出版社：American Society for Biochemistry and Molecular Biology
摘要：During the differentiation of human enterocytes, the secretion of apolipoprotein B is switched from the apoB-100 form seen almost exclusively in fetal cells to the apoB-48 form seen almost exclusively in adult cells. This switch is accomplished by the post-transcriptional editing of the messenger RNA for apoB-100. We report that a similar switch occurs during the differentiation of the human colonic carcinoma cell line, Caco-2, and that this is accomplished by the same mRNA editing mechanism. Caco-2 cells cultured on Millipore filters developed confluent electrically resistant monolayers, and on Western blot analysis, using a monoclonal antibody directed against the amino terminal region of human apoB-100 (Mab C1.4), secreted greater than 50% apoB-48 (of total apoB-100) into culture media, while Caco-2 cells grown on plastic secreted greater than 95% apoB-100. To assess whether mRNA editing was responsible for the switchover from apoB-100 to apoB-48, apoB cDNA fragments spanning nucleotides 6504-6784 of apoB mRNA were synthesized using RNA isolated from Caco-2 cells grown on filters and Caco-2 cells grown on plastic. The appropriate oligonucleotide primers and Moloney murine leukemia virus reverse transcriptase were used. The resulting cDNA fragments were amplified by the polymerase chain reaction (PCR), and PCR products were subcloned and sequenced. A single cytosine/thymine base change occurred in 8/20 clones of cDNA derived from Caco-2 cells grown on filters (corresponding to a cytosine/uridine change in mRNA) and in 1/25 clones of cDNA derived from Caco-2 cells grown on plastic. PCR products of genomic sequences from Caco-2 cells did not contain the stop codon.( TRUNCATED AT 250 WORDS)
