Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1983;2(12):2369–2373. doi: 10.1002/j.1460-2075.1983.tb01748.x

The isolation of cDNA clones for human apolipoprotein E and the detection of apoE RNA in hepatic and extra-hepatic tissues.

S C Wallis, S Rogne, L Gill, A Markham, M Edge, D Woods, R Williamson, S Humphries
PMCID: PMC555459  PMID: 6199196

Abstract

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.

Full text

PDF

Images in this article

2370Fig. 2.
on p.2370
2372Fig. 5.
on p.2372

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Agarwal K. L., Brunstedt J., Noyes B. E. A general method for detection and characterization of an mRNA using an oligonucleotide probe. J Biol Chem. 1981 Jan 25;256(2):1023–1028. [PubMed] [Google Scholar]
  2. Auffray C., Rougeon F. Purification of mouse immunoglobulin heavy-chain messenger RNAs from total myeloma tumor RNA. Eur J Biochem. 1980 Jun;107(2):303–314. doi: 10.1111/j.1432-1033.1980.tb06030.x. [DOI] [PubMed] [Google Scholar]
  3. Aviv H., Leder P. Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose. Proc Natl Acad Sci U S A. 1972 Jun;69(6):1408–1412. doi: 10.1073/pnas.69.6.1408. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Basu S. K., Brown M. S., Ho Y. K., Havel R. J., Goldstein J. L. Mouse macrophages synthesize and secrete a protein resembling apolipoprotein E. Proc Natl Acad Sci U S A. 1981 Dec;78(12):7545–7549. doi: 10.1073/pnas.78.12.7545. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Basu S. K., Ho Y. K., Brown M. S., Bilheimer D. W., Anderson R. G., Goldstein J. L. Biochemical and genetic studies of the apoprotein E secreted by mouse macrophages and human monocytes. J Biol Chem. 1982 Aug 25;257(16):9788–9795. [PubMed] [Google Scholar]
  6. Blue M. L., Ostapchuk P., Gordon J. S., Williams D. L. Synthesis of apolipoprotein AI by peripheral tissues of the rooster. A possible mechanism of cellular cholesterol efflux. J Biol Chem. 1982 Sep 25;257(18):11151–11159. [PubMed] [Google Scholar]
  7. Blue M. L., Williams D. L., Zucker S., Khan S. A., Blum C. B. Apolipoprotein E synthesis in human kidney, adrenal gland, and liver. Proc Natl Acad Sci U S A. 1983 Jan;80(1):283–287. doi: 10.1073/pnas.80.1.283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Breslow J. L., McPherson J., Nussbaum A. L., Williams H. W., Lofquist-Kahl F., Karathanasis S. K., Zannis V. I. Identification and DNA sequence of a human apolipoprotein E cDNA clone. J Biol Chem. 1982 Dec 25;257(24):14639–14641. [PubMed] [Google Scholar]
  9. Breslow J. L., Zannis V. I., SanGiacomo T. R., Third J. L., Tracy T., Glueck C. J. Studies of familial type III hyperlipoproteinemia using as a genetic marker the apoE phenotype E2/2. J Lipid Res. 1982 Nov;23(8):1224–1235. [PubMed] [Google Scholar]
  10. Brown M. S., Kovanen P. T., Goldstein J. L. Regulation of plasma cholesterol by lipoprotein receptors. Science. 1981 May 8;212(4495):628–635. doi: 10.1126/science.6261329. [DOI] [PubMed] [Google Scholar]
  11. Böyum A. Isolation of mononuclear cells and granulocytes from human blood. Isolation of monuclear cells by one centrifugation, and of granulocytes by combining centrifugation and sedimentation at 1 g. Scand J Clin Lab Invest Suppl. 1968;97:77–89. [PubMed] [Google Scholar]
  12. Chang J. C., Kan Y. W. A sensitive new prenatal test for sickle-cell anemia. N Engl J Med. 1982 Jul 1;307(1):30–32. doi: 10.1056/NEJM198207013070105. [DOI] [PubMed] [Google Scholar]
  13. Cheung P., Chan L. Nucleotide sequence of cloned cDNA of human apolipoprotein A-I. Nucleic Acids Res. 1983 Jun 11;11(11):3703–3715. doi: 10.1093/nar/11.11.3703. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
  15. Edge M. D., Green A. R., Heathcliffe G. R., Meacock P. A., Schuch W., Scanlon D. B., Atkinson T. C., Newton C. R., Markham A. F. Total synthesis of a human leukocyte interferon gene. Nature. 1981 Aug 20;292(5825):756–762. doi: 10.1038/292756a0. [DOI] [PubMed] [Google Scholar]
  16. Gait M. J., Singh M., Sheppard R. C., Edge M. D., Greene A. R., Heathcliffe G. R., Atkinson T. C., Newton C. R., Markham A. F. Rapid synthesis of oligodeoxyribonucleotides. IV. Improved solid phase synthesis of oligodeoxyribonucleotides through phosphotriester intermediates. Nucleic Acids Res. 1980 Mar 11;8(5):1081–1096. doi: 10.1093/nar/8.5.1081. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Ghiselli G., Gregg R. E., Zech L. A., Schaefer E. J., Brewer H. B., Jr Phenotype study of apolipoprotein E isoforms in hyperlipoproteinaemic patients. Lancet. 1982 Aug 21;2(8295):405–407. doi: 10.1016/s0140-6736(82)90439-1. [DOI] [PubMed] [Google Scholar]
