GENOMICS

7,65-74

(1990)

cDNA Cloning of Human Oxysteroi-Binding Protein and Localization of the Gene to Human Chromosome 11 and Mouse Chromosome 19 DITSA LEVANON,*

CHIH-LIN HSIEH,~ UTA FRmcKE,t’$ PAUL A. DAWSON,* MICHAEL 5. BROWN,* AND JOSEPH L. GOLDSTEIN*

NEALE D. RIDGWAY,*

*Departments of Molecular Genetics and Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75235; and tHoward Hughes Medical Institute and $Departments of Genetics and Pediatrics, Stanford University Medical Center, Stanford, California 94305-5428 Received

December

I, 1989;

Press.

Inc.

INTRODUCTION

Cellular cholesterol homeostasis in mammalian cells is maintained by feedback regulation of the low-density lipoprotein (LDL)’ receptor and enzymes in the choSequence data from EMBL/GenBank Data 1 Abbreviations used: sterol-binding protein; hydroxy-3-methylglutaryk

January

9, 1990

lesterol biosynthetic pathway (Goldstein and Brown, 1984). This sterol-mediated regulation is achieved at the level of gene transcription of the LDL receptor and HMG CoA synthase and at both the transcriptional and post-transcriptional levels for HMG CoA reductase (Metherall et al., 1989; Nakanishi et al., 1988). Physiologically, the major regulator of this system is cholesterol derived from plasma LDL. LDL enters cells by receptor-mediated endocytosis and is delivered to lysosomes, where its cholesteryl esters are hydrolyzed and the liberated cholesterol becomes available for regulatory actions (Brown and Goldstein, 1986). Indirect evidence suggests that the cholesterol derived from the lysosomal hydrolysis of LDL may not directly mediate the feedback regulatory events described above. Rather, the active agent may be a polar derivative of cholesterol formed in the cell (Kandutsch and Chen, 1978). Oxygenated sterols, such as 25hydroxycholesterol, 20-hydroxycholesterol, or 7-ketocholesterol, are potent transcriptional repressors of the genes for the LDL receptor, HMG CoA synthase, and HMG CoA reductase when dissolved in solvents and added externally to cultured cells (Osborne et al., 1988; Smith et al., 1988). In addition to their transcriptional regulatory actions, oxysterols can act post-transcriptionally to enhance the degradation of HMG CoA reductase (Gil et al, 1985) and to activate acyl-coenzyme A:cholesteryl acyltransferase, the enzyme that synthesizes cholesteryl esters within cells (Brown et aZ., 1975). The proteins that mediate the action of oxysterols remain to be identified. One possible candidate is the oxysterol-binding protein (OSBP), a low-abundance cytosolic protein whose specificity of sterol binding in vitro is correlated with the specificity of sterol suppression of HMG CoA reductase activity in cultured cells (Taylor et aZ., 1984). We have recently purified OSBP to homogeneity from hamster liver cytosol (Dawson et

Cellular cholesterol metabolism is regulated primarily through sterol-mediated feedback suppression of the activity of the low-density lipoprotein receptor and several enzymes of the cholesterol biosynthetic pathway. We previously described the cloning of a rabbit cDNA for the oxysterol-binding protein (OSBP), a cytosolic protein of 809 amino acids that may participate in these regulatory events. We now use the rabbit OSBP cDNA to clone the human OSBP cDNA and 5’ genomic region. Comparison of the human and rabbit OSBP sequences revealed a remarkably high degree of conservation. The cDNA sequence in the coding region showed 94% identity between the two species, and the predicted amino acid sequence showed 98% identity. The human cDNA was used to determine the chromosomal localization of the OSBP gene by Southern blot hybridization to panels of somatic cell hybrid clones containing subsets of human or mouse chromosomes and by RFLP analysis of recombinant inbred mouse strains. The OSBP locus mapped to the long arm of human chromosome 11 and the proximal end of mouse chromosome 19. Along with previously mapped genes including Ly-Z and CD20, OSBP defines a new conserved syntenic group on the long arm of chromosome 11 in the human and the proximal end of chromosome 19 in the mouse. o 1990 Academic

revised

this article have been deposited with the Libraries under Accession No. 504757. LDL, low-density lipoprotein; OSBP, oxyPCR, polymerase chain reaction; HMG, 3SDS, sodium dodecyl sulfate. 65

