Cloning and characterization of a member of the rat multidrug resistance (mdr) gene family

Gene. 106 (1991) 229-236 ic) 1991 Elsevier

GENE

Science

Publishers

B.V. All rights reserved.

229

0378-l 119/91/$03.50

06069

of a member of the rat multidrug resistance (mdr) gene family

Cloning and characterization (P-glycoprotein;

recombinant

Jeffrey A. Silverman,

DNA;

PCR;

rodents;

mouse and human

genes; gt vector;

aflatoxin)

Hannu Raunio, Timothy W. Gant and Snorri S. Thorgeirsson

Laboratory of Experimental Carcinogenesis, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 (U.S.A.) Received by J. Piatigorsky: 19 May 1991 Revised/Accepted: 23 May/l0 June 1991 Received at publishers: 30 July 1991

SUMMARY

The rat mdr gene family [genes encoding P-glycoprotein (Pgp)] was characterized and the complete sequence of a rat mdr cDNA was determined based on seven independent cDNA clones that correspond to the same gene. The longest of these clones contains a 4.3-kb insert which represents a full-length rat mdr cDNA. The longest open reading frame of this sequence is 3933 bp; the first ATG is at 103 bp, making the deduced protein 1277 amino acids long (141 kDa). This correlates well with previously identified Pgp. The sequence of this gene has a very high, > 900/6, degree of identity to the mouse mdrlh gene (also known as the mdrl gene) therefore, we designate it the rat mdrlb gene. Transcription of this gene begins at a single start point 151 nucleotides upstream from the start codon. We show here that the rat gene family is comprised of three members, which is consistent with previous data on other rodent species.

INTRODUCTION

The mdr gene family is comprised of two members in humans, and three members in hamsters and mice (Endicott and Ling, 1989; Ng et al., 1989; Van der Bliek and Borst, 1989). Complete cDNA clones of the human and mouse mdr genes and partial clones of the hamster mdr genes have been isolated and characterized (Chen et al., 1986; Endicott et al., 1987; Gros et al., 1986a,b; 1988; Van der Bliek et al., 1988). The sequences of these genes are

Correspondence to: Dr. J.A. Silverman, Bethesda,

Tel. (301)496-0731; Abbreviations:

Fax (301)496-0734.

aa, amino acid(s); 2-AAF,

base pair(s); cDNA, encoding cytochrome

transcription

Pgp,

2-N-acetylamino-fluorene;

bp,

DNA complementary to RNA; c.vcP450, gene(s) P45Os; DMSO, dimethylsulfoxide; kb, kilobase

or 1000 bp; mdr, gene(s) deoxyribonucleotide; reaction;

Bldg. 37, Rm. 3C25, NCI, NIH,

MD 20892 (U.S.A)

encoding

Pgp; nt, nucleotide(s);

ORF, open reading

P-glycoprotein(s)

start point(s).

oligo, oligo-

frame; PCR, polymerase

(alternative

abbreviation:

Mdr);

chain rsp,

highly conserved which suggests a critical role for their protein products in normal cellular physiology. Analysis of these sequences indicates that Pgp is comprised of two very similar halves connected by a less well conserved linker region (Chen et al., 1986). Each half of the protein contains six putative membrane-spanning regions and one nt-binding site. Although the function of nt binding in drug resistance is unknown, its integrity is critical to Pgp function; metabolic inhibitors such as azide and vanadate decrease drug resistance (Horio et al., 1988; Van der Bliek and Borst, 1989). Further, mutation of one or both of these sites destroys the ability of the protein to pump out drugs (Azzaria et al., 1989). In addition to the high degree of sequence identity between mdr genes, there is also a significant amount of sequence homology between the mdr genes and other transport proteins. Noteworthy among these proteins are bacterial transport proteins such as HlyB, MalK, HisP and OppD (Chen et al., 1986). A structural similarity to the recently isolated cystic fibrosis transmembrane conductance regulator gene (CFTR) and Hum1 and Hum2 genes, which may be important in antigen presentation in cells, has

230 also been described (Deverson et al., 1990; Riordan et al., 1989; Spies et al., 1990). Although the coding regions of the mdr genes are highly conserved between species, there are significant sequence differences particularly in the flanking regulatory regions (Hsu et al., 1990; Raymond and Gros, 1990; Ueda et al., 1987b). In our previous investigations we have demonstrated a functional regulation of the rat mdr gene(s) (Burt and Thorgeirsson, 1988; Gant et al.. 1991; Thorgcirsson et al., 1987). Increased r& expression was observed in rat livers following surgical partial hepatectomy and after administration of carcinogens. Further, exposure of isolated rat hepatocytes in primary culture to xcnobiotics results in increased levels of I~M/TmRNA and Pgp. To further investigate the specific mechanisms of regulation of thcnrdrgenes by xenobiotics, it was necessary to isolate and characterize members of the rat mdr gene family. In this investigation we describe the cloning and characterization of a member of the rat mdr gene family. This rat gene has a high degree of homology to the mouse mdrlh gene (also known as mdrl ; see below for a discussion of mdr gene family nomenclature) therefore, we designated it the rat rndrlh gene. We further demonstrate that the rat rlldr gene family is comprised of three members, as are the other rodent species examined to date.

RESULTS

AND

DISCUSSION

(a) Isolation and characterization of a rat mdr cDNA We initially screened a 1.gtlO phagc cDNA library that was constructed using mRNA isolated from the liver of a rat which was previously treated with aflatoxin Bl. Three clones, RDR2b13, 14, 15, were first identified which hybridized to the human pH DRSA plasmid. The largest of these clones. RDR-14, was approximately 3.2 kb (Fig. 1). The inserts of these clones were partially sequenced and found to correspond to the same gene. We tentatively identified this rat gene by searching the GenBank database with the FASTA program and using the Besttit program for an optimal alignment (Devereux et al., 1984). The highest degree of similarity was to the 3’ end of the human MDRl gene (data not shown). To obtain clones which contained the 5’ end of the gene we rescreened the cDNA library with a rat mdr probe corresponding to this region of the gene. This probe, 830 (Fig. l), was obtained by PCR amplification of a rat cDNA pool that was constructed with poly(A) + mRNA isolated from primary rat hepatocytes which were treated with cycloheximide. We have previously shown that treatment of rat hepatocytes in vitro with cycloheximide increases the expression of mdr RNA (Gant et al., 1991). This cDNA pool was PCR-amplified to obtain a rat DNA fragment

CLONES RDR15 RDR2bl3 RDR14 RDRB4 RDRBl ATG

Rat m&lb 3 I

I

PROBES

I-l

I

(No.)

Rat

830

3594

-

pHDR5A

Human Fig. I. Structure

of nrdr cDNA

clones and probes.

Five cDNA

corresponding

to the same rat mdr gene were isolated

cDNA

Also shown

library.

aligned

clones

from a rat liver

to the clones is a schematic

of the

full-length rat m&/b gene; the ATG start codon and TGA stop codon are marked. Three probes used in the library screening and Southern-blot analysis fragments, described

are aligned PCR

below the gene. Probes

amplified

from

rat

RNA

830 and 3594 are DNA (830) or DNA

in sections a and b. The human MDRI plasmid,

a gift from M.M. Gottesman

and has been previously

(3594)

pHDRSA, described

as was

(Chen

et al., 1986). B, Bg/II; P, Psri; S. SncI.

corresponding to nt position 830-2 182 in the human A4DRl gene which is farther upstream than any of the isolated rat cDNA clones. Screening of the cDNA library with this probe resulted in the identification of four additional clones. Upon subsequent sequence analysis, two of these clones were identified as the same gene as the previously isolated clones (Fig. 1). The largest of these new clones, RDR-Bl, was approx. 4.3 kb long (Fig. 1). The sequence of RDR-Bl is presented in Fig. 2. This clone contains 4254 nt with the longest ORF of 3933 nt. The first ATG in this ORF is 103 nt from the beginning of this clone. The assignment of this position as the first aa in the encoded protein is based on a comparison to the mouse and human mdrl genes. It is noteworthy that, unlike the mouse mdrl gene, there is no purine 3 nt upstream from this site to correspond with the eukaryotic initiator consensus sequence of Kozak (1987); there is, however, a G at position + 4 which has also been indicated to be important for eukaryotic initiation and may compensate for the lack of a purinc at nt position -3. There are multiple stop codons in both alternate reading frames such that no peptide over 75 aa would be translated. Starting at the ATG at nt position 103, the ORF would contain 3831 nt encoding a protein containing 1277 aa (141236 Da). The first stop codon, TGA, encountered in this reading frame is at position 3832; it is followed by a 321-bp noncoding region.