  18. Ish-Horowicz D., Burke J. F. Rapid and efficient cosmid cloning. Nucleic Acids Res. 1981 Jul 10;9(13):2989–2998. doi: 10.1093/nar/9.13.2989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Mahley R. W., Hui D. Y., Innerarity T. L., Weisgraber K. H. Two independent lipoprotein receptors on hepatic membranes of dog, swine, and man. Apo-B,E and apo-E receptors. J Clin Invest. 1981 Nov;68(5):1197–1206. doi: 10.1172/JCI110365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Markham A. F., Edge M. D., Atkinson T. C., Greene A. R., Heathcliffe G. R., Newton C. R., Scanlon D. Solid phase phosphotriester synthesis of large oligodeoxyribonucleotides on a polyamide support. Nucleic Acids Res. 1980 Nov 25;8(22):5193–5205. doi: 10.1093/nar/8.22.5193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  22. Messing J., Vieira J. A new pair of M13 vectors for selecting either DNA strand of double-digest restriction fragments. Gene. 1982 Oct;19(3):269–276. doi: 10.1016/0378-1119(82)90016-6. [DOI] [PubMed] [Google Scholar]
  23. Rall S. C., Jr, Weisgraber K. H., Innerarity T. L., Mahley R. W. Structural basis for receptor binding heterogeneity of apolipoprotein E from type III hyperlipoproteinemic subjects. Proc Natl Acad Sci U S A. 1982 Aug;79(15):4696–4700. doi: 10.1073/pnas.79.15.4696. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Rall S. C., Jr, Weisgraber K. H., Mahley R. W. Human apolipoprotein E. The complete amino acid sequence. J Biol Chem. 1982 Apr 25;257(8):4171–4178. [PubMed] [Google Scholar]
  25. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Schneider W. J., Kovanen P. T., Brown M. S., Goldstein J. L., Utermann G., Weber W., Havel R. J., Kotite L., Kane J. P., Innerarity T. L. Familial dysbetalipoproteinemia. Abnormal binding of mutant apoprotein E to low density lipoprotein receptors of human fibroblasts and membranes from liver and adrenal of rats, rabbits, and cows. J Clin Invest. 1981 Oct;68(4):1075–1085. doi: 10.1172/JCI110330. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Shoulders C. C., Baralle F. E. Isolation of the human HDL apoprotein A1 gene. Nucleic Acids Res. 1982 Aug 25;10(16):4873–4882. doi: 10.1093/nar/10.16.4873. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Talmadge K., Stahl S., Gilbert W. Eukaryotic signal sequence transports insulin antigen in Escherichia coli. Proc Natl Acad Sci U S A. 1980 Jun;77(6):3369–3373. doi: 10.1073/pnas.77.6.3369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Thomas P. S. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5201–5205. doi: 10.1073/pnas.77.9.5201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Utermann G., Hees M., Steinmetz A. Polymorphism of apolipoprotein E and occurrence of dysbetalipoproteinaemia in man. Nature. 1977 Oct 13;269(5629):604–607. doi: 10.1038/269604a0. [DOI] [PubMed] [Google Scholar]
  31. Wallace R. B., Johnson M. J., Hirose T., Miyake T., Kawashima E. H., Itakura K. The use of synthetic oligonucleotides as hybridization probes. II. Hybridization of oligonucleotides of mixed sequence to rabbit beta-globin DNA. Nucleic Acids Res. 1981 Feb 25;9(4):879–894. doi: 10.1093/nar/9.4.879. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Weisgraber K. H., Rall S. C., Jr, Mahley R. W. Human E apoprotein heterogeneity. Cysteine-arginine interchanges in the amino acid sequence of the apo-E isoforms. J Biol Chem. 1981 Sep 10;256(17):9077–9083. [PubMed] [Google Scholar]
  33. Woods D. E., Markham A. F., Ricker A. T., Goldberger G., Colten H. R. Isolation of cDNA clones for the human complement protein factor B, a class III major histocompatibility complex gene product. Proc Natl Acad Sci U S A. 1982 Sep;79(18):5661–5665. doi: 10.1073/pnas.79.18.5661. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Zannis V. I., Breslow J. L. Human very low density lipoprotein apolipoprotein E isoprotein polymorphism is explained by genetic variation and posttranslational modification. Biochemistry. 1981 Feb 17;20(4):1033–1041. doi: 10.1021/bi00507a059. [DOI] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES