Isolation of a cytochrome P-450e gene variant and characterization of its 5′ flanking sequences

Vol. 144, No. 1, 1987

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Pages 258-263

April 14, 1987

ISOLATION OF A CYTOCHROME P - 4 5 0 e

'CHARACTERIZATION

GENE VARIANT AND

OF ITS 5' F L A N K I N G

SEQUENCES

P.N.Rangarajan, H.Ravishankar, and G.Padmanaban D e p a r t m e n t of B i o c h e m i s t r y , I n d i a n I n s t i t u t e of S c i e n c e , B a n g a l o r e 560 012, INDIA

Received February 25, 1987

A c y t o c h r o m e P-450e gene v a r i a n t h a s b e e n i s o l a t e d from t h e r a t l i v e r genomic l i b r a r y . It i s a t y p i c a l e gene c l o n e but u n i q u e i n h a v i n g b - l i k e s i n g l e b a s e s u b s t i t u t i o n s at s p e c i f i c s i t e s i n t h e 5' f l a n k i n g r e g i o n . It a l s o a p p e a r s to h a v e c e r t a i n a d d i t i o n a l r e s t r i c t i o n s i t e s i n t h e i n t r o n s . W h e n c o m p a r e d w i t h t h e c y t o c h r o m e P-450b gene, t h e e gene has some of t h e r e p e t i t i v e motifs i n t e r r u p t e d i n t h e 5' f l a n k i n g r e g i o n . In a d d i t i o n , t h i s r e g i o n i s c h a r a c t e r i z e d b y t h e p r e s e n c e of a l t e r n a t i n g p y r i m i d i n e purine stretch, s t e r o i d hormone r e g u l a t o r y elements, consensus e u k a r y o t i c e n h a n c e r s e q u e n c e and s e q u e n c e s i n v o l v e d i n g e n e r a l amino a c i d r e g u l a t i o n . SUMMARY:

© 1987 Academic Press, Inc.

The of

cytochrome

are

drug

P-450,

b

phenobarbitone and

the

some

of

rat

essentially

liver[i,2).

The

d i s t i n c t gene p r o d u c t s .

them

turn

may

out

gene

EcoRI-genomic

library

and

characterized.

gene v a r i a n t ,

to

be

clone its

This

h a v i n g some b - l i k e

psuedogenes[3). has

been

5'flanking clone

appears

In

isolated

sequence to

be

b

two

and

species

e

species

It h a s b e e n s u g g e s t e d

P-450e gene f a m i l y may c o n t a i n 9 - i l

P-450e

been

in

induces

cytochrome

cytochrome

has

e,

h i g h l y homologous but a r e

that

a

prototype

members, the

present

from upto a

although

the

-800

liver

base

pairs

cytochrome

s i n g l e b a s e s u b s t i t u t i o n s in the

study,

rat

P-450e

5'flanking

r e g i o n and i t a l s o c o n t a i n s many p o t e n t i a l r e g u l a t o r y s e q u e n c e s .

MATERIALS

AND METHODS

A charon 4A-rat liver genomic library (kindly provided by T . D . S a r g e n t , R . B . W a l i a c e and J . B o n n e r ) was s c r e e n e d w i t h n i c k - t r a n s l a t e d c y t o c h r o m e P-450e p r o b e , pP-450e91 c o v e r i n g e x o n s s i x to n i n e ( 4 ) , b y B e n t o n - D a v i s p r o c e d u r e ( 5 ) . About 300,000 p l a q u e s were a n a l y s e d and two p r o m i n e n t s i g n a l s were o b t a i n e d . One of t h e m , k P-450e5, was p l a q u e p u r i f i e d and t h e DNA was i s o l a t e d from l y s e d E . c o l i (LE392) c u l t u r e s . Tile DNA was d i g e s t e d w i t h EcoRI and a i 4 k b f r a g m e n t m i g r a t i n g b e t w e e n t h e l e f t and r i g h t a r m s was e l u t e d from 0.5~ a g a r o s e g e l s . T h i s f r a g m e n t was f u r t h e r c h a r a c t e r i z e d b y s o u t h e r n b l o t t i n g and r e s t r i c t i o n m a p p i n g ( 6 ) . The DNA f r a g m e n t s were s u b c l o n e d i n M i 3 m p i 0 and M 1 3 m p l l and s e q u e n c e d by dideoxy procedure(7). 0006-291X/87 $1.50 Copyright © 1987 by Academic Press, Inc. All r~,hts of reproduction in any form reserved.

258

BIOCHEMICAL AND BIOPHYSICAL RESEARCH C O M M U N I C A T I O N S

Vol. 144, No. 1, 1 9 8 7

RESULTS

The up

14 kb

in southern

(Fig.la). gels

insert isolated from

hybridization

BamHl

reveal

digestion

three

with of

fragments

reveal

k P-450e5

similar

to the clone pgP-450pb6

A

exons

detailed

covering HpaIl,

exens

was

the

i.0

fragment

the

and

five

2.5 and

in the

5

kb

of

BamHI-EcoRI

six

reveals

introns

and

it is very The

flanking

fragment

additional

in addition

agarose

analysis

et el(8).

5'

lights

(Fig.lb).

sequence

clone

the

0.8%

2.5 kb

partial

Mizukami

kb

on

and

P-450e

by

DNA,

probe,pP-450e91

analysis

and

cytochrome

about

cDNA

4.5 kb

fragments

described

and

the

Avail

kb

first

sequence

the

the

5'

in

identity

substitutions

at

and

and

with

pairs

BamHI-SstI

intron

sequenced

sequence base

of

digests of k P-450e5

sequence.

of

sites

to having

clone

kP-450e5

for" Sau961, all the

sites

for pgP-450pb6(Fig.2).

The of

six

four,

and

of

a typical

to

analysis

Bglll

reported

analysis

one

EcoRI

the nick-translated

the

restriction

to be

DISCUSSION

of size 7.0 kb,

Gross

contains

AND

the

about

data

except

1

800

are

flanking

case

four

fragment,

sites

2

of

3

of

in

sequence

information

5'flanking

Fig.3.

P-450b has

a

portion

flanking

region

A cemparision available

and

region.

exon,

5'

e

typical

1

4

first the

presented

tP-450e5 the

the

nucleotides

cytochrome

that in

covering

genes(9)

b-like

The

of

upto

reveals

single

cytochrome

this -450

base P-450e

2

23.1

b

,~

~

--212

!~

_7,4

:iD

-5.6 --4.9

i~,~

- 3.5

~ I

9.4 6.6 4/.,

2,3 2.0

(a) F i g . 1.

(b)

R e s t r i c t i o n e n z y m e d i g e s t i o n and s o u t h e r n a n a l y s i s of XP-450e5 DNA.(a) l a n e l - u n c u t DNA; l a n e 2 - E c o R I d i g e s t e d DNA; l a n e 3 - S o u t h e r n a n a l y s i s w i t h n i c k - t r a n s l a t e d c y t o c h r o m e P-450e cDNA p r o b e ; l a n e 4 Size m a r k e r s . (b) l a n e l - B a m H I d i g e s t i o n of t h e 14 k b i n s e r t ; lane2-size markers. 259

BIOCHEMICAL A N D BIOPHYSICAL RESEARCH C O M M U N I C A T I O N S

Vol. 144, NO. 1, 1 9 8 7

P-450b 51 ] I I

[

BEE

1

23

4 56

78

•

II

•

II

II

9 "~I t

•

I

I

I

I

I

II

]

I

I

I

I

B

B

B

B

E

EB

E

E

E

E

E

P-450e

I

1

23

4 56

I

II

I I'

I

E

1

B Ss

9

78 "

I

I

B

E

I

I

II

II

lkb

EBB

E

"1 I

F~

l

B HAH

kP-450e5

l

I

I

I

S

S

A

H

I

I

23

•

II

I

E

I~

I

PvBg

4 56 II

•

I

B Ss

j

I

l

B

E

l-.-J lkb

k_.

r

]

I

I~

I I

I

B HA HSS

I~ L . ~

S H B g Pv A

I I

I I

I

S H

AA

H

I~

Pv Bg

I

I

H

E

r CCGGACCTTTTCCCTCCTAAGTTCATTCTCCAGCCAGGTCCGTGGGTGGGAAGAGAA•

2.

variants

significant

but

less

gene(3),

to

e

base

the

restriction

contribute

cytochromo reported

two

a

high

with

but

sites

is

the

-,

characterized

by

in t h e

features

to

the

differences

in

and

e

response

Suwa

et

genes

al(9),

in the

the

length

region.

introns,

in

5'flanking

efficiency

to

of

having It

although

of

also

study typical

appears

some of t h e s e earlier.

region,

expression

phenobarbitone. alternating

regions

present

been depicted

the

region showing

corresponding in t h e

5'flanking

the

exons

described

P-450b

by

the

gene may not h a v e

interesting

over

remaining

to

kP-450e5

in

homology

the

homology

gene

substitutions

in t h e c y t o c h r o m e P - 4 5 0 e

are

show

eight,

P-450e

additional

There may

to

and

substantially

identical single

have

sites

reported

seven

cytochrome

b-like to

are

exons

appears

•

Restriction map analysis of kP-450e5 in c o m p a r i s i o n with those of c y t o c h r o m e P-450b and e genes. The restriction m a p of t h e BamHI-EcoRI fragment covering exons four, five and six(presented in line three from the top) is from the cytochrome P-450e gene clone, pgP-450pb6, described by Mizukami et ai ( 9 ). Restriction sites: B-BamHI; E-EcoRI ; Ss-SstI ; H-HpaII; A-AvaII ; $-Sau96I ; Pv-PvuII; Bg-BglII.

spanning

of t h e

I00 bp

1

Fig.

gene

100bp

E

As

which of

the

already

pyrimidine-purine

sequence

in t h e

region

-255 to -290 w h i c h can a s s u m e a Z-DNA c o n f o r m a t i o n ,

is

longer

in

cytochrome

much

gene

[ (ca)5].