GCTCCCATCT TCGAGGCTCA GCTCMCTCA

GAGCTACTTC TTCCAAATTC

-!EF CTTAACGGM L N G

GAGCAGACM R A 0

CTGTGCATGG

EEG

6

AAAAGAGTAA AAAGGAGAAG GAGAAGAMC K K S K K E K E K K

CTGCTGTTGG CATATTCGGG ATGTTTCGCT P A Y G I F G N F R

ATGCAGATTG GCTTGACAAG Y AD Y LDK

CTCTGGGAAC TCTCGCTGCT ATCATCCACG A L G T L A A 1 I H

GAACCCTGCT

TCGGATACAT GACAGATAGT TTTACCCCM F G Y N T D S F T P

GCAGAGACCC

CGAGCGATTA R Al

CTAATCMAG T N Q

CCGTCAGCGA CACGAGTCTG GAGGAGGACA T V S D EED T S L

TGGCCATGTA HAHY

TGCCTACTAT AYY

TTGGTGCCGG

I

126

GTTGCCTACA V A Y

TCCAGGTTTC ACTTTGGTGC CTGGCAGCTG 10 v s L Y c L A A

GGAGACAAAT 6 R 0

TTTTCCATGC F F HA

CATCATGAAT CAGGAGATAG IM N 9 E I

GCTGGTTTGA CGTGAATGAC G Y F D V N D

498 166

GClGGGGAGC A G E

TCAACACCCG TCTCACAGAT GACGTCTCCA L N T R L T D D" S

AAATTAATGA CGGAATTGGT GACAAACTTG K I N D G I G D K L

GAATGTTCTT TCAGTCCATA ACGACATTTT G N F F T T F 0 S 1

CAGCCGGTTT TATAATAGGA S A G F I I G

618 206

TTTATAAGTG F I s

GTTGGAAGCT AACCCTTGTA ATTTTGGCCG G Y K L T L V IL A

TCAGCCCTCT TATTGGGTTG 1CATCTGCCA Y s P L I G L S 5 A

TGTGGGCAAA "YAK

GGTACTGACT TCATTTACTA V L T S F T

ATAAGGAACT CCAGGCTTAT N K E L 0 A Y

738 246

GCGAAAGCTG A K A

GAGCAGTTGC CGAAGAAGTC TTAGCAGCCA G A V A E E V L A A

TCAGMCTGT I R T

GATTGCGTTT CGAGGACAAA I A F G G 9

AGMGGMCT K K E

TGAMGGTAC MTAAMATT N K N E R Y

TAGMGMGC L E E

858 286

GGCATAAAGA

MGCCATCAC

TTGCCTACCT

GTTGGTCTAT

CACTGGCATT

CTGGTATGGG

TCCTCTCAA,, TGAATATTCT

L

c

n

GIK

G

9

ATAATTGATA I

TGAMTCAAC S E IN

ID

AGTACACATA S T H

GGCCMCATT

KAIT

ATTGGACAAG 1

GAACTTCTCA MGATGGGCA K N F 5 K" G

18

TACATCTTGG CGGACTTCGC GAAGGAAACC CGGAGTGTTA CGTGAGGTCC TGATGGAGTT TGAAGAGGX

A

N

TCCATAGGTA 1

SIG

G

I

T

A

L

Y

TCCCCTCCTG L

P

L

ATGCTGGTGT L

H

L

v

ACACMGATT AGGCAGAAGT I H K I R 0 K

V

L

L

v

GCGTCTTATG Y

AS

Y

A

L

A

L

F

U

Y

TACACGGGCA Y

T

G

ACCTCCTTGG G

T

S

L

5

II 0

IG

V

A

L

S

GCATTCTGAC P

H

s

258 0

TGTGCTCATC G

v

L

N

E

Y

86 378

TAAMGAGTT K R V

A

978 5

326

TGCTTACCGT CTTCTTCTCT ATTTTATTGG V L T V F F S I L L

GGACTTTCAG TATTGGACAT TTAGCCCCAA G T F S I G H L A P

ACATAGAAGC CTTTGCAAAT GCAAGAGGGG N I E A F A N AR G

CAGCCTATGA AATCTTCAAG A A Y E I F K

1098 366

ATGAGCCAAG

AGGGACACM

ATTTGGMTT

ACCCATCACG

V

1218 406

N

C

CATCGACAGC

P

S

MGATCTTGA K IL

AGGGCCTCM KG L

N

GAGGGCGAGG E Gt

TCAGTATTGA V S ID

ATTCGCTATG IK Y (IGpACAF

ID

TTCTCMCCA s

F

S

T

KG

H

ATAATG6G.U S

G

L

E

TMAAATGTT F

K

N

TACTTCAACT V

Y

F

N

Y

P

S

AAGTGAAGTT R

S

E

CGACCCCATA D P I

1338 446

TCAATGTGAG GTATCTGCGG GAMTCATTG IN V R Y L R E I I

GGGTGGTGAG GVVS

TCAGGAACCC QEP

GTGCTGTTTG VLF

CCACCACGAT ATT,

TGCCGAAPAC A E N

1458 486

GCCGAGAAAA CGTCACCATG GATGAGATAG V T ,, G R E N 0 E I

AGAAAGCTGT CMGGMGCC E K A V K E

MTGCCTATG N A Y

ACTTCATCAT GAAACTGCCC CACAAATTTG D F I " H K F K L I'

ACACCCTGGT TGGTGAGAGA 0 T L V G E R

1578 526

TysGG;GGGACAGAAACAG QKQ

AGGATCGCCA RIA

TTGCCCGGGC CCTGGTCCGC AACCCCMGA IA A A L v If N P K

TCCTTTTGTT GGATGAGGCC ACGTCAGCCT I L L L DE A T S A

TGGACACAGA AAGCGAAGCC L D T E S E A

1698 566

CCGCTCTGGA TMGGCTAGA GMGGCCGGA AA L D K A R E G R

CCACCATTGT GATAGCTCAC CGCTTGTCTA T T I V IA H R L S

CAGTGCGCAA TGCTGACGTC ATTGCTGGTT T V R N A D V IA G

TTGATGGTGG TGTCATTGTG F 0 G G v I v

1818 606

ATCATGAAGA N HtE

GCTCATGMA GAGAAGGGCA L" K E KG

TTTACTTCM IY F

K

ACTTGTCATG ACACAGACTA L v n T 0 T

GAGGAAATGA MTTGAACCA GGAMTMTG R G N E IE P G N N

CTTATGMTC A Y E

S

D

1938 646

ACTGGTGCCT T G A

CTGAGIIGAC TTCAGAAGM TCAA4ATCTC S E L T S E E 5 K 5

CTTTMTAAG P L I

R

GAGATCMTT CGCAGAAGTA R S I R R S

TCCACAGAAG ACAAGACCAG GAGAGAAGAC I H R R ERR 0 D 0

TTAGTTCGM L S S

K

AGAGGATGTG E D V

20% 686

GATGAAGAIG D E 0

TGCCTAIGGT TTCCTTTTGG v P H v SFU

AGCTAAATAT KLNI

TAGTGAATGG CCCTATTTAG S E U P Y L

TTGlGGGTGl ACTTTGlGCT GTTATAAATG V V G V L C A V IN

GGTGCATACA G C ID

ACCAGTGTTT P V F

2178 726

GCCATAGTGT AlV

TTTCAMGAT F S K

GTMCTTGTT C N L

F

TTCCCTTCTC TTTCTGGTCA s L L F L V

TGGGMTGAT H G HI

TTCTTTTGTT 5 F v

2298 766

1CTICAMlC V F K

S

CAIGCTGCGA CAGGATATAA H L R Q D I

GCTGGTTTGA S Y F"

TGACCATAAA D H K

2418 606

a

GAGCAAGGAA E

9

6

I

9

D

ATCAGGACCA 1 R T

CAGATCCTM QIL

TGTAGGGGTT TTTTCMGAG V G V F S R

ACGTACTTCl T Y F

TTCAAGGCTT CACATTTGGC AAAGCTGGAG F Q G F T F G K A G

AAYF"";"

G;TC~Tr,TTAC~~;