OBBB-7543/90$3.00

0 1990 by Academic Press., Inc. All rights of reproduction in my form reserved. Copyright

LEVANON

ET

AL. -1 120

240

360

480

600

720

840

960

1080 GStKDoCCSCKGDHSDEDDEWEPr 1200

1320

M~Ar(;Cnc~~~T~~Ml~cAtG~G~tAG~c~a~~~~~t~~c~nl~~ 441NEP LS?ILoR LTED .

1440 LC .

YHE

LLDRAAKCCW

5

.

.

GmfCMXXAAATAccXtCcAttATaCcclctXT

MirCA~A~~~~G~a~C;ACCArGCTGhCltCAMMfCM 521SWHPPAI’AHHACSKWGUtLROEIKITSKFRGKYLSIHPLG

561t

AMA~AnGTATmCCAtaMCIGGGCIKChCtllCMnGCM;;T I WC IFHATGHWYTYKKVTTtVHN

6OlVW

CIGMtCIKMGACI~GtMt~T~~~t~tt WKTGDKCN

1680

1800 I At~~t~C~Al~~~ Y 5 YF

LKFVP

?GGAAMGGAAtCCt&CGMGMtaAGMMC 661WKRWP LP KWACWC(Y

LEOLCtVAAFt

IVCKLWf

DQSGEID

I

-AGA~Atc~~

1920

SROVARKVTCCVTOP

AttGThCln;TtCTCffiMiCTt~?CtdhCtCT~MT~ YF SE LALt

SGKVHF

l

LWAWC

tGGCUtaCCCCK;K;l;hCiXtt~:CtGAC CTAPTDSRLRPD

2160

-‘II

cumcmcmmt~~7c~mmmta&ummamaacta 72lORL?lENCRWDEANAEKOR GaAcAcc~TAtGAtc&ATNlGa~ot 761GTP YDP

YK

-

-AAAGACtTtCCACMAGMCMC LCEKORLSRKKRkRCAHK

~AAGAA;kACCtGTtACcAAGGAGttMCccATAtttAt ALWFERKRDP VT

KE

LTH

I

2280

AGOfXbAT-CtMAOMMACPaACtUi2400 YROCYYECKEKODW G~cTTACCMG~

~~~~~~~~~ffiT~~~~AtATM?~~~~AG~t~

AtcAMGCcACAGAkGAT ATED

3’

2517

~01 Ii

FIG. 1. Nucleotides

Nucleotide sequence and deduced are numbered on the right. Amino

amino acids

acid sequence are numbered

of human OSBP and comparison with the sequence of rabbit OSBP. on the left. Amino acid residue 1 is the putative initiator methionine.

GENE

MAPPING

OF

OXYSTEROL-BINDING

al., 198913) and cloned the rabbit liver cDNA (Dawson et al., 1989a). In transfected cells this cDNA encoded a protein whose sterol-binding specificity matched that of authentic OSBP. Rabbit OSBP is an Bog-aminoacid protein that migrates as a doublet of 96 and 101 kDa on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. On gel chromatography the protein behaves as a multimer of 280 kDa. The deduced amino acid sequence was suggestive of the presence of a leutine zipper, a recently recognized structural motif that mediates protein dimerization (Landschulz et al., 1988). In the current study, we report the isolation and cloning of the human OSBP cDNA and the 5’ genomic sequences. We also describe the chromosomal localization of the OSBP gene to the long arm of human chromosome 11 and the proximal end of mouse chromosome 19. MATERIALS

AND

METHODS

General Methods Standard molecular biology techniques were used (Maniatis et al., 1982). Restriction fragments or B&lgenerated deletion fragments derived from the cDNA clones were inserted into Ml3 and Bluescript KS11 (Stratagene) vectors and sequenced by the dideoxy chain termination method (Sanger et al., 1977) with either the Ml3 universal sequencing primer or specific oligonucleotides. Sequencing reactions were performed using a modified bacteriophage T7 DNA polymerase (Tabor and Richardson, 1987) or Taq DNA polymerase (Innis et al., 1988) with 35S-labeled nucleotides for manual sequencing or fluorescently labeled universal primer on an Applied Biosystems Model 370A DNA sequencer. Sequences were analyzed using programs obtained from the University of Wisconsin Genetics Computing Group.

Isolation

of Human

OSBP cDNA Clones

The polymerase chain reaction (PCR) was used to analyze several human cDNA libraries to determine which ones contained the OSBP cDNA (see below). This screen revealed the presence of OSBP clones in an oligo(dT)-primed human kidney cDNA library in XgtlO that was selected for inserts > 2 kb (Bell et al., 1986) (kindly provided by Graeme I. Bell). Plaques (360,000) from the human kidney cDNA library were screened using 32P-labeled probes derived from the 3’ and the 5’ end of the rabbit OSBP coding region (Daw-

PROTEIN

67

5

-AUG *

--(AJn Probe

3

FIG. 2. Sl nuclease analysisof in the humanOSBP gene. A uniformly

the transcriptioninitiation site labeled ‘OP probe was prepared as described under Materials and Methods. The stippled box at the bottom represents the 5’ end of the OSBP gene; the major sites of transcription initiation are denoted by the vertical arrow. The solid box represents the OSBP coding sequence in exon 1; exon 1 ends at nucleotide position +362. The position of the oligonucleotide used for sequencing and preparation of the Sl probe is denoted by the asterisk, the extended “P-labeled probe is denoted by the solid line. The mRNA is indicated by the wavy line. ‘*P-labeled Sl probe (l2 X 10’ cpm; 4-8 pmol) was added to 35 pg of SV40-transformed human fibroblast RNA (lane 1) or 35 pg of wheat germ tRNA (lane 2), heated to 90°C, slowly cooled to 60°C, allowed to hybridize for 12-16 h, and then digested with 570 units of Sl nuclease (Sigma Chemical Co.) for 30 min at 37’C. The Sl nuclease-resistant products and the Sl probe alone (lane 3) were analyzed on a 7 M urea/8% polyacrylamide gel containing 20% formamide. To align the Sl nuclease-protected fragments with the genomic DNA sequence, parallel dideoxy DNA sequencing reactions were performed using Taquence (United States Biochemical Corp.) with the same oligonucleotide and Ml3 template used to generate the Sl probe. The sequence is shown as the RNA strand. The DNA sequencing reactions and Sl nuclease-protected products were resolved on the same gel. The gel was dried and exposed to X-ray film for 72 h at -70°C with an intensifying screen.

