The isolation of cDNA clones for human apolipoprotein E and the detection of apoE RNA in hepatic and extra-hepatic tissues.
We have isolated cDNA clones coding for apolipoprotein E (apoE) from a cDNA library prepared from adult human liver mRNA. Mixtures of 128 different oligonucleotides, 17 residues long were synthesised to be complementary to regions of the mRNA corresponding to amino acids 1-6 and 151-156. Five independent apoE clones were selected by direct screening of 5000 recombinants with the two oligonucleotide mixtures. Two overlapping clones contain the 3'-untranslated sequence, the entire coding sequence and an additional 30 bases 5' to the amino terminus of the mature protein. The DNA sequence has been determined spanning the known sites of amino acid substitutions which account for the observed protein polymorphism of apoE. Using the clones as probes in Northern blot analysis of total human liver and kidney RNAs and leucocyte poly(A)+ RNA we have detected a single species of mRNA in liver and kidney of 1.2 kb and two larger species in leucocyte RNA. The level of expression of the mRNA in kidney is approximately 10% of that in liver while the level of apoE RNA sequences in the leucocytes is less than 1% of that in the liver.
D000328:Adult; D000595:Amino Acid Sequence; D001053:Apolipoproteins / Q000235:genetics*; D001057:Apolipoproteins E; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000302:isolation & purification*; D004262:DNA Restriction Enzymes; D006801:Humans; D007668:Kidney / Q000378:metabolism; D008079:Lipoproteins, VLDL / Q000235:genetics*; D008099:Liver / Q000378:metabolism*; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D011061:Poly A / Q000235:genetics; D012313:RNA / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics*
D001053:Apolipoproteins; D001057:Apolipoproteins E; D008079:Lipoproteins, VLDL; D012333:RNA, Messenger; D011061:Poly A; D012313:RNA; D004247:DNA; D004262:DNA Restriction Enzymes