"":"";&A"," CTTCTMTGT A S N

V

2538 846

ClCATTGTCT L IV

TGGGTGGAAT TATTGAAATG AAACTGTTGT L G G I I E H K L L

CTGGTCAAGC CTTGMGGAC S G 9 A L K D

2658 686

CTTCCGCACT GTTGTCTCTT F R T v v s

TGACTCGG6A GCAGAAGTTT GAAACTATGT E T H L T R E P K F

ATGCCCAGAG CTTGCAGATA Y A 9 5 L 9 I

2778 926

CTTCACCCAG

ATTTTTCCTA TGCTGCTTGT Y F S Y A AC

GTGCCTACTT GAYL

2898

TAGAGATCTC LEIS

CMTTWAAA A I E

R

ATGCTTTGM N A L

CCATACAGM P

Y

K

GAAAGCACAC GTCTTTGGGA K A H V F G

TCACCTTCGC 1TFA

S

ACCTTGGCAC AGGMTTATC N L G T G I I

E

N

0

GGCTTGCTGT AGTTACCCAG AATGTAGCAA N V A R LA V V T Q

MGAAAGAGC

GCTACAGAAG A T E

CCAAAGTGAC

ATGGGCTCCA N G S

TAAAGGGGCT K GA

TACTTGTAGT MTTAIACCA L L v v I IP

TGGGAAGATC GKI

CAACGGMTT QRN

AGATCCTCAC CMGCGACTC CGATACAIGG R Y N E I L T K R L

TCTTAGTCTA TGGCTGGCAG CTTACACTTT v L v Y L T L G U 0

K

A

ACGACGACCA TGAAACCAM D D 0 H tTK

llAlCCTTAG L 5 L

K

MCAGTGGCT NSG

N

AGAGGCTCTA QRLY

G

CCTGGTTGGC LVG

IN

CAGCTGCTGC QLL

V

CGGACAGGAC

0

CACMCTGTC TTY

V

AGACGGTAGC DTVA

P

GTGGGMAAG CGKS

GTGGTTCACG

CCTGMGGTG MGAGCGGGC KSG L K V

ACCAGACAGT K

F

T

9

GCCATGATTT A H I

TTCCGGTTCG FRF

GGTGGCACGA V AR

966

GMCTCATGA E L H

CGTTTGAAAA TGTTATGTTG GTATTTTCTG T F E N v n L V F 5

CTGTTGTCTT TGGTGCCATG GCAGCAGGGA A V V F G A H A A G

ATACCAGTTC ATTCGCTCCT GACTACGCGA N T S S F A P D Y A

AGGCCAAAGT CTCGGCATCC K A K V 5 A 5

3018 1006

CACATCATTG H I 1

GGATCATTGA GMAATCCCC GAGATIGACA K I P E ID G I I E

GCTACAGCAC GGAGGGCTTG MGCCTMTT K P N S Y S T E G L

GGTTAGMGG U L E

AAATGTGAAA TTTAATGGAG N V K F N G

TCAAGTTCAA CTATCCCACC Y P T V K F N

3138 1046

CGACCCAACA R P N

TCCCAGTGCT TCAGGGACTG AGCTTCGAGG I P v L S F E 0 G L

TGAAGAAGGG VKKG

GCAGACGCTC DTL

GCAGCAGTGG GSSG

CTGCGGGAAG CGK

TCCAGCTGCT VPLL

3258 1086

TACMCCCCA Y N P

TGGCTGGMC NAG

AAATAAAGCA ETKD

ACTCMTGTC CAGTGCGTCC L N V PC V

GCCGAGAACA A E N

TCGCCTACGG AGACMCAGC CGTGTCGTGT I A Y G 0 N S R V V

AGAGTGGGAG R V G

ACAMGGGAC DKGT

TCAGCTGTCG QLS

ACGGAGAGTG T E S

AAAAGGTCGT EKVV

CCAGGAAGCG PEA

AACGGCCAGG

AGCACGGTW; STY

CGAGCGCTTC E R F

GCGCACTGGG CATTGTGTCC CAGGAGCCCA I v 5 R A L G D E P

TCCTGTTTGA CTGCAGCATC I L F D c s 1

,126

CTCATGAGGA GATCGTGAGG GCCGCCAGGG 5 H E E 1 V R A AR

AGGCCMCAT E AN

CCACCAGTTC ATCGACTCAC IDS 1 HP F

TGCCTGAGAA ATACMCACC Y N T L P E K

3498 1166

GGCGGGCAGA GGQ

AGCAGCGCAT KQRI

CGCCATCGCG CGCGCCCTCG AI A R A L

TCAGACAGCC TCACATCTTA CTTCTGGATG H I L L L D V R q P

AAGCGACATC AGCTCTGWIT E AT 5 AL 0

3618 1206

CTGGACMAG LDK

CCAGGGAAGG AREG

CCGCACCTGC GTTGTGATCG R T C v v I

CGCACCGCCT GTCCACCATC CAGAACGCAG A H R L S T I P N A

ACTTGATCGT GGTGATTCAG D L I V V I9

37.38 1246

TCAAGGAGCA UXCACCCAC V K E H GTH

CAGCAGCTGC DQL

TGGCCCAGAA AGGCATCTAT TTCTCGATGG G I Y F S H L A Q K

TTCAGGCTGG AGCAMGCGC V Q A G AKR

TCATGAGCTG S-

GGAGTATTTG

AGGTGCTAAG

3858 1277

TATTTCTAAT

ATTGGTGTTC

AAACATGGCA

CGTAACCAM

GTTAAAAGGT

TAAAAGCACT

GTTAMGGTA

ATTTCATCM

GACG%AAGC

CTTCAGAGAC

TTCATPATTA

AATGAACCGA

3978

,UTTG&UM

AAAATCATTA

AACAGGGCCA

CATTTTTTM

TTGTATTATG

TGATTCMGA

GAACATATAG

TTTTTTTAAA

AAGAAATGTG

TAGTTTTGTT

TCAGTTTTTT

TAATTTCTAC

4098

CCTATTCCCT

TAAATGATCA

TAMGGCTGT

AMMGCACT

ATTTTTTTGC

GGC

N

Fig. 2. Nucleotide

G

9

sequence

T

AGTGTTTCTA GATGGCAAAG V F L D G K

CGCCTGGTGG RLV

G

of the rat mdrlb

cDNA

and aa sequence.

231

138 46

4151

The nt sequence

is numbered

in a 5’-to-3’

orientation

starting

at the ATG start

codon and ending at the first in-frame stop codon (bold underline). The deduced aa sequence is shown below the nt sequence. The aa symbols are aligned with the third nt of each codon. The aa corresponding to the nt-binding site motif A and ATP-binding active transporter consensus sequence are underlined. This sequence has been submitted to the GenBank database under accession No. M62425.

232 TABLE

I

Comparison

of the rat, mouse,

hamster

and human

mdr/MDR gene sequences

Human

Genes”

Mouse

MDRl

Hamster

MDR2

mdrla

mdrl b

mdr2

mdrla

79.1

71.5

82.5

90.4

69.6

78.1

80.4

75.1

47.7

76.8

83.4

77.9

82.0

100.0

100.0

nldrlb

Yb nt homology” Rat mdrlh Hamster Hamster

mdrlb’ ipgp2J mdrla’ (pgpl)

83.5

64.2

89.3

17.9

65.0

Mouse mdr2

71.1

86.1

71.1

69.4

100.0

Mouse mdrlb (mdrl)

78.7

70.6

84.2

100.0

Mouse mdrla (mdr3) Human MDR2 (MDR3)

82.2

71.6

100.0

14.9

100.0

“ Alternative h Comparison PCGene accession

gene designations of the rat, mouse,

package numbers

are in parentheses. hamster

(Intelligenetics, of the sequences

and human

mdr/MDR gene sequences.

Palo Alto, CA). The complete used for this comparison

sequences

The y0 identity

of these genes, coding

are as follows: hamster

was calculated and noncoding,

mdrla, Ml7897 (Endicott

using the NALIGN et al., 1987); hamster

(Endicott et al., 1987); human MDRl, Ml4758 (Chen et al., 1986); humanMDR2, M23234 (Van der Bliek et al., 1988); 1990); mouse mdrlb Ml4757 (Gros et al., 1986a). and mouse mdr2, JO3398 (Gros et al., 1988). L Note that the hamster

mdrla and mdrlb sequences

program

was used for this comparison,

of the The

mdrlb, Ml7896

mouse mdrla, M33581 (Hsu et al.,

are incomplete.

is an imperfect polyadenylation signal, AATTAAA, at position 3964. It is unlikely, however, that this represents the authentic signal which is probably not contained in this clone. The predicted size of the rat Pgp is in good agreement with previously described Pgps from other species (Endicott and Ling, 1989). Comparison of this rat mdr gene with the other known mdr sequences reveals a high degree of sequence conservation. The homology of this clone to the other mammalian mdr genes is shown in Table I. The entire available sequences of these genes, coding and noncoding regions, was used for this comparison (Chen et al., 1986; Devault and Gros, 1990; Endicott et al., 1987; Gros et al., 1986a; 1988; Van der Bliek et al., 1988). This table lists also the alternate names by which certain mdr genes are known. These genes were originally named as they were isolated. There

However, a functional nomenclature system has recently been proposed which divides these genes into those that are associated with drug resistance, class 1 genes, and those not yet associated with drug resistance, class 2 genes (Scotto et al., 1986). This nomenclature system also facilitates cross species comparison. In mice and hamsters two genes, mdrla and mdrlb, have been associated with drug resistance which corresponds to a single gene in humans. All three species also have a highly homologous gene, mdr2, which has yet to be ascribed a function in drug resistance. It is possible that the protein product of these class-2 genes transports yet unidentified substrates in a similar fashion to Pgp. The highest degree of similarity of the rat RDRBI cDNA is to the mouse mdrlb gene (also referred to as the mdrl

gene) with an overall identity of 90.4 y0 (Table I). Thus, we have designated the clone presented here the rat mdrlb gene. Clearly, the mdr genes are highly conserved between species but as indicated in Table I the rodent genes have a higher degree of homology to each other than to the human gene. This indicates sequence divergence since the speciation of rodents from primates. The least identical region of the rat mdrl b gene from the mouse mdrl b gene is in the 3’-untranslated region with a homology of 69:; (data not shown). The predicted protein of this rat mdr gene contains the important structural features identified in the Mdr proteins from other species (Endicott and Ling, 1989; Gottesman and Pastan, 1988). The bilateral symmetry of the molecule with each half containing several membrane spanning regions and one nt-binding region is evident in the predicted protein of this gene. A hydropathic plot for the rat Mdr 1b protein demonstrates a near-identical plot to that of the mouse Mdrlb (Fig. 3) and human MDRl proteins (data not shown). The presence of six membrane-spanning domains indicates that these proteins are integral membrane proteins. Such a localization has been previously confirmed using epitope-specific antibodies to various domains of Pgp (Yoshimura et al., 1989). The sequences surrounding the nt-binding regions are particularly conserved between species and are characteristic of the ATPbinding cassette family of active transport proteins. This has led to the hypothesis that the Pgps are members of this superfamily of membrane transport proteins which now includes over 30 members (Hyde et al., 1990). Members of this superfamily of transport proteins are

233

1

-5e-,,,,,,,,, 1

Fig. 3. The hydropathy computer

program

are negative

400

ZBB

,,,,,,,,, 208

bat3

,I,,,,,,, 480

which implements

along the ordinate.

The numbers

,,,,I,,,, 888

The hydropathy

the analysis method of Kyte and Doolittle along the abscissa

represent

found in both prokaryotes and eukaryotes and are thought to be important in ATP-dependent transport of a diverse range of compounds. These proteins include the bacterial transporters HlyB, which is involved in the movement of hemolysin across the bacterial membrane, and MalK and HisP, which are important in metabolite transport. In yeast, the STE6 gene is important in the transport of mating factor a and is a member of this family (McGrath and Varshavsky, 1989). The recently isolated cystic fibrosis transmembrane conductance regulator gene and the HAM1 and HAM2 genes, which may be important in the antigen recognition system of the MHC locus are also members of this gene superfamily (Deverson et al., 1990; Riordan et al., 1989; Trowsdale et al., 1990). The high degree of evolutionary conservation of this sequence motif indicates an important

iem3

,,,,,,,/, 608

plot for the rat (A) and mouse (B) Mdrl b proteins.

package

808

m38

III,,, leaa

plots were obtained (1982). Hydrophobic

the aa positions

aa

,I,,,Tr lzaa

aa

using the SOAP program

in the PCGENE

regions are positive and hydrophilic

regions

in the proteins.

role in normal cellular physiology and possibly common ancestral gene (see section c).

indicates

a

(b) Mapping of the transcription start point (tsp) Primer extension analysis was used to map the distance of the tsp from the start of translation. A 25mer oligo corresponding to nt positions 17-41 relative to the ATG start codon was used as a primer for the reverse transcription. A 192-nt extension product was observed in RNA extracted from isolated hepatocytes maintained in primary culture (Fig. 4, lane 1). We also observed an identical size extension fragment in RNA isolated from hepatocytes treated with 2-AAF and from the livers of rats previously treated with that agent (lanes 2 and 3, respectively). The greater intensity of the band in the drug-treated

234

1

23

1

GATC

2

3

4

5

6

151 nt-

Fig. 5. Southernblot

analysts

DNA was digested

with BarnHI

lowing electrophoresis Magnagraph Fig. 4. Primer extension

analysis to determine

was hybridized

to a “P-labeled

myeloblastosis

virus

reaction urea

gel and

(DMSO 2-AAF

products

treated

ladder (G,A,T,C)

oligo probe and transcribed transcriptase

were electrophoresed

then

treated)

reverse visualized

hepatocytes;

the tsp. Total RNA (50 ng) (Archer

et al., 3985). The

in an So, polyacrylamide/7

by autoradiography. 2,2-AAF

with avian

treated

Lanes:

M

rat liver. A dideoxy

was used to measure

the size of the extension

3

-

2

1)N.A. Gcnonuc

(lanes 1-3) or EcoRI (lanes 4-6). Fnlin OX?,, agarosc gels the DNA was transferred to

membranes

(Micron

Separations.

to a single exon wdr probe,

Inc.. Westboro, 3594, described

in

section E. Lanes: I and 4, rat DNA; 2 and 5, mouse DNA; 3 and 6, human DNA. .4 1-kb ladder margin.

(BRL, Gaithersburg,

MD) is marked

on the right

1. control

(20 PM) hepatocytes;

(five days at 20 mg/kg)

nylon

MA) and hybridized

of rat, mouse and human

-

3.

sequencing products.

samples indicates that this agent increased the expression of the mdrlb gene in the liver. These data support our previous observation of enhanced mdr expression with this agent (Burt and Thorgeirsson, 1988; Gant et al., 1991). These data further show that the tsp is 15 1 nt 5’ to the start codon. No difference in the tsp was observed between the RNA isolated from the rat liver and the RNA from the isolated hepatocytes. Thus, the rat 5’-untranslated region is similar in length to that observed for the mouse mdvl b gene (Raymond and Gros, 1989). Also indicated by these data is that the rat gene, like the mouse mdrlb gene, has a unique t.rp. This is in contrast to the human MDRl and murine

mdrlo genes for which two tsp have been described in drug-resistant and normal cell lines (Hsu et al., 1990): Ueda et al., 1987a,b). Isolation and characterization of a genomic clone containing the upstream sequences of this rat gene will permit us to further characterize this regulatory region.

(c) Identification of the members of the rat mdv gene family Previous investigations have demonstrated that the mdr gene family consists of two genes in humans and other primates and three genes in mice and hamsters, however, no data are yet available on the rat gene family (Chin et al.. 1989; Ng et al., 1989). To identify the number of genes in rats, genomic DNA was isolated from rat liver, digested with the indicated restriction endonuclease and examined