son et al., 1989a). Filters were hybridized at 37°C in 50% formamide hybridization buffer containing 1 X lo6 cpm/ml of single-stranded Ml3 probe and washed in 2X SSC (1X SSC = 150 mM sodium chloride and 15 mM sodium citrate, pH 7) and 0.5% (w/v) SDS at 5O’C. Clones positive for both the 5’ and the 3’ probes (38 of 68) were characterized initially by PCR (see below) and then plaque purified. Phage DNA was prepared (Miller, 1987), and cDNA inserts were subcloned into Bluescript KS11 and Ml3 vectors for restriction mapping and DNA sequencing.

Nucleotide position +l is assigned to the A of the ATG codon. The translated 807-amino-acid sequence of the human OSBP protein is shown beneath the nucleotide sequence. Dots above the human nucleotide sequence denote bases that differ from the corresponding rabbit nucleotide sequence (6). Amino acid residues that differ from the rabbit protein are enclosed in boxes and the corresponding amino acids in the rabbit sequence are shown below the human sequence. The putative leucine zipper region is underlined.

68

LEVANON

ET

AL.

ATGGCGGCGACG~~~CC~~GCCAG 1MAATELRGVVGPGPAAIAALGGGGAGPPVVGGGGGRGDAG CCAU;CPCCGGCGCCGCGTCAB 41PG SGAASGTVVAAAAG

121

AGqtaactgcttccgg~Cgctgtcgct R

120

TCGCGGCGGCGGCGCGAGCCCCCGGCCCGGGGGCCGGGGC GP GP GAGGVAAAG

PAPAP

P

CPXGGGCWGGGC TGGS G

240

388

3

FIG. 3. Nucleotide sequence of the first exon and 5’ flanking sequence of the human OSBP gene. Nucleotides are numbered on the right. Amino acids are numbered on the left. Nucleotide position +l is assigned to the A of the ATG codon. The cluster of transcription initiation sites determined by Sl nuclease mapping is denoted by the heavy underline. The sequence of the entire first exon is shown. Intron sequences are denoted by lowercase letters.

DNA (50-100 ng) isolated from different libraries was used as a template for PCR (Saiki et ah, 1988) with pairs of oligonucleotide primers bordering fragments at the 3’ or 5’ coding region of the rabbit OSBP cDNA. The amplification reaction was prepared as described (Gil et al., 1988) and carried out sequentially for 2 min at 36”C, 2 min at 5O”C, 3 min at 72”C, and 1 min at 94°C for 25 cycles. The PCR product was isolated and subjected to Maxam-Gilbert sequencing as described (Gil et al., 1988). To identify human clones that extended to the most 5’ region of the cDNA, plaques were analyzed by PCR using a 30-mer oligonucleotide corresponding to nucleotides 77-106 of the rabbit OSBP cDNA (Dawson et aZ., 1989a) in combination with primers corresponding to the left and right arms of XgtlO.

al. (1979). To prepare a uniformly labeled probe for Sl nuclease analysis, a 1.2-kb BamHI-SmaI fragment of genomic DNA that extends 5’ from the SmaI site at position +173 in the human OSBP cDNA was subcloned into Ml3 (Fig. 2). A 30-mer oligonucleotide complementary to nucleotides +3 to +32 in the cDNA sequence was annealed to the Ml3 clone and extended as described (Reynolds et al., 1985), except that annealing was performed at 55°C for 15 min followed by incubation at room temperature for 10 min and [a32P]dATP was used. A 297-nucleotide single-stranded 32P-labeled fragment was isolated by MluI digestion followed by 7 M urea/5% polyacrylamide gel electrophoresis. Sl nuclease analysis was performed as described (Reynolds et aZ., 1985).

OSBP Genomic Clones

Somatic Cell Hybrids

