Proc. Nati. Acad. Sci. USA Vol. 81, pp. 508-511, January 1984 Genetics Localization of the structural gene for human apolipoprotein A-I on the long arm of human chromosome 11 (atherosclerosis/high density lipoprotein/apolipoprotein C-Ill/cell hybrid/Southern blot analysis) PETER CHEUNG*, FA-TEN KAOt, MARTHA LIAO LAWt, CAROL JONESt, THEODORE T. PUCKt, AND LAWRENCE CHAN* *Departments of Cell Biology and Medicine, Baylor College of Medicine, Houston, TX 77030; and tEleanor Roosevelt Institute for Cancer Research, The Lita Hazen Laboratory and the School of Medicine, University of Colorado Health Sciences Center, Denver, CO 80262 Contributed by Theodore T. Puck, September 19, 1983 ABSTRACT Apolipoprotein A-I (apo A-I), the major apo- tide sequence of a nearly complete apo A-I cDNA clone (12). lipoprotein in human high density lipoproteins, is involved in The genomic DNA for apo A-I has been isolated and its se- the disease atherosclerosis. Cloned apo A-I cDNA (pAl-3) was quence has been determined (24). There is evidence that the used as a probe in chromosome mapping studies to detect the apo A-I gene is located close to another apolipoprotein gene, human apo A-I structural gene sequence in human-Chinese apo C-III (25), the latter being located approximately 2.6 ki- hamster cell hybrids. Southern blot analysis of 13 hybrids lo- lobases (kb) below the 3' end of apo A-I on the same cloned calized the gene to human chromosome 11. Confirmation of genomic fragment. The chromosomal identifications and the chromosomal assignment was obtained by analysis of a hy- mapping positions of both genes have not been described brid (J1) containing a single human chromosome, no. 11. Re- previously. gional mapping was achieved by using deletion subclones of J1 We have used cloned apo A-I cDNA as a hybridization that localized the human apo A-I structural gene to the region probe to map the human apo A-I gene by Southern blot anal- llql3-+qter. Since the human apolipoprotein C-Ill (apo C- ysis of human-Chinese hamster somatic cell hybrids. By this III) structural gene is closely linked to apo A-I, it can be as- technique, the human apo A-I structural gene has been local- signed to the same region on the long arm of chromosome 11. ized on chromosome 11. Regional mapping was carried out, By extension of methods previously described, it now appears indicating that apo A-I is on the long arm of chromosome 11, possible to carry out fine-structure analysis of this and related in the region encompassed by llql3-*qter. Thus, the apo C- gene regions on chromosome 11 and to study the biochemical III structural gene can also be assigned to this region on concomitants of these genes and of genes on other chromo- chromosome 11. The further steps now possible that permit somes for analysis of their role in atherosclerosis. genetic-biochemical analysis of the role of this and other genes important in atherosclerosis are indicated. High density lipoproteins (HDL) are a heterogeneous group of lipoproteins important in atherosclerosis and distin- MATERIALS AND METHODS guished from other lipoproteins by their flotation properties. Cell Culture. The Chinese hamster cell line CHO-K1 and Plasma HDL are inversely correlated with the atherogenic the human cell line HT-1080 (26, 27) were cultured in F12 process (1-4), although the mechanism by which they confer medium/8% fetal calf serum. The various human-CHO-Ki protection against atherosclerosis is unclear. In vitro experi- cell hybrids used were prepared from fusions between sever- ments involving isolated erythrocytes and cultured cells sug- al different auxotrophic mutants of CHO-K1 cells and vari- gest that HDL may be involved in delivery of cholesterol ous human cells as described (26-28). The following cell hy- from the peripheral tissues to the liver for disposal (5-8). brids were analyzed for the presence or absence of the hu- Metabolic studies in man have suggested an inverse correla- man apo A-I gene by the Southern blotting procedure tion between the size of the total body cholesterol pool and (numbers in parentheses indicate human chromosomes car- the plasma HDL level (9). It is also possible that HDL may ried in each hybrid): CP3 (2, 3, 4, 5, 7, 8, 11, 12, 14, 16, 18, simply be a marker for a certain pattern oflipid transport and 19, 20, 21, X), CP5 (1, 3, 4, 5, 6, 8, 9, 10, 12, 14, 15, 17, 18, metabolism that confers protection against the development 21, 22, X), CP6 (3, 4, 6, 7, 9, 10, 12, 14, 17, 18, 19, 20, 21, 22), of atherosclerosis (10). CP12 (2, 