235 by Southern-blot analysis. DNAs were also analyzed analysis. obtained

Human and mouse genomic to serve as controls for the

The probe used was a 200-bp rat gene fragment by PCR that corresponds to exon 28 based on the

human and mouse mdrl genes (Fig. 1) (Chen et al., 1990; Raymond and Gros, 1989). Upon digestion with each BamHI and EcoRI we observed three restriction fragments in the rat DNA samples (Fig. 5, lanes 1 and 4). Similar results were obtained with XbaI and Hind111 (data not shown). We also observed three restriction fragments in the mouse (lanes 2 and 5) and two fragments in the human DNA samples (lanes 3, and 6). This confirms previous investigations which examined the number of genes in mice and humans (Chin et al., 1989; Ng et al., 1989). Together these data suggest that these rodent species have three members of the mdr gene family whereas primates have two. The presence of three genes in rats, mice and hamsters, while primates only have two, indicates that the third gene may have arisen after the divergence of rodents from primates. Of the two rodent genes associated with drug resistance the mdrl a gene has been suggested to be more closely associated with the human MDRI gene (Chin et al., 1989). The close homology of the sequence of mdrlb to that of mdrlu further indicates that this third gene may have resulted from a later gene duplication. Previous work by us and others demonstrates that mdr genes are co-regulated with other genes involved in the protection of cells by xenobiotics, e.g., members of the cycP4.50 gene family (Burt and Thorgeirsson, 1988; Fairchild et al., 1987). For example, exposure of isolated hepatocytes to 2-AAF or 3-methylcholanthrene causes a significant increase in the expression of mdrlb and cycP450-IA mRNAs and in Pgp levels (Gant et al., 1991). This increased expression is, at least in part, mediated by increased transcription of the mdr gene(s) (T.W.G., J.A.S., S.S.T., in preparation). The isolation and characterization of the rat mdr gene presented here and the yet to be cloned other members of the rat gene family will permit further investigation of the regulatory mechanisms of these genes in this important model system. (d) Conclusions (1) A cDNA encoding a complete rat mdr gene has been isolated and characterized. This gene has a high degree of sequence identity to the mouse mdrlb gene, thus it was designated the rat mdrlb gene. (2) The tsp for this gene is 15 1 nt upstream from the start codon. (3) The rat mdr gene family has three members. This is similar to the mouse and hamster gene families but in contrast to the human gene family which has two members.

ACKNOWLEDGEMENTS

We

acknowledge

Dr.

P.A.

Marino

and

Dr.

M.M.

Gottesman for their comments on this manuscript and A.D. Olson for his assistance with the computer analyses.

REFERENCES Archer,

T.K., Tam, S.P., Deugau,

K.V. and Deeley, R.G.: Apolipoprotein

CII mRNA levels in primate hepatocarcinoma

cell

liver. Induction

line.

HepG2.

by estrogen

J. Biol.

in the human

Chem.

260 (1985)

1676-1681. Azzaria,

M., Shurr, E. and Gros, P.: Discrete mutations

predicted

to confer multidrug Burt,

resistance.

R.K. and Thorgeirsson,

resistance

of MDR-1 multidrug

P-450 genes in rat liver by xenobiotics.

J.

Inst. 80 (1988) 1381-1386.

Chen, C., Chin, J.E., Ueda, K., Clark, D.P., Pastan, and Roninson,

LB.: Internal

duplication

proteins

cells. Cell 47 (1986) 381-389.

Chen, C., Clark, D., Ueda, Roninson, LB.: Genomic (MDRI)

I., Gottesman,

and homology

in the mdrl (P-glycoprotein)

transport resistant

resistance

in the

Mol. Cell. Biol. 9 (1989) 5289-5297.

S.S.: Coinduction

and cytochrome

Natl. Cancer

introduced

sites of the m&l gene abolish its ability

nucleotide-binding

K., Pastan, organization

M.M.

with bacterial

gene from multidrug-

I., Gottesman, M.M. and of the human multidrug

gene and origin of P-glycoproteins.

J. Biol. Chem.

265 (1990) 506-514. Chin, J.E., SoBir, R., Noonan,

K.E., Choi, K. and Roninson,

I.B.: Struc-

of the human MDR (P-glycoprotein)

ture and expression

gene family.

Mol. Cell. Biol. 9 (1989) 3808-3820. Devault,

A. and Gros,

confer multidrug

of the mouse mdr gene family

P.: Two members

resistance

with overlapping

but distinct drug specili-

cities. Mol. Cell. Biol. 10 (1990) 1652-1663. Devereux,

J., Haeberli,

sequence

P. and Smithies,

analysis

programs

0.: A comprehensive

for the VAX. Nucleic

Acids

set of Res. 12

(1984) 387-395. Deverson, E.V., Gow, I.R., Coadwell, J., Monaco, J.J., Butcher, G.W. and Howard, J.C.: MHC class II region encoding proteins related to the multidrug

resistance

family of transmembrane

transporters.

Nature

348 (1990) 738-741. Endicott, J.A. and Ling, V.: The biochemistry of P-glycoprotein mediated multidrug resistance. Annu. Rev. Biochem. 58 (1989) 137-171. Endicott,

J.A., Juranka,

P.F., Sarangi,

and Ling, V.: Simultaneous in drug-sensitive

F., Gerlach,

expression

Chinese hamster

J.H., Deuchars,

of two P-glycoprotein

K.L. genes

ovary cells. Mol. Cell. Biol. 7 (1987)

4075-4081. Fairchild,

CR.,

Ivy, S.P., Rushmore,

T., Lee, G., Koo, P., Goldsmith,

M.E., Myers, C., Farber, E. and Cowan, K.H.: Carcinogen-induced mdr overexpression is associated with xenobiotic resistance in rat preneoplastic liver nodules and hepatocellular Nat]. Acad. Sci. USA 84 (1987) 7701-7705. Cant,

T.W., Silverman,

and Thorgeirsson,

Carcin. Gottesman,

Proc.

J.A., Bisgaard,

H.C., Burt, R.K., Marino,

P.A.

S.S.: Regulation

of 2-acetylaminofluorene

and

3-methylcholanthrene family expression

carcinomas.

induction in primary

ofmdr and cytochrome

hepatocyte

cultures

P45OIA gene

and rat liver. Mol.