A human fetal liver partial HueIII-AluI genomic library in XCharon 4A (Lawn et al., 1978) was screened by in situ plaque hybridization with a probe derived from the 5’ end of the human OSBP cDNA. Eleven clones were identified out of 1 X lo6 recombinants, and one clone, XhOSBP, was further characterized. A 4.5kb EcoRI fragment from XhOSBP hybridized to the 5’ OSBP cDNA probe and was analyzed by restriction enzyme analysis and Southern blotting. Fragments corresponding to the 5’ exon and 5’ flanking sequences were subcloned into Ml3 and Bluescript for DNA sequence analysis.

Primary chromosomal assignment of the human OSBP locus was carried out with 11 hybrid clones that derived from five independent fusion experiments between Chinese hamster and human cell lines (for summary, see Yang-Feng et al., 1986). For regional mapping of OSBP on human chromosome 11, two Chinese hamster x human hybrid clones of series XXI derived from human cells with a t(11;15)(pll;p12) translocation (Francke and Francke, 1981) were used. For mapping in mouse, 12 Chinese hamster X mouse and one rat X mouse hybrid clones or subclones derived from four different series of hybrids (Francke et al., 1977; Francke and Taggart, 1979; Joyner et al., 1985) were used. DNA from five inbred mouse strains (AKR/J, C3H/HeJ, C57BL/6J, C57/J, and DBA/2J) and from the BXD set of recombinant inbred mouse strains were purchased from the Jackson Laboratory, Bar Harbor,

Sl Nuclease An.ulysis of OSBP Transcription Initiation Sites Total RNA was prepared from cultured SV40-transformed human fibroblasts as described by Chirgwin et

GENE

MAPPING

Maine. The progenitor strains for the BXD C57BL/6J (B) and DBA/BJ (D). Restriction

OF

OXYSTEROL-BINDING

set are

69

PROTEIN 1

2

3

4

5

6

7

6

9

10

11

12

13

Enzyme Digestion

DNA (10 pg) from hybrid or parental cells was digested with a fourfold excess of Hind111 in medium salt buffer. For polymorphism screening in mouse strains, 5 pg of each inbred mouse strain DNA was digested with each of the six enzymes, EcoRI, BarnHI, BglII, HincII, PstI, and HindIII. For linkage analysis in mouse, 5 )cg of DNA from each of the 26 BXD strains was digested with P&I. Southern

Hind III

Blot Hybridization

DNA fragments were separated by agarose gel electrophoresis and transferred to Hybond-N membranes (Amersham) by the method of Southern (1975). The probe used in this study was a 2.5-kb fragment of the human OSBP cDNA extending from nucleotide position 1490 to 3942 (probe pB+2.5). The probe was labeled with [32P]dCTP by oligolabeling (Feinberg and Vogelstein, 1983). The filters were prehybridized in 5X SSC, 10X Denhardt’s solution, 0.5% (w/v) SDS, and 100 pug/ ml sheared salmon sperm DNA at 65°C for 4 h and then hybridized in 4X SSC, 5X Denhardt’s solution, 10 mM sodium phosphate (pH 7), 1% SDS, 10% (w/ v) dextran sulfate, and 100 pg/ml sheared salmon sperm DNA with 32P-labeled probe at 65°C for 16 h. After hybridization, filters were rinsed in 2X SSC/O.5% SDS, and washed in 2X SSC/O.5% SDS at room temperature for 15 min and in 1X SSC/l% SDS at 65°C for 15 min. All filters were autoradiographed at -70°C for varying lengths of time with Kodak X-Omat AR film. RESULTS

cDNA

Cloning of Human

OSBP

A human kidney cDNA library was screened with 32P-labeled single-stranded Ml3 probes derived from the 3’ and 5’ ends of the rabbit OSBP coding region. Three positive clones containing the most 5’ and 3’ sequences were subcloned into Ml3 and Bluescript KS11 vectors for DNA sequencing. Figure 1 shows a comparison of the human and rabbit OSBP nucleotide and protein sequences. The human OSBP cDNA encodes a protein of 807 amino acids, 2 amino acids shorter than the deduced sequence of the rabbit OSBP. Overall, the human protein is 98% identical to the rabbit OSBP. In the coding region, the nucleotide sequence of the human cDNA is 94% identical to that of the rabbit. The human coding region is preceded by a 5’ untranslated region of 87 nucleotides and a 3’untrans-

FIG. 4. Mappingthe human OSBP gene. Hybridization of 32Plabeled human pBC2.5 probe to a Southern blot of HindIII-digested DNA from Chinese hamster X human hybrid cell lines and controls. Lane 1, Chinese hamster cells V79/380-6; lane 2, human diploid lymphoblastoid cells; lanes 3-13, Chinese hamster X human hybrid cell lines. Lanes 3 and ‘7-11 are positive for the 17.0-kb human OSBP fragment; the 2%kb fragment was also present in these lanes on the longer exposure autoradiogram. Lanes 4-6, 12. and 13 contain only Chinese hamster OSBP sequences and were scored as negative.

lated region of 1433 nucleotides (data not shown). The isolated clones did not extend to the poly(A) tail. Transcription Initiation

Site and 5’ Flanking Region

A fragment of genomic DNA encompassing the putative transcription start site was isolated from a bacteriophage h library as described under Materials and Methods. To map the human OSBP transcription start sites, we performed an Sl nuclease analysis with human fibroblast RNA (Fig. 2). The uniformly 32P-labeled single-stranded genomic DNA probe extended 265 nucleotides upstream of the initiator ATG codon. The end of the protected fragment was localized by simultaneous electrophoresis of a dideoxynucleotide-terminated primer-extended product. The primer that was used for the sequencing reaction was the same as the one used for the Sl nuclease probe. The Sl nuclease digestion gave a cluster of protected fragments that terminated at a position that was 107 to 111 nucleotides upstream of the initiator ATG (Fig. 2). Primer extension analysis of the same RNA yielded similar results (data not shown). The initiation site at position -107 is 20 nucleotides beyond the 5’ terminal end of the human OSBP cDNA. The nucleotide sequence of the first exon and 5’ flanking sequence of the human OSBP gene is shown in Fig. 3. No obvious TATA-box sequence is observed within 100 bp of the transcription start site. However, a sequence GCACCAAT, resembling the CCAAT con-

70

LEVANON

ET

TABLE Correlation

of Human

OSBP

Sequences

with

Human

AL.

1

Chromosomes Human

Hybridization/ chromosome

12

34

5 3 5

+/+ -/+/-

2 4 4

120 3 22 5 34

-/+

11

32

Discordant Informative

hybrids hybrids

Note. The numbers for each chromosome. excluded.

5

6

11

10

of hybrids Hybrids

8

9 0

14

33 24

3 4

2 5

4 5

2

3

10

1412

121

66 10

67

6 8

10

that are concordant in which a particular

45 11

9

5

6 7

8

9

10

11

12

14

4 3

13

14

16

17

3 13

2

2 4

3

3

4

4

4

4

10

12 10

5

14

15

18 3 13

19

20

21

3

3 4

4 3

121

2

3

3

3

2

12

6

0

4

9

7

7

5

4

5

5

4

3

11

9

11

11

9

11

10

10

9

11

11

10

15 16

Hind III the OSBP gene in the mouse. Hybridization FIG. 5. Mapping of 32P-labeled human pB+2.5 probe to a Southern blot of KndIIIdigested DNA from Chinese hamster X mouse hybrid cell lines and controls. Lane 1, mouse 3T3 cells; lane 2, Chinese hamster cells V79/380-6; lanes 3-14, Chinese hamster X mouse hybrid cell lines; lane 16, rat X mouse hybrid cell line; lane 16, rat Morris hepatoma 7777 cells. Lanes 3,6,8,11, and 16 contain only Chinese hamster or rat OSBP sequences and were scored as negative. Lanes 4,5, ‘7, 9,10, and 12-14 contain mouse OSBP sequences as well as Chinese hamster OSBP sequences and were scored as positive. The 3.4-kb fragment was absent in lanes 4, 5, and 7 due to polymorphism.

and discordant was structurally

(+/or -/+) rearranged

22

X

43 10 31

8

(+/+ or -/-) chromosome

13

12

10

The human OSBP gene was mapped with a panel of 11 Chinese hamster X human somatic cell hybrids with reduced numbers of human chromosomes. After hybridization of the 32P-labeled human OSBP cDNA probe pBf2.5 to HindIII-digested genomic DNA, two restriction fragments of 17.0 and 2.8 kb were detected in human control DNA (Fig. 4, lane 2) and several 3 4

Cell Hybrids

chromosome

3

5 0

Somatic

6

Mapping of Human OSBP Gene

2

11

X Human

11

sensus sequence, is located 55 nucleotides 5’ of the transcription initiation site. No sequencesresembling a sterol regulatory element (SRE-1) (Smith et aZ.,1988) were observed in the 5’ flanking region.

1

10

