Here, two DNA constructs were designed so that the promoter region of the gene for the pro-alpha 1(I) chain of human type I procollagen drove expression of the human type II procollagen gene in mouse NIH 3T3 cells, a culture line that normally synthesizes type I procollagen but not any cartilage-specific protein such as type II procollagen. Both constructs were expressed as both mRNA and protein. In clones expressing the construct at high levels, the steady-state levels of mRNA and the production of type II procollagen were comparable to the mRNA levels and production of type I procollagen from the endogenous mouse genes. Comparison of clones containing the two constructs demonstrated that sequences extending 80 base pairs beyond the major polyadenylation signal of the gene are not in themselves sufficient for correct termination and 3' processing of RNA transcripts. The results strongly suggest that specific sequences present in a downstream 3.5-kilobase SphI/SphI fragment determine the termination of the transcription. Of special importance is that the system will make it possible to examine the consequences of mutations in the human type II procollagen gene on the processing of RNA transcripts and on the functional properties of the protein simply by using the genomic DNA from leukocytes or other non-cartilaginous sources.
Ala-Kokko
L
L
Department of Biochemistry and Molecular Biology, Jefferson Institute of Molecular Medicine, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107.
Hyland
J
J
Smith
C
C
Kivirikko
K I
KI
Jimenez
S A
SA
Prockop
D J
DJ
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D003001:Cloning, Molecular; D003488:Cyanogen Bromide; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation; D006801:Humans; D051379:Mice; D010957:Plasmids; D011347:Procollagen / Q000096:biosynthesis / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D014158:Transcription, Genetic; D014162:Transfection
D011347:Procollagen; D012333:RNA, Messenger; D003488:Cyanogen Bromide