Isolation and characterization of the human apolipoprotein A-II gene: Electron microscopic analysis of RNA:DNA hybrids, nucleotide sequence, identification of a polymorphic MspI site, and general structural organization of apolipoprotein genes

Y. K. Tsao, C. F. Wei, D. L. Robberson, Antonio Gotto, L. Chan

Research output: Contribution to journalArticle

30 Scopus citations

Abstract

Three cloned apolipoprotein A-II genes were isolated from a human genomic cosmid library constructed in our laboratory. An ~3-kilobase HindIII insert containing the entire gene was analyzed by RNA:DNA hybridization and electron microscopy. The apo-A-II gene was found to consist of 4 exons and 3 intervening sequences (IVS), and the lengths of each exon and IVS were estimated by direct observation of the hybrids. The entire ~3-kilobase HindIII insert was sequenced. The 5' end of the gene was determined by primer extension. The DNA sequence confirms the presence of 4 exons and 3 IVS: exon 1, 34 nucleotides; exon 2, 76 nucleotides; exon 3, 133 nucleotides; exon 4, 230 nucleotides; IVS-I, 169 nucleotides; IVS-II, 299 nucleotides; and IVS-III, 396 nucleotides. A 'TATA box' is located at position -29 from the CAP site. A 'CAT box' is presented at position -78. A 'TG' element consisting of (TG)19 is identified at the 3' end of IVS-III. Furthermore, an enhancer core sequence, CTTTCCA, is identified at position -355 in the 5' flanking sequence. At positions -497 to -471 upstream from the CAP site is a stretch of 27 nucleotides that show high homology to stretches of 5' flanking sequences in the apo-C-II, apo-A-I, apo-E, and apo-C-III genes. An Alu dimer sequence is located approximately 300 nucleotides from the 3' end of the gene. Within this Alu sequence, we have identified a polymorphic MspI site. Restriction fragment length polymorphism involving this site has been previously shown to correlate with apo-A-II levels and high density lipoprotein structure. Analysis of conformation by Chou-Fasman analysis and by the helical hydophobic moment of Eisenberg et al. indicates that in all of the 5 apolipoproteins characterized at the nucleotide level to date, i.e. apo-C-II, apo-A-II, apo-E, apo-A-I, and apo-C-III, the 2 IVS within the peptide coding regions of the gene tend to occur at regions corresponding to the surface of the polypeptide chain and divide the protein into distinct functional domains.

Original languageEnglish (US)
Pages (from-to)15222-15231
Number of pages10
JournalJournal of Biological Chemistry
Volume260
Issue number28
StatePublished - 1985

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Fingerprint Dive into the research topics of 'Isolation and characterization of the human apolipoprotein A-II gene: Electron microscopic analysis of RNA:DNA hybrids, nucleotide sequence, identification of a polymorphic MspI site, and general structural organization of apolipoprotein genes'. Together they form a unique fingerprint.

Cite this