in Rodent

52 10

5

with the human OSBP sequence are given or present in fewer than 10% of cells were

fragments were observed in Chinese hamster control DNA (Fig. 4, lane 1). In hybrids containing human chromosome 11, the human signals were present as well as the Chinese hamster signal (Fig. 4, lanes 3, 711). No human signal was detected in hybrids that lacked human chromosome 11 (Fig. 4, lanes 4-6, 12, and 13). The presence of human OSBP sequences showed perfect concordance with the presence of human chromosome 11 in hybrid cell lines (Table 1). All other human chromosomes were excluded by at least two discordant hybrids. With two hybrids that contained defined regions of human chromosome 11 without an intact chromosome 11, we were able to localize the human OSBP gene to region llpll+qter of chromosome 11. Mapping of Murk OSBP Gene The mouse OSBP gene was assigned with a panel of 13 mouse X rodent somatic cell hybrids having reduced numbers of mouse chromosomes. After hybridization of the 32P-labeled human cDNA probe pB+2.5 to HindIII-digested genomic DNA, two fragments of 6.3 and 3.4 kb were detected in mouse control DNA (Fig. 5, lane l), several fragments were observed in Chinese hamster control DNA (Fig. 5, lane 2), and 8.8- and 3.8kb fragments were detected in rat control DNA (Fig. 5, lane 16). In hybrids containing mouse chromosome 19, the mouse signals were present as well as the Chinese hamster fragments (Fig. 5, lanes 4,5, 7,9, 10, and 12-14). No mouse signal was detected in hybrids not containing mouse chromosome 19 (Fig. 5, lanes 3, 6, 8, 11, and 15). The presence of mouse OSBP sequences showed perfect concordance with the presence of mouse chromosome 19 in hybrid cell lines (Table 2). All other mouse chromosomes were excluded by at least two discordant hybrids. Polymorphism in Inbred Mouse Strains After hybridization of the 32P-labeled human cDNA probe to BgZII-, PstI-, and H&&II-digested mouse ge-

GENE

MAPPING

OF

OXYSTEROL-BINDING

TABLE Correlation

2

of Mouse-Specific OSBP Sequences with Mouse Chromosomes in Rodent X Mouse Somatic Cell Hybrids Mouse

Hybridization/ chromosomes

+/+ -/+/-/+ Discordant Informative

hybrids hybrids

Note. The numbers for each chromosome. excluded.

71

PROTEIN

1

2

3

4

5

6

7

8

9

6 2 2 2

8 3 2 0

6 5 2 0

5 5 2 0

3 4 3 0

4 4 2 12

6 3 12

6 5

2 4 5 1112

4 12

2 13

2 13

2 12

3 10

of hybrids that are concordant Hybrids in which a particular

3 11

0 3 12

2 13

(+/+ or -/-) chromosome

nomic DNA, two distinct patterns were observed (Fig. 6). Pattern 1 (Fig. 6, lane 1) shows 6.4-, 2.1-, and 0.9kb fragments with BgZII; 6.3-, 5.4-, 4.9-, 4.3-, and l.lkb fragments with P&I; and 6.3-, and 3.4-kb fragments with HindIII. Pattern 2 (Fig. 6, lane 2) shows 6.4-,

6 12

chromosome 10

11

12

3 4 4

0 4 8

5 3 3

5 12

9 13

5 13

and discordant was structurally

13 3 4 4 11 5 12

14 3 4 5 6 13

15

16

7 3 13 2 3 13

4 3 2 5 12

17 7 3 13 2 3 13

18

19

X

4 5 0

8 4 0 0

5 2 2 3

3 12

0 12

5 12

(+/or -/+) with the mouse OSBP sequence are given rearranged or present in fewer than 10% of cells were

2.1-, and 2.0-kb fragments with BgZII; 6.3-, 5.4-, 4.9-, 4.6-, and l.l-kb fragments with P&I; and 6.3-, and 3.1kb fragments with HindIII. Mouse strains AKR/J and C57BL/6J exhibit pattern 2, while C3H/HeJ, C57/J, and DBA/2J exhibit pattern 1. Genetic Analysis

with Recombinant

Inbred Strains

The strain distribution pattern of the BXD set of recombinant inbred mouse strains is shown in Fig. 7 and summarized in Table 3. A comparison of the strain distribution pattern of the OSBP gene with that of Ly1 and of Ly-10 (Tada et aZ., 1982) reveals a close linkage. One recombinant between Ly-1 and OSBP (strain 11) and three recombinants between Ly-10 and OSBP (strains 01, 11, and 30) were found among the 25 strains compared (Table 3). Thus, OSBP must be located near Ly-1 and Ly-10 at the proximal end of mouse chromosome 19. DISCUSSION

0.9-m 6gl II

Pst I

Hindlll

