Molecular cloning and sequence analysis of the rat liver carnitine octanoyltransferase cDNA, its natural gene and the gene promoter

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Biochimica et Biophysica Acta 1264 (1995) 215-222

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Molecular cloning and sequence analysis of the rat liver carnitine octanoyltransferase cDNA, its natural gene and the gene promoter Sun J. Choi a,b, DOO H. Oh a, Chung S. Song b, Arun K. Roy b, Bandana Chatterjee U,c,. a Bioproducts Research Center, Yonsei University, Seoul, South Korea b Department of Cellular and Structural Biology, The Universi~ of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drit,e, San Antonio, 7X 78284, USA c Audie L. Murphy Memorial VA Hospital, San Antonio, 7?( 78284, USA

Received 2 May 1995; revised 29 June 1995; accepted 30 June 1995

Abstract

The full-length cDNA and the natural gene for rat peroxisomal carnitine octanoyltransferase (COT) have been isolated and sequenced. The 2681 bp long cDNA contains an open reading frame for 613 amino acids, resulting in a protein with a deduced molecular weight of 70 301, and a C-terminal peroxisomal targeting sequence (Ala-His-Leu). The isolated COT cDNA has 51 bp of the 5' untranslated region (UTR), 79l bp of 3' UTR, two putative polyadenylation sites, and a poly(A 19_23 ) tail. Screening of a rat genomic DNA library in the A phage with the COT cDNA probe resulted in the isolation of seven overlapping clones, together containing the complete COT gene with seventeen exons. All of the exon-intron boundary sequences conform to the GT-AG rule. The COT gene appears to spread over 40 to 60 kbp region of the rat genome. The transcription initiation site of the COT gene was determined through primer extension, and the promoter sequence up to the position - 1 1 4 0 was established. The promoter lacks the canonical TATA box and a promoter-reporter construct containing the sequence encompassing - 1140 to + 84 base positions and the firefly luciferase reporter cDNA yielded about 100-fold increase in promoter activity in transfected hepatoma cells. Some of the consensus sequences for putative cis elements present in the promoter sequence are: the two CCAAT motifs for CTF/NF1/CBP binding (at - 2 8 4 and -93), two GC boxes for Spl binding (at - 160 and -68), two AP2 sites (at - 3 5 9 and -25), a half site (TGACCT) for the peroxisome proliferator activated receptor (PPAR) binding at - 7 3 7 within a partial palindromic sequence region. Potential regulatory elements, such as several palindromes and repeat motifs for five different sequence segments, are also identified. Keywords: Carnitine octanoyltransferase; cDNA; Promoter; Peroxisome; Peroxisome proliferator activated receptor

1. Introduction

Peroxisomes are single-membraned cytoplasmic organelles, and the sites of metabolism for a variety of endobiotic and xenobiotic compounds [1-3]. Fatty acids longer than C18 are almost exclusively metabolized in peroxisomes through the fl-oxidative pathway. Furthermore, fatty acids, which are to-oxidized in the endoplasmic reticulum, are subsequently transported into peroxisomes for chain shortening. Therefore, peroxisomal /3-oxidation

~ The sequence data reported in this paper have been submitted to the EMBL/GenBank Data Libraries under the accession numbers U26033 (COT cDNA) and U26034 (COT promoter sequence). * Corresponding author. Fax: + 1 (210) 5673803. 0167-4781/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSD10167-4781(95)00146-8

is characterized by a broad substrate specificity, and unlike mitochondrial fl-oxidation, can operate independently of the energy status of the cell. Such an arrangement is suitable for elimination of poorly metabolizable compounds without coupling to oxidative phosphorylation [4]. An obligatory step in peroxisomai fl-oxidation is the transport of the chain-shortened, activated fatty acids from the peroxisomes to the mitochondria for complete oxidation. Carnitine and carnitine acyltransferases are believed to play important roles in the acyl CoA transport [5]. However, peroxisomal fatty acid /3-oxidation is carnitine independent, and thus the mechanism of the transfer process itself is not clearly understood. In addition, peroxisomal carnitine acyltransferases are likely to ensure that the local acyl C o A / C o A ratio is maintained in the physiological range so that the organelle function continues

216

S.J. Choi et al. / Biochimica et Biophysica Acta 1264 (1995) 215-222

normally [6,7]. Three different forms of carnitine acyltransferases with overlapping substrate specificity catalyze the following reversible reaction: L-( -- )carnitine + acyl CoA = acyl carnitine + CoASH Carnitine acetyltransferase (CAT), with maximum specificity for the short chain acyl (C2-C 4) group, is present both in the mitochondria and peroxisomes [7,8]. Carnitine palmitoyltransferase (CPT), with specificity for C ~0 to C ~8 fatty acids has been localized to both microsomal and mitochondrial compartments [9,10], whereas carnitine octanoyltransferase (COT), showing the highest activity with C 8 to C16 chain length substrates, is present mostly in the peroxisomes, and to some extent in microsomes [5,11,12]. However, controversy remains as to whether the peroxisomal COT and the microsomal/mitochondrial CPT represent the same enzyme [11]. Normally, /3-oxidation in peroxisomes occurs at a very low level. Upon exposure to either very long chain fatty acids or a group of structurally diverse xenobiotics such as hypolipidemic agents (e.g., clofibrate, ciprofibrate, and (4-chloro-6-(2,3-xylidino)-2-pyrimidinyl)thioacetic acid) and plasticizers (e.g., dioctylphthalate), peroxisomal /3oxidation is markedly induced in several rodent tissues, most remarkably in the liver and kidney [13-15]. The enhanced oxidative activity is due to drug mediated transcriptional induction of the enzymes involved in the /3oxidation pathway [16-21]. Peroxisome proliferator activated receptors (PPARs) of the steroid receptor superfamily have been implicated to play a major role in this induction process [22,23]. In our earlier study, we reported that the carnitine octanoyltransferase (COT) mRNA level increases by 40fold or higher in response to peroxisome proliferators [21]. Because of this large inductive response, the study of the transcriptional regulation of the COT gene by peroxisome proliferator activated receptor (PPAR) can prove especially advantageous. We had previously reported on cloning of a fragment of the COT cDNA from the rat liver [21]. In continuation of this study, we now report the sequence of the full-length COT cDNA, structure and organization of the natural gene for COT, and characterization of the COT promoter.

library in the lambda ZAPII vector was screened to isolate additional clones in order to confirm the sequence of the COT cDNA retrieved from the primary library. The rat genomic library in the lambda EMBL3 S P 6 / T 7 vector (Clontech, Palo Alto, CA) was used for isolation of COT genomic clones. 2.2. Screening of cDNA and genomic libraries

Libraries were screened by colony hybridization with a 1642 bp COT cDNA fragment as the probe. This cDNA fragment was previously isolated in our laboratory from a Agtl 1 expression library using a rabbit antiserum to the rat COT [5,21]. The probe was labeled by random priming using [ a- 32P]dCTP. Positive recombinant phage plaques were purified through three rounds of screening of the phage particles plated at increasingly higher dilutions. DNAs from positive clones were prepared according to standard procedures, and subsequently purified by phenolchloroform extraction and ethanol precipitation [24]. 2.3. Construction of a reporter plasmid for analysis of promoter activity

The COT promoter activity was investigated by testing its ability to direct the expression of a downstream luciferase reporter gene. A recombinant plasmid (pl.2kbp COT-Luc) was constructed by cloning a segment of the COT promoter ( - 1 1 4 0 to + 84 bp) into the luciferase vector pGL2 basic (Promega, Madison, WI). The 1224 bp COT promoter fragment was isolated by polymerase chain reaction (PCR) amplification of the DNA template of the plasmid pCOT2NRI containing 4.0 kbp of the COT genomic DNA insert. PCR amplification was performed in the presence of a vector-specific sense primer and a COT gene-specific 27-mer oligonucleotide (5'-CTCCTGCACCCGGCTTGGCTCTCTGCA-3'), spanning positions + 30 to +4, as the antisense primer. The PCR product was verified by nucleotide sequencing, and was cloned upstream of the luciferase cDNA in the reporter vector pGL2 basic (Promega, Madison, WI). The COT promoter activity was analyzed by determining its ability to direct luciferase expression from the reporter plasmid pl.2kbp COT-Luc in transfected HepG2 (human hepatoma) cells.

2. Materials and methods

2.4. Cell transfection

2.1. Animals and DNA libraries

HepG2 cells (seeded at 1 • 1 0 6 for each T25 flask) were cultured overnight as a monolayer under 5% CO 2 in DMEM-F12(I:I) medium supplemented with 10% fetal bovine serum. The next day, following a change into fresh medium, cells were cultured for 2 h and then transfected with 10 ~g of the plasmid DNA using the calcium phosphate method [25]. For transfection, the cells were exposed to the calcium phosphate-DNA mixture for 4 h, then subjected to 15% glycerol shock for 3 min, and finally

Male Sprague-Dawley rats ( ~ 200 g) maintained on a diet of Purina Rat Chow were used for this investigation. In some cases, dioctylphthalate was added to the diet at 0.2% for 2 weeks prior to killing. The COT cDNA was initially isolated from a rat liver cDNA library that we had constructed in the lambda Uni-ZAP TM XR vector (Stratagene, San Diego, CA). Subsequently, a rat liver cDNA

S.J. Choi et al. / Biochimica et Biophysica Acta 1264 (1995) 215-222

they were cultured for additional 24 h before harvesting. The cell lysate was assayed for luciferase activity and protein concentration as described earlier [26].

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Primer extension was carried out according to a previously described protocol [27]. The transcription start site was confirmed in two independent primer extension analyses using two different primers. One primer is a 28-mer synthetic oligodeoxynucleotide (5'-TGATTTTCCATGGTATAGTCACAGTAAG-3'), complementary to the COT mRNA sequence from positions +62 to +35 and the second primer is a 30-mer oligodeoxynucleotide (5'-TGGAATGTTCGTTCTTCAATTGACTTAGCC-3'), complementary to the COT mRNA sequence from positions + 95 to +66. The primers were labeled at the 5' end with [T-32p]ATP and T4 polynucleotide kinase prior to their annealing to COT mRNAs. Both total RNAs and poly(A) + RNAs were analyzed for transcription start site determination. Primers were extended in the presence of the Superscript reverse transcriptase (Gibco-BRL, Bethesda, MD). Total liver RNAs were isolated from the frozen liver tissue by guanidinium thiocyanate-phenol-chloroform-isoamyl alcohol procedure, and poly(A) enriched mRNAs were selected from total RNAs by oligo(dT) affinity column chromatography [28,29]. An M13 DNA sequencing product was run in parallel as a reference for size determination of the primer-extended product.

3. Results and discussion

3.1. Isolation and characterization of the full-length COT cDNA The cDNA clone for COT was initially isolated from a primary rat liver cDNA library in the lambda Uni-ZAP XR vector. Subsequently, the same clone was retrieved from a second library in the lambda ZAPII vector that was purchased from a commercial source (Stratagene, CA). Isolation of the same cDNA clone from two different libraries prevented the possibility of accidentally isolating cloning artifacts as cDNA inserts. The restriction map and sequencing strategies for the full-length COT cDNA (2681 bp) are depicted in Fig. 1. The overlapping DNA fragments were sequenced using the dideoxy chain termination procedure [30], and the complete cDNA sequence is presented in Fig. 2. The sequence data reveals 51 bases of the 5'-untranslated region, an open reading frame (ORF) of 1839 bases, predicting 613 amino acid residues for a protein of 70301, and a 3'-untranslated region of 791 nucleotides preceding the 19 to 23 residue long poly(A) tail. Two polyadenylation signals (AATAAA) are located at positions + 2622 and + 2663. The AUG initiator codon of the predicted ORF for the 2681 bp COT cDNA is

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2.5. Primer extension analysis

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Fig. 1. Restriction map and DNA sequencing strategy for the rat liver COT cDNA. X, XbaI; B, BamHI; R, EcoRI.

preceded by the base 'A' at position - 3 and the base 'C' at position - 2 . This conforms to the Kozak consensus sequence of CC(A/G)CCAUG for the eukaryotic translation initiation site [31 ]. Thus, the isolated cDNA fragment represents the full-length COT mRNA sequence. It should be noted that in earlier studies, we had isolated a 1642 bp COT cDNA fragment (clone 2A x) that was able to express an immunoreactive epitope specific for the rat COT protein [21]. The cDNA insert of clone 2A x was used as the hybridizing probe to isolate the full-length COT cDNA described in the present report. In our earlier communication, we had also reported on the isolation of additional clones that overlap clone 2A x at the 5' end [21]. Those additional clones, along with clone 2A×, provided a composite nucleotide sequence for 2143 bp of the COT cDNA. Upon comparison of the composite sequence with the full-length COT cDNA sequence spanning 2681 nucleotide residues, as reported here, we found that a DNA segment within the earlier reported 2143 bp sequence was assigned to an inappropriate position. Thus, the nucleotides + l to + 485 of the composite sequence actually represents positions + 1707 to + 2191 of the full-length 2681 bp COT cDNA. Evidently, the hgtl 1 expression library used in our initial study contained ligation artifacts for cDNA inserts. Upon examination of the predicted ORF for the 2681 bp full-length COT cDNA, a 781 bp long 3' untranslated region is evident. Sequences of comparable size have also been observed in the mRNAs encoding other peroxisomal enzymes. For example, acyl-CoA oxidase contains 1800 bases of a 3' untranslated region, and enoylCoAhydratase:3-hydroxyacyl-Coa dehydrogenase and catalase contain 700 to 800 bases of 3' untranslated segments [32,33]. It should be noted that the predicted molecular weight of the COT enzyme, as derived from the cDNA sequence, is different from the 64 to 66 kDa of the COT molecular weight established through size analysis of the purified enzyme [21,34,35]. The cDNA sequence deduced primary structure of the COT protein reveals a consensus peroxisomal targeting sequence (PTS), i.e., Ala-His-Leu at the carboxy-terminal. The consensus PTS (Ser/AlaLys/Arg/His-Leu-COOH) has been reported for several peroxisomal enzymes [36,37]. The deduced molecular weight of 70301 may indicate that the enzyme is synthesized as a preprotein with an N-terminal mitochondrial targeting sequence (MTS) and a C-terminal peroxisomal targeting sequence (PTS). The N-terminal MTS in the

S.J. Choi et al. / Biochimica et Biophysica Acta 1264 (1995) 215-222

218

+i

GAG TGC AGA GAG CCA AGe eGG GTG HAG GAG TTT TCT TAC TGT GAC

+1621

CTG TTT GAG GAT CCA CTT TTC T e e AGA AGT GGA GGA GGT GGG hAT Leu Phe G l u Asp P r o Leu Phe S e t A r g S e t G l y G l y G l y G l y ASh

+46

TAT Ace ATG GAA hAT CAA TTG GeT hAG TeA ATT GAA GAA GGA ACA ~ H Glu Ash Gln Leu AIs Lye Set Ile Glu Glu Arq Thr

+1666

TTT GTG CTG TeA ACA AGT CTG GTT GGT TAC TTA CGA ATT HAG GGA Phe Val Lau Ser Thr Ser Leu Val Gly Tyr Leu Arg Ile Gln GIy

+91

TTC CAG TAC CAG CAC TCT CTT CCG CCC TTG c e c GTT CCT TCG CTT P h s G l n T y r G i n ASp S e t Leu P r o P r o Leu P r o V s l P r o S e t Leu

+1711

GTC GTG GTT eec ATG GTA CAT hAT ~ A TAC GGC TTT TTC TAC CAC Val Val Val Pro Met; Val H i S Ash Gly Tyr GI¥ Phe Phe Tyr His

+136

GAA GAA TeA CTG hAG AAG TAC CTT GAG TeA GTG AAG CCA TTT GCA GIu GIu Set Leu LFS Lye Tyr Leu Glu Set Vsl Lye Pro Phe Ala

+1756

ATe AGA HAT GAC AGG TTT ~ GTG ACA TGT TeA Tee TGG AGG TeA I l o Arq Asp Asp A r g Ph@ V a l V a l T h r t ' y s S e t S e t T r p A r g S e t

+181

hAT GAA GAS GAA TAC AAG AAA ACT C~A GAA ATA GTT CAA AAG TTT ASh Glu Asp Glu Tyr Lye Lye Thr Glu G1u Ils Val Glo Lye Phe

HIS01

TGT CTT GAG ACT GAT GCA GAA AAG TTA GTG GAG ATG ATT TTT CAT Cy8 LeU GIu Thr Asp Ala GIu Lys Leu Val Glu Met Ii@ Phe His

+226

CAA CAT GGA GTT GGC hAG ACA TTG CAT HAG hAG TTA CTT ~ AGG GIn Asp GIy Vel GIF Lye Thr Leu His GIn Lye Leu Leu Glu Arg

+1846

~ T TTC CAC GAT ATG ATA CAT CTG ATG hAC ACG GeT CAT CTT TAG AIa Phi His Asp Met Ile Hi8 Leu Met Ash Thr AIa His Leu*

+271

GeT AAA GGA AAA AGA AAC TGG CTG GAA GAG TGG TGG CTC AAT GTC Ala Lye Gly Lyw Arg Aen Trp Leu Glu GIu Trp Trp Leu Aen Val

+1891

AGA CTC AGA GAC ATA HAG GTC ACA GAA ACT GGG TAC GGA GAA TGG

+316

GCC TAC TTG CAT GTG CGT ATT CCA TeA CA~ CTG AAC GTG AAC TTT Ale Tyr Leu ASp Val Arg Ile Pro Ser Gln Leu ASh Val Ash Phe

+361

GTG GGT CCG TCT CCC CAC TTT GAA CAe TAC TGG CCT GCA AGG GAA Val Gly Pro Ser Pro Hie Phs Glu Hie Tyr Trp Pro Ale Arg Glu

+406

GGC ACT HAG TTG GhA AGA GGA AGe ATA eTA CTG TGG CAC AAe TTG Gly Thr Gin Leu GIu Arq Gly Ssr lls Leu Leu Trp His Asn Leu

+451

AAC TAC TGG HAG CTG eTA AGA AGA GAA AAA TTG CCT GTA CAT AAA ASh Tyr Trp Glo LeU Leu Arg Arg GIu Lye Leu Pro Val His Lys

+496

TCT GGA hAT ACT CCT eTA GAC ATG AAC CAA TTC eGG ATG CTG TTT Ser Gly Aen Thr Pro Leu ASp Mst Aen Glo Phe Arg Met Leu Phe

+541

TCT ACe TGC AAG GTT CCG GGA ATe ACT AGA GAT TCG ATT ATG AAT Set Thr Cys LFI Vel Pro GIF Ile Thr Arq Asp Set Ill Met Ash

+586

TAT TTT AAG ACT GAG AGe GAG GGG CAT TGT CCG Ace CAC ATT Gee Tyr Phe Lys Thr GIu Set Glu Gly Hie e y e Pro Thr His Ile Ala

+631 +676 +721 +766

GTG CTG TGT CGA GGC AGA GCG TTT GTC TTC HAT GTC CTC CAT CAC V a l Leu Cye A r g G l y Arg A l a P h s V a l Phe Asp V a l L e u H i s ASp GGT TGT TTG ATe Ace CCA CCA C~%A CTT CTC ACA CAA CTG ACA TAC G l y Cys LOU I l e T h r P r o P r o G l u Leu Leu A r g G l n Leu T h r T y r ATe TAC HAG AAA THe TGG AAT GhA CCT GTT GGG CCC AGT ATA GCG Ile Tyr Gin Lye eye Trp ASS Glu Pro Val GIy Pro Set Ile Ala GCA TTA Ace AGT GAG GAG CGA ACT eGG TGG GCG AAG GCA AGA GAA Ala Leu Thr Ser Glu Glu Arg Thr Arq Trp Ala Lye Ala Arq GIu

+811

TAT CTG ATT GGT CTT HAT CCA GAG AAC TTG ACT TTA TTA GAA AAA Tyr Leu Ila Gly Leu Asp Pro Glu Asn Leu Thr LOU Leu Glu Lys

+856

ATT CAA Tee AGT TTA TTT GTG TAT TCC ATA GAA GAS ACe AGT CCA If@ Gln Set Set Leu Phe V81 Tyr Se~ I18 Glu Asp Thr Ser Pro

+901

CAT GCA Ace CCA GhA AAT TTT TCT CAG GTC TTT GAA ATG CTT CTT HAS Ala Thr Pro Glu Ash Ph@ Set Gin Val Ph@ Glu Met Leu Leu

+946

GGT GGA HAT CCA GCA GTG GGC TGG GGT GAC AAG Tee TAT AAT CTG Gly Gly Asp Pro Ala Val Arg Trp Gly Asp Ly8 Set Tyr Asn Leu

+991

ATT Tee TTT GeT hAC GGA ATA TTT ~ TGT AGe TGT HAT CAT Ile Ser Ph@ Ala Ash Gly Ile Phe GIF CyJ set Cys Asp His Ala

+1036

CCT TAT GAT GCA ATG CTT ATG GTG AAC ATT GeT CAe TAT GTT GAT Pro Tyr Asp Ala Met LeU Met Val ASh Ile Ala His Tyr V81 Asp

+1091

GAG hAG CTC eTA GAG ACG GAA GGG AGA TGG AAG GGT TeA GAA AAA GIU LF8 Leu Leu Glu Thr Glu Gly Ar~ Trp Ly8 GIF Set Glu Lye

+1126

GTC eGG HAT ATA CCG TTG CCA GAG GAG CTG GeT TTC ACT GTG HAT Vel Arg Asp Ile Pro Leu Pro Glu Glu Leu Ala Phe Thr Val Asp

+1171

GAG AAG ATA CTG AAT GAC GTC TAC CAA Gee AAA Gee CAA CAC CTC Glu Lye lie Leu Aen Asp Vel Tyr GI8 Ala Lye AIa Gln Hie Leu

+1216

AAA GCA GCA TCT GAT TTA HAG ATA GCA GCA TCT Ace TTC ACA TCT Lye Ale Ala Set Asp Leu Gln Ile A18 Ala Set Thr Ph@ Thr Set

+1261

TTT GGC AAA AAG CTC ACT AAG AAG GAG Gee CTT CAC CCT GAC Ace Ph@ Gly Lye Lye Leu Thr Lye Lye Glu Ala Leu Hie Pro Asp Thr

+1306

TTT ATT ~ G CTC GeT CTT HAG CTC GCC TAC TAC AGA CTT CAT GGA Phe Ile Gln Leu Ale Leu GID Leu Ale Tyr Tyr Arg Leu Hie Gly

+1351

CGC ece GGT TGC TGC TAT ~ ACA GeT ATG ACA AGA TAC TTT TAC Arg Pro GIF eye eye Tyr Glu Thr Ala Met Thr Arg Tyr Phe Tyr

+1396

CAT GGC CGA ACA GAG ACT GTG CGA TCT TGT ACA GTG GAG Gee GTC Mie Gly Arg Thr Glu Thr Val Arg SeE eye Thr Val Glu Ala Val

+1441

AGG TGG TGC HAG Tee ATG HAG HAT CCT TCT GCC AGT CTC CTT GAA Arg Trp eye Gin Ser Met Gln Asp Pro Set Ale Set Leu Leu Glu

41486

CGT HAG CAA hAG ATG TTA GAC GeT TTT ~ AAG CAT AAC AAG ATG Arg Gln GIN Lye Mot Leu Asp Ale Phe AI8 Lye H~S Ass Lye Mst

+1531

ATG AGA HAT TGT Tee CAT GGA AAA GGA TTT GAC CGT CAC CTT TTA Met Arg Asp eye Ser Hie Gly Lye GIF Phe Asp Arg HAS Leu Leu

+1576

GGC CTT TTG CTC ATA GCA AAA GAG GAA GGC CTC CCT GTT CCA GAA GI¥ Leu IAU LeU I l e ASs L y s G l u G l u G I y LeU P r o V81 P r o GIu

+1936

CAT GGT HAT ACG ACA TGG AAG GAA TGT TGA CTT AAA GGA AAC CTG

+1981

TTA ATG HAG GGA TTA GAG AGG GAT GeA CTC TAG ATT TAT TCT ACe

+2026

TTA AAG CCT TCT GTT GCA ACA GCA ATG CAA ACT HAG ACA TAG TGA

+2071

ATA GAA eTA THe AAT GTT TTA AGe CTC AAC AAT GCA eAT CTG TAT

+2116

ATT TT~ ACA ATA CAA ATC eTA CTC TAA TGT TAA AAT ATT TTT GTT

+2161

GGC ACA TGT GTA GGT TGC AAG Tee TCT GTG AAC ATA ATT ATA GAG

+2206

TAT TTC TeA AGe ACT TTA ATA CTT TCT AAT GGC HAG AGG GTA TAA

+2251

AAC CCA TGG TTA GAT GCT hAT TTc CCT GAC ATe AGT Gee TTC TAC

+2296

ATe HAG CAC AGG AGT ACA AGe eTA TGA GAT TTC ATG GGA AAA CCA

+2341

eTA TTG TTC AAT ATT GAT eTA AAA TAG CTC CTT TGA ACA GAC AAA

+2386

AGT ATe AAG TTG TAT TAG AAA AGA ATA TTA GCA AAA CTe ATT ATG

+2431

ATA TGT TGT AAT TAA TTT TGT GAA TAT AAA ATe AAA ACA CTT CCA

+2476

TTT AA~ TCT ACT TGG TAG AGT TAG TGG CTT TAA AGG GTT AAA TGT

+2521

CGA GTA TC~ TTC TeA GAA CTT TAT AAT TAT TTC CCA CTG TTA TTC

+2566

AAA ATG TTA GCA TAT AGA CAT TCT CCC ATT GTA ATT HAG TGT TTA

+2611

TAT TCT CAA AGA ATA AAG CAT CCA GAA Tee TTG TAA TTT CTC ATT

+2656

TAT TTT CAA TAA AAA TGA TTC CTG AT

Fig. 2. cDNA and predicted amino acid sequences for the rat liver COT, The ATG translation start site at position + 52 yields an open reading frame of 1839 bases. The TAG stop codon is indicated with an asterisk. The two poly-adenylation signals (AATAAA) are underlined.

S.J. Choi et al. / Biochimica et Biophysica Acta 1264 (1995) 215-222

(Ill)

(115) (1121 (1151 (li~1 (941 (I/6) (l(r~) I l l )

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Fig. 3. Exon-intron organization of the natural COT gene. The DNA sequences of all seventeen exons match with the cDNA sequence of the entire open reading frame. The intron sizes have not been determined. The seven overlapping A clones, which together contain the entire natural gene for COT are depicted. The seventeen exons of the COT gene, interrupted by sixteen introns, are spread over at least 40 kbp of the rat genomic DNA.

preprotein leaves open the possibility that the translation product of the COT gene has suitable structural features for both mitochondrial and peroxisomal targeting. This dual targeting could provide a mechanism for peroxisomal as well as mitochondrial localization of COT and/or CPT [11,38]. 3.2. Isolation and characterization of the natural gene for COT Seven independent genomic clones for the rat COT were isolated from a lambda EMBL3 SP6/T7 rat genomic DNA library following screening with the full-length COT cDNA (2681 bp). A physical map of the COT gene was constructed on the basis of restriction enzyme digestion patterns combined with sequence analysis of the individual exons (Fig. 3). For this sequence analysis, rat COT genomic DNA fragments were subcloned in plasmid vectors and small fragments containing the exons were subsequently sequenced. Fig. 3 summarizes the exon-intron organization of the COT gene encompassing seventeen exons. Additional hybridization experiments, using probes specific for the promoter region and the polyadenylation sites of the rat COT gene, established the positions of the first and last exon, respectively. A systematic analysis of all seven lambda clones demonstrated that a single large gene of at least 40 kbp encodes rat COT. This result is in good agreement with the Southern blot of rat genomic DNA probed with 2681 base pairs of the COT cDNA (data not shown). Thus, the complete COT gene comprises sixteen small exons and one large exon. For exons 1 through 17, the corresponding sizes in bp are 30, 136, 125, 182, 125, I09, 94, 126, 102, 84, 108, 131, 124, 79, 94, 120, and 912. All exon/intron boundaries display canonical splice consensus sequences [39,40], with each intron sequence beginning with GT at the donor splice site and ending with AG at the acceptor splice site (Fig. 4). The transcription start site of the COT gene was determined by primer extension analysis (Fig. 5). As shown in Fig. 5, a 62 bp primer extended product was generated

either from total liver RNAs (lane l, panel A) or from poly(A) enriched liver mRNAs (lane 2, panel A) in the presence of the 28 nucleotide long antisense primer (5'TGATTTTCCATGGTATAGTCACAGTAAG-3'). Upon comparison of the nucleotide sequences of the COT cDNA (Fig. 2) and the COT genomic DNA (described later under Fig. 6), the transcription start site can be assigned at the G residue (marked by an arrow) of the genomic DNA sequence depicted on the far right side of Fig. 5. The transcription initiation site was further confirmed with a second 30 nucleotide long antisense primer (5'-TGGAATGTTCGTTCTTCAATTGACTTAGCC-3') that yielded a 95 bp primer extended product. Thus, as is the

Exon

1

GGTGCAGGAG ~rt gaggattctg t t t g g t t t t t +30

ag TTTTCTTACT

Exon 2

+31

Exon

2

CTTGAGTCAG ~rt atgttcataa caatttttat lg TGAAGCCATT +166 +167

Exon 3

Exon

3

AAGAAACTGG gt atttatttgt cattcccttt ag CTGGAAGAGT ÷291 +292

Exon 4

Exon

4

TGCTAAGAAG gt gagtg1:gctc +473

cttttttaat ag AGAAAAATTG +4?4

Exon S

Exon

5

TTTAAGACTG gt aagtaa:gac gtccttttgc ag AGAGCGAGGG +598 +sgg

Exon 6

Exon

6

~L'I~I~G

Exon

7

GTGGGCG~G g t +801

Exon

8

TTTTTCTCAG g t +927

Exon

9

TAGCTGTGAT qt gaqtatgttg ttctcatttt ag CATGCTCCTT +1029 +1030

Exon 10

Exon I0

GAGATGGAAG gt atgtcaggc¢ ttctattttc ag GGTTCAGAAA +1113 +1114

Exon Ii

Exon 11

CCTCAAAGCA gt aggtttagtg gttctcagtt ag GCATCTGATT +1221 +1222

Exon 12

TTCATGGACG gt a a g c c g t t ~ +1352

aaactatttt ag CCCCGGTTGC +1353

Exon 13

Exon 13

TTCTGCCAGT gt ga~cattgga Ettgtgacac ag CTCCTTGAAC +147& +1477

Exon 14

Exon 14

CATGGAAAAG gt acccttgtta a~tttcttgc ag GATTTGACCG +ISSS +lSS6

Exon 15

Exon 15

TCrCCAGAAG gt a&tttgcGct tgtgcctcac ag TGGAGGAGGT +184% +1650

Exon 16

Exon 26

GAGATGACAG ~ aag¢ctactc tgtatttcac sg GTTTGTGGTG +I769 e277o

Exon 17

Exon

12

gt ctttaatcca +707

atcttccata

a g ACJU~CTGACA +708

Exon 7

taggagtccc

Cttgctcttt

a g GCJJ~GAGAAT +802

Exon 8

aacttttgtt

aatgttttat

a g GTCTTTGAAA +928

Exon 9

Fig. 4. Sequences at the exon-intron junctions for the COT gene. The junction positions are numbered with respect to the + 1 transcription initiation site.

S.J. Choi et al. / Biochimica et Biophysica Acta 1264 (1995) 215-222

220

5'

ACGT

A CGT 1 2

G G

3

vector

c

B

A

5'

+1 site

sequence

T

) AG A * ~

<95~94

<62 gDNA

G T G C A G A G A G C C

G T G C A G A G A G C C

A

A

A G

A G

C G G G T

C G G G T

C

3'

C cDNA

3'

Fig. 5. Primer extension analysis of the transcription start site of the COT gene. The primer extended product in panel A resulted from the 28-mer antisense oligonucleotide spanning positions + 62 to + 35 of the COT mRNA. For panel B, extension was initiated from a 30-met antisense primer ( + 95 to + 66). In panel A, both total liver RNAs (lane 1) and poly (A) enriched RNAs (lane 2) were used for primer extension. For the second antisense primer only total liver RNAs were used (lane 3). The sequences of the COT genomic DNA (gDNA) and eDNA are in complete agreement. The arrow marks the + 1 site. The lack of two terminal base residues at the 5' end of the COT eDNA accounts for the nonconformity in sequences between the genomic and eDNA clones at the two terminal bases. Several base residues from the vector sequence for both genomic and eDNA clones are also shown.

-1140

TATAACGTTATGGAGAATGTAGCACTTACATACTATGGAGGGGAAAAAAAGTTCTTTAGC

-1080

AGT CACTTTGACAAGCTTAGAGAGATCAGATTGATTATGAAACACCTCCa%GTAAGCTGTA

-I020

case with almost all class II genes, transcription o f the C O T g e n e initiates at the purine (G) residue.

CAGTCATAAAACATAGTAATTCTC~-AAGAAACAAGACTATTTACACCGAGTCCTTCAATG et

-960

A ~ ~ T A G ~ " T C I ~ ~1~~ G ~ I ~ T I ~ A ~ I ~ A ~ T F I ~ *e*

-900

AG~-iiTx'AATATACTAATTTGAAATGAAGTCTTTAAAt~I~ TGT C G A A ~ T G T

-840

AAAATA~'I-x'x*A*CCTTCCACGAAAATCAAACAGAAGAI~__IZTkAACTGTCTATAAGCTTA

~'~2~.AG

C

-780

~TTGT~

-720

TTGACCTCATAGTCTTCCTCTAGAACTTGCTAGCTTTCTGGATTAAAAATGCTACTTCTT

-660

AGACT~L~TTCTGGCCAGCTCAAAGTTTGAAGt~FI~AACATCGCTAGAGA

-600

CTCCAAGGGCTTGCAGCTGGC CAAATCTTAGTCT CAAAGAGG CGACGAGGCCAAACCGGA

C - ~ L T T T A ~C A I ~ C ~ T ~ T I ~ A ~ I ~ . A ~ r

C~C~ ' G ~

T~

-540

T CCATTCTGCTGCTATTCTCTTGCCAG CACACAGGGTAGACAAAATAAAATGTCTCAGTA

-480

CAACTC~GTAAAAATTCTTG CTTGGGCCAT~d~%AGGAGGCTCGCACTCGCAAGGGGGA

-420

GCGACTGGGAAGAAAGAACACAGCGCCAAGC~CCTAAGACTTCGGTAAAAAGCAGG CTGA

-160

G~G~GGG~CCTCCTTACCCAGCCA CAGGG CTGTGCCGGAGCGTGGGCAGAGCCGCCTAC

-300

AT~CCTAGG

-240

G~GAGCTAATCGGATTTTCTGCTCGCGTTCCTGGGGTCCGCAGC CTGGGGCGCAACC

~ T ~ T C C C G C A G C A T C CGGA~GGUGCTGCTGTAGCCGGGACT

-180

AGAGCCAGGGAGACCGG~ACCTGC~GACTCGACAGCCACCCCTTCCAGCG

-120

~G~I~GGACCCTCGCC~ C T T T C ~ T ~ . ~ A T C C C G T T T C C C G A C G C G C ~ L ~ e*eee

-60 +I +35

CCCTC~CGGCTTTCCGTTCTAGTTCGG~AACCCGGGT~ eQeet *Qe GAGTC~-A~&G~C.,t'TAAGCCC_.C.~TGeA~C~AG~ g a g g a t t ¢ t ~ t t g ~ t t t t a g

3.3. Nucleotide sequence and promoter function o f the 5' flanking region o f the C O T gene

TTTT

t~TACTGTGA~TATAt~_ATG~AAAAT

Fig. 6. The nucleotide sequence of the COT gene 5' flanking region. The continuously underlined sequence at the 3' end (positions + 1 to + 60) denotes exon 1, the first intron (in lower cases) and part of exon 2. The consensus sequences for various transcription factors are indicated in bold faces and their positions are described in the text. The five different repeat motifs are underlined with 1 through 5 asterisks underneath. Repeat for each motif is indicated by the same number of asterisks at the bottom of the underline. Interrupted underlining mark partial palindromes within a long stretch of DNA sequence.

T h e nucleotide s e q u e n c e o f the C O T g e n e p r o m o t e r up to the position - 1 1 4 0 is s h o w n in Fig. 6. The C O T p r o m o t e r lacks canonical T A T A e l e m e n t s w h i c h are generally f o u n d 20 to 30 bp u p s t r e a m f r o m the transcription initiation sites o f m a n y genes. H o w e v e r , potential binding sites for a n u m b e r o f transcription factors can be identified within this p r o m o t e r [41]. F o l l o w i n g is a representative list o f potential regulatory elements. T w o S p l binding sites (GGGCGGG) at p o s i t i o n s -160 and -68, two CTF/NF1/CBP binding C C A A T sites at positions - 284 and - 9 3 , two consensus A P 2 sites [41,42] at positions - 3 5 9 and - 2 5 , an O c t l site at - 4 5 1 , two P E A 3 sites, one at - 263 and the other at - 702 in the n e g a t i v e strand, two G A T A 1 sites, one at - 2 8 9 ( C C A A T C T C , c o n f o r m ing to M Y W A T C W Y , [43]) and the other at - 6 5 6 ( T G A T A A , [41]). Finally, the s e q u e n c e T A T T I ' G T w h i c h is the potential binding site for the hepatocyte nuclear factor H N F - 5 [44], is present in the n e g a t i v e strand at position - 7 9 8 . There are five different repeat s e q u e n c e motifs: T A A A C T G T at positions - 8 6 7 and - 7 9 9 , C T C C A G T A A at - 1 0 3 5 and - 4 7 7 , C T G C A A G A at - 9 9 9

S.J. Choi et al. /Biochimica et Biophysica Acta 1264 (1995) 215-222

221

Table 1 COT gene promoter activity in HepG2 cells (human hepatoma) for luciferase reporter expression Plasmid

Luciferase activity (mV/20/~1 extract)

Promoter strength (activity/mg protein)

Fold stimulation

Experiment 1: pl.2kbpCOT-Luc

1044, 1078, 812.8

92.6

pGL2-basic (promoterless)

8.4, 11.1, 15

474.5, 492.2, 388.9 mean: 451.9 3.6, 5.02, 6.02 mean: 4.88

Experiment 2: pl.2kbpCOT-Luc

1024, 1177, 1084

133.3

pGL2-basic

9.8, 3.3, 8.5

436.1,485.7, 510.2 mean: 477.3 4.55, 2.32, 3.86 mean: 3.58

Two different batches of plasmid preparations were used for experiment 1 and experiment 2. For each plasmid, transfection was performed in triplicates and the mean values were used to calculate fold stimulation of normalized luciferase activity in the presence of the COT promoter.

and - 1 5 1 , T T C A G G G C at - 9 4 1 and - 3 2 , and GCGCPuGGGPyGGGACCCTCPuCGGCTTTCCGT at - 1 2 3 and - 7 3 . In addition, palindromes of potential regulatory function are present from - 7 8 0 to - 7 6 6 (AAATTGTAACAATTT) and from - 1 5 to - 7 (ACCCGGGT). Partial palindromes are: CAATTTATTTCAAAAGGTAAATTAAAAAATGCATTGACCTGTGAAAATAAATTG ( - 7 7 1 to - 7 1 8 ) , GGCAGAGCCGCCTACATCTCTGGACCTAGGCCAATCTCCC ( - 3 1 5 to - 2 7 6 ) , and AGGCCAATCTCCGCAGCATCCGGAAGGAAGCTGCTGTAGCCGGGACT ( - 288 to - 2 4 1 ) . Partial dyad symmetries within the above-mentioned sequences are indicated by underlines. Interestingly, the palindrome at - 771 to - 718 contains the half site for the putative PPAR binding sequence (TGACCT, [45]). It will be important to determine if the - 7 7 1 / - 718 site does indeed include a binding sequence for the PPAR, a transcription regulatory protein of the steroid receptor superfamily [46]. The 1140 bp COT gene 5' flanking sequence displays a strong promoter function as determined from its ability to direct luciferase reporter gene expression from the plasmid pl.2kbp COT-Luc in transfected HepG2 cells. As shown in Table 1, luciferase expression driven by - 1140 bp of the COT promoter was found to be higher by approx. 100-fold, or more, compared to the level of reporter expression from the parent plasmid pGL2 basic, in the absence of any eukaryotic promoter. In conclusion, this report describes isolation and sequence characterization of the full-length cDNA for rat liver carnitine octanoyltransferase (COT). The cDNA deduced molecular weight is higher than the molecular weight determined from size analysis of the purified enzyme (70301 versus 66000). A mitochondrial targeting sequence for the COT protein at the N-terminal, that may be cleaved to yield the mature COT product targeted to mitochondria, may explain the discrepancy in the molecular weight. In addition, the peroxisomal targeting sequence Ala-His-Leu at the C-terminal is likely to account for

peroxisomal localization of the COT protein. We have also isolated the entire natural gene for COT and shown that the gene is split into 17 exons. All of the 16 exon-intron junctions follow the canonical GT-AG rule, with the 5'splice donor site of the intron starting with the sequence GT and the 3' splice acceptor site ending with the sequence AG. The COT gene sequence up to - 1 1 4 0 bp reveals a TATA-Iess promoter with consensus binding sites for a number of transcription factors such as S p l , C T F / N F I / C B P , AP2, OCT1, PEA3 and HNF5. Finally, the isolated COT 5' flanking sequence displays a strong promoter function in transfected liver cell lines and efficiently directs the expression of the downstream reporter gene. The TGACCT sequence at position - 7 3 7 , residing within a partial palindrome spanning positions - 7 7 1 to - 7 1 8 , represents a half site for the peroxisome proliferator response element that has been identified for several peroxisome proliferator-inducible genes [45,47]. Future investigations are directed to explore the regulatory significance of the TGACCT element, as well as other palindrome and repeat elements identified in the present study.

Acknowledgements We are grateful to Ms. Nyra White for secretarial assistance. A.K.R. is the recipient of a MERIT award from NIH (R37 DK-14744). B.C. is a VA career scientist and is the recipient of a VA Merit-Review support.

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