4, 8, 9, 11, 12, 13, 14, 16, 18, 22, X), CP14 (2, 4, 5, 9, Apolipoprotein A-I (apo A-I), the major apoprotein in 12, 14, 17, 18, 19, 20, 21, 22, X), CP15 (2, 3, 4, 5, 9, 11, 12, HDL, is a polypeptide of 243 amino acids (11, 12) and has a 14, 17, 19), CP16 (3, 5, 7, 14, 16, 17, 19, 20, 21, X), CP17 (1, Mr of -28,000. It is a co-factor for lecithin-cholesterol acyl- 4,5,9,12,14,15, 18, 19,21,22),CP18(1,3,8,11,13,14,15, transferase, a plasma enzyme that catalyzes the conversion 16, 17, 18, 19, 20, 21, 22), CP20 (2, 3, 4, 5, 8, 9, 14, 17, 19, of cholesterol and phosphatidylcholine to cholesteryl esters 21), CP26 (1, 4, 5, 6, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19, 21), and lysophosphatidylcholine (13). This protein was found to CP27 (2, 3, 4, 5, 6, 7, 12, 13, 14, 16, 18, 21, X), and CP28 (1, be a necessary component in mixtures with phospholipid 3, 4, 5, 6, 9, 16, 18, 19, 20, 21, 22). The preparation of each that can remove cholesterol from ascites cell membranes hybrid and its human chromosome content have been de- (14, 15). scribed (27, 28). In addition to the isozyme analysis conduct- Apo A-I is synthesized mainly in the liver and small intes- ed previously for identification of the human chromosomal tine in mammals (16-20), with the synthesis in the latter pre- content of each hybrid, we have also analyzed the hybrids by sumably regulated by dietary lipid ingestion (20, 21). Recent- cytogenetic techniques using trypsin banding (29) and the ly, Cheung and Chan (12), as well as Shoulders and Barelle Giemsa 11 differential staining (30) in sequential steps (31). (22) and Breslow et al. (23), have cloned the cDNA for hu- The cell hybrid clone J1, which contains a complete CHO- man apo A-I. Cheung and Chan also determined the nucleo- K1 genome together with a single human chromosome 11 (32), and three of its deletion mutant subclones, J1-7, J1-11, The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: apo, apolipoprotein; HDL, high density lipopro- in accordance with 18 U.S.C. §1734 solely to indicate this fact. teins; CHO, Chinese hamster ovary; kb, kilobase(s). 508 Downloaded by guest on September 27, 2021 Genetics: Cheung et aL Proc. NatL Acad. Sci USA 81 (1984) 509 and J1-23 (33, 34), were cultured in F12 medium. In clone J1- C H 1 2 3 4 5 6 7 8 9 1011 12 13 7, part of the short arm including 11pter--p1l has been de- leted, in Ji-11 most of the long arm including llql3--qter has been deleted, and J1-23 has a deletion in the short arm including llpter-*p13, as described (33, 34). -23.2 Preparation of DNA from Cultured Cells and Cell Hybrids. Cells were grown in 150-mm dishes to confluency and har- vested by trypsinization. The cells were treated with protein- ase K after washing and DNA was isolated using described procedures (26, 35). .41'~~~~~~~~~. Digestion and Gel Electrophoresis of DNA. Aliquots (10 Mg) of DNA were digested with restriction enzymes in 30-gl (fi- nal) volumes under conditions recommended by the suppli- ers (HincIl, Amersham; BamHI, HindIII, Kpn I, EcoRI, Sst FIG. 1. Hybridization of pAl-3 to HincII-digested DNA from I, New England BioLabs). Digestions were carried out for 6 human-Chinese hamster somatic cell hybrids and their parental hr in the presence of excess enzyme (3-5 units/Mg of DNA). cells. Lanes: C, Chinese hamster cell CHO-KI; H, human cell HT- The DNA were at 1080 (a similar pattern was obtained from DNA from human leuko- digested samples electrophoresed 60 V for cytes); 1-13, to HincIl-digested DNA from somatic cell hybrids 16 hr on 1% agarose gels in a horizontal gel apparatus (Be- CP3, CP5, CP6, CP12, CP14, CP15, CP16, CP17, CP18, CP20, thesda Research Laboratories). The gel was stained with CP26, CP27, and CP28, respectively. Molecular weight markers ethidium bromide and the DNA was visualized with ultravio- consisted of HindIll-digested X phage DNA. *, Position of human- let light. The DNA was transferred to nitrocellulose paper specific band hybridizing to pAl-3; **, position of CHO-specific (Schleicher & Schuell) by the method of Southern (36). band cross-hybridizing to pAl-3. Lanes 1, 4, 6, 9, and 11 represent Hybridization of DNA. Cloned apo A-I cDNA (pAl-3) was positive hybrids showing the presence of the human band as defined isolated from the recombinant plasmid by Pst I digestion. in lane H. The cDNA consists of the complete coding and 3'-noncoding analysis in the 13 hybrids revealed high concordance be- sequence together with part of the 5'-noncoding region of tween the apo A-I gene and human chromosome 11.