FIG. 6. Polymorphism of OSBP gene in the mouse. Hybridization of s2P-labeled human pB+2.5 probe to a Southern blot of BglII, P&I, or HkdIII-digested DNA from inbred mouse strains. Lane 1 in each panel shows restriction enzyme digestion pattern 1. Lane 2 in each panel shows restriction enzyme digestion pattern 2. Mouse strains C3H/HeJ, C57/J, and DBA/2J exhibited pattern 1; strains AKR/J and C57BL/6J exhibited pattern 2.

The similarity between the nucleotide and the protein sequences of the human and rabbit OSBP is striking and suggests that this protein performs a vital function in cellular cholesterol metabolism. This high degree of conservation is usually reserved for proteins that participate in protein-protein interactions, such as cytoskeletal proteins (actin, tubulin, etc.). To date, OSBP has not been shown to bind to other proteins. All of the major structural features previously identified in rabbit OSBP are conserved in the human enzyme (Fig. l), including the glycine/alanine/proline-rich NH,-terminal domain (amino acids l-80), the cluster of regularly spaced leucine residues suggestive of a leutine zipper motif (amino acids 207-242), and the two clusters of negatively charged residues with nearby serines and threonines (amino acids 189-198 and 351-

72

LEVANON 01 02

05

06

08

09

11 12

13

14

15

16

ET

AL. 19 20

18

21

22

23

FIG. ‘7. Patterns of OSBP polymorphism in PstI-digested DNA from 26 strains of BXD bridization of ‘*P-labeled human pB+2.5 probe to a Southern blot of PstI-digested DNA from progenitor strains of the BXD set are C57BL/6J (B pattern) and DBA/2J (D pattern). Strain 06 is an example of D. The strain distribution pattern is summarized in Table 3.

TABLE Inbred

(BXD)

Strain

Distribution

Pattern BXD

Locus

00000011111111222222222333 12568912345689012345789012

Ly-1” Ly-10” OSBP

BBBDDBBBDBBBBBDBBDDB DBBDDBBBDBBBBBDBBDDB BBBDDBDBDBBBBBDBBDDBD

’ Data

from

Tada

et al. (34).

Strain

27 was uninformative

30 31 32

set of recombinant inbred BXD recombinant inbred number 01 is an example

mouse strains. Hymouse strains. The of B; strain number

(Lalley et al., 1989; Tada et al., 1982; Tedder et al., 1988; Glaser et al., 1989). The homologous human loci to Ly-1 (CD5), Cd-20, Fth, and Pygm have been assigned to chromosome 11, region q12-q13 (Junien and McBride, 1989). The localization of these genes and OSBP to the long arm of human chromosome 11 and the proximal end of mouse chromosome 19 indicates a new conserved syntenic group between human and mouse. As previously reported, human chromosome 11 has extensive syntenic conserved groups on mouse

358) suggestive of a potential casein kinase II phosphorylation site (Dawson et al., 1989a). We have mapped the human OSBP gene to the long arm of human chromosome 11 by the method of somatic cell genetics. Using somatic cell hybrids and recombinant inbred mouse strains, we have also mapped this gene to the proximal end of mouse chromosome 19 in the same region with genes for lymphocyte cell surface antigen Ly-1, Ly-10, Cd-20, Fth (ferritin heavy chain), and Pygm (muscle glycogen phosphorylase)

Recombinant

24 25 27 28 29

3 of Chromosome strain

19 Loci

in 26 Mouse

Strains

number

-BBDDB -BBBDB B and not used to determine

linkage.

B

D

D

B

GENE

MAPPING

OF

OXYSTEROL-BINDING

chromosomes 2,7, and 9 (Barton et aZ., 1988; Lalley et aZ., 1989). Furthermore, several genes on the distal end of mouse chromosome 19 have homologous loci on the long arm of human chromosome 10 (Lalley et al, 1989). Although the OSBP shows a binding specificity that is consistent with a role in transducing the regulatory actions of oxygenated sterols, to date no functional evidence has been obtained to confirm that OSBP plays such a role. It is hoped that the information being gathered from biochemical as well as genetic studies will allow the assignment of a function to this highly conserved protein.

nylate kinase-l

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