(1991) in press. M.M. and Pastan,

I.: The multidrug

transporter,

edged sword. J. Biol. Chem. 263 (1988) 12163-12166. Gros, P., Croop, J. and Housman, D.: Mammalian multidrug gene: complete rial transport

cDNA proteins.

sequence

indicates

Cell 47 (1986a)

strong homology 371-380.

a double resistance to bacte-

236 Gros, P., Neriah, Y.B., Croop, J.M. and Housman, D.E.: Isolation and expression of a complementary DNA that confers multidrug resistance. Nature

323 (1986b) 728-731.

Cell. Biol. 8 (1988) 2770-2778. Horio, M., Gottesman, M.M. and Pastan, I.: ATP-dependent of vinblastine in vesicles from human multidrug-resistant

transport cells. Proc.

Hsu, S.I.-H., Cohen, D., Kirschner, L.S., Lothstein, L., Hartstein, M. and Horwitz, S.B.: Structural analysis of the mouse mdrla (P-glycoprotein) promoter

reveals

the basis for differential

transcript

hetero-

5774.2 cells. Mol. Cell. Biol. 10 (1990)

3596-3606.

fibrosis,

model ofATP-binding

multidrug

resistance

proteins

and bacterial

associated transport.

with cystic Nature

346

(1990) 362-365.

Kyte, J. and Doolittle, thic character

R.F.: A simple method

of a protein.

for displaying

the hydropa-

1224-1232. M. and

correlation cation

D., Blanck,

G., Mellins,

complex class II region controlling the class 1 antigen pathway. Nature 348 (1990) 744-747.

Thorgeirsson,

S.S., Huber,

Gottesman,

B.E., Sorrell.

M.M.: Expression

S., Fojo, A.T., Pastan,

of the multidrug-resistance

and regenerating

preI. and

gene in

rat liver. Science 236 (1987)

1120-l 122. Trowsdale,

J., Hanson,

I., Mockridge,

Kelly, A.: Sequences related

encoded

I., Beck, S., Townsend,

in the class II region

to the ABC superfamily

Ueda, K., Clark, Pastan, cloning

of transporters.

A. and

of the MHC

Nature

D.P., Chen, C., Roninson,

348 (1990)

P.: Mammalian

of exon organization

of an ancestral

multidrug-resistance

with structural

gene. Proc. Natl. Acad.

domains

gene: and dupli-

Sci. USA 86 (1989)

Raymond, M. and Gros, P.: Cell-specific activity of &-acting regulatory elements in the promoter of the mouse multidrug resistance gene mdrl. Mol. Cell. Biol. 10 (1990) 6036-6040. J.R., Rommens,

J.M., Kerem,

B.-S.. Alon, N., Rozmahel,

I.B., Gottesman,

1.: The human multidrug resistance 1 (mdrl) and transcription initiation. J. Biol. Chem.

Ueda, K., Pastan, promotor

I. and Gottesman,

region of the human

Van der Bliek, A.M. and Borst,

M.M. and gene. cDNA 262 (1987a)

M.M.: Isolation multidrug

quence

of mdr3 cDNA

ofthe

(P-glycoprotein)

17432-17436.

P.: Multidrug

resistance.

Adv. Cancer

P.M., Schneider,

encoding

a human

C. and Borst,

P-glycoprotein.

P.: SeGene 71

(1988) 401-411. Yoshimura,

A., Kuwazuru,

Y., Sumizawa,

Uda, T. and Akiyama,

S.: Cytoplasmic

structure of the multidrug transporter, with sequence-specific antibodies. 16282-16291.

R.,

and sequence

resistance

Res. 52 (1989) 165-203. Van der Bliek. A.M., Kooiman,

Gros,

6488-6492.

Riordan,

patibility sentation

S., Arnold,

R.: A gene in the human major histocom-

gene. J. Biol. Chem. 262 (1987b)

340 (1989) 400-404.

Ng, W.F., Zastawamy, R.L., Veinoe-Dreot, L. and Ling, V.: Identification of members of the P-glycoprotein multigene family. Mol. Cell. Biol. 9 (1989)

M., Bahram,

and expresin mammalian

505-508.

J. Mol. Biol. 157 (1982) 105-132.

McGrath, J.P. and Varshavsky, A.: The yeast STE6 gene encodes a homologue of the mammalian multidrug resistance P-glycoprotein.

Raymond,

Spies, T., Bresnahan,

resistance

741-743.

Kozak, M.: An analysis of 5’noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 15 (1987) 8125-8148.

Nature

sion of genes associated with multidrug cells. Science 232 (1986) 751-755.

of

of complemen-

tary DNA. Science 245 (1989) 1066-1073. Scotto, K.W., Biedler, J.L. and Melera, P.W.: Amplification

hepatocarcinogenesis

Hyde, S.C., Emsley, P., Hartshorn, M.J., Mimmack, M.M., Gileadi, U., Pearce, S.R., Gallagher, M.P., Gill, D.R., Hubbard, R.E. and Higgins, CF.: Structural

J., Lok, S., Plavsic, N., Chou, J.-L., Drumm,

M.C., Collins, F.S. and Tsui, L.-C.: Identification

E., Pious, D. and DeMars,

Sci. USA 85 (1988) 3580-3584.

geneity in multidrug-resistant

Z., Zielenski,

M.L., Iannuzzi,

the cystic fibrosis gene: cloning and characterization

Gros, P., Raymond, M., Bell, J. and Housman, D.: Cloning and characterization of a second member of the mouse tndr gene family. Mol.

Natl. Acad.

Grzelczak,

T., Ichikawa, orientation

M., Ikeda,

S.,

and two-domain

P-glycoprotein, demonstrated J. Biol. Chem. 264 (1989)