This

the is

true

of

P-450e kP-450e5 260

gene as

[(ca)19] well.

than In

in

the

addition,

P-450b certain

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 1 4 4 , No. 1, 1 9 8 7

1

)I

[-. 8

t.. P

I H

I H

i P

i Ss F

t

700 bp -800 ggatccat t t t c t c a a t t c a a g t t c t c t c c t c t c a a a t ~ _ a c t _ c t a g c t t g t a t c a t g t t g a c a c a a a a g t t a g a c c c g g g 8 -700 ccccaacttcttctctgccaggtttcagcctctctctccat tt~actcctgagcctcactgctcctgaatctcttgccttt

-600

•cttt•tttt••t••••att•tc•ca••t•t•••tatcat•••••aga•a•tgaaatg•g•a•t•t••a•aaatt••at• tggtagactt t tcaaagacagggacaggaaccaacagacggagacaagcacaggaatcat ggga-tSaOcOtattctt gtcaa b b ctcaaacataatcacatgtacccaggacacaaaaaacatacagagaagccccaataatt_taagattatacatgtaaata -400

b

b

taccc tagacat_scaagaaaagaccacccagt_gcatctagactcagacaaagaaatt tacatcggtacgtttatatcag -300 aaatgatct t tcacatag8aaaagcatatagaacacgcacacacacacacacacacacacacacacacacacacaat -200 cccatgccctagtaagtaaacagagctgacaaaactgagttgacaagtgcacacccatttacataaaacaagaggcct -100

aagtcccagtgcccttt~C~gtgtatctgtttcgtgg~tgccaacatctatggtgtgggtaagggaatgagga gtgaatagccaaagcaggaggcgtgaacatctgaagttgcataactgagtgtaggggcagattcagcataaaagatcct 1 8ctggagagcatgc

ACTGAAGTCTACCGTGGTTACACCAGGACCATGGAGCCCAGTATCTTGCTC 100 CTCCTTGCTCTCCTTGTGGGCTTCTTGTTACTCTTAGTCAGGGGACACCCAAAGTCCC GTGGC

AACTTCCCACCAGGACCTCGTCCCCTTCCCCTCTTGGGGAACCTCCTGCAGTTGGACAGAGGA 2OO GGCCTCCTCAATTCCTTCATGCAGgtgagatattcacagggcctggagctc F i g . 3. DNA s e q u e n c e analysis of the BamHI-Ssti fragment. The 1.0kb f r a g m e n t c o v e r i n g t h e f i r s t e x o n , a p o r t i o n of t h e f i r s t i n t r o n and a b o u t 800 n u c l e o t i d e s of t h e 5 ' f l a n k i n g r e g i o n was s e q u e n c e d b y the d i d e o x y chain t e r m i n a t i o n method. The sequencing s t r a t e g y is i n d i c a t e d b y a r r o w s . R e s t r i c t i o n s i t e s : B-BamHI ; P - P s t I ; H - H i n c I I ; Ss-SstI. The cytochrome P-450b-like single base substitutions are underlined. The steroid recognition elements are boxed. The eukaryotic core enhancer-like s e q u e n c e and the putative heiner e g u l a t o r y domain are i n d i c a t e d by a wavy line. The d a s h e d lines indicate general aminoacid regulatory sites.

repetitive but gene.

are

motifs

depicted

interrupted Repetitive

by motifs

in

single in

Table base the

I

are

seen

in

substitutions

5'flanking

cytochrome

in

region

the

P-450b

cytochrome

offering

sites

Table I R e p e t i t i v e m o t i f s in t h e 5 ' f l a n k i n g region of t h e c y t o c h r o m e P-450e and b g e n e s . T h e n u m b e r s i n d i c a t e t h e f i r s t n u c l e o t i d e p o s i t i o n s in t h e 5 ' f l a n k i n g r e g i o n of t h e c y t o c h r o m e P - 4 5 0 e gene

Cytochrome

P-450e gene

Cytochrome

-170 -352 CCCAGTGC, C C C A G C G C -123 -419 -474 ACATCT, ACATGT, ACATGT -359 -457 AAGAA, AAAAA

CCCAGTGC, ACATGT, AAGAA,

261

P-450b gene

CCCAGTGC

ACATGT, AAGAA

ACATGT

gene P-450e

for

the

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

VoI. 144, No. 1,1987

binding

of

strength the

regulatory

of

proteins

promoter

higher

level

of

are

considered

elements(10). expression

These

of

the

to

contribute

differences

cytochrome

may

P-450b

to

the

total

contribute gene

to

than

that

of t h e e gene i n r e s p o n s e to p h e n o b a r b i t o n e .

There region a

of

steroid

and in

are

a

potential

sequence

element

metaliothionin

genes(11].

This

the

5'

flanking

seen

The

orientation,

in

the

e x a m i n e d for

characterized

enhancer

viral

in

can

also

be

-315)

consensus

5'

seen

-157

hormone

and

P-450(C21)

resembles

region

by

group

TTT It

the AAA TGTGGTTTG

in

in

kP-450e5

of

Finally,

binding

sites

specific

transcription

the

is

also

regulatory factors

seperated shown

cytochrome

regulation(10) for

been

since the

upstream

which

activation

heine r e g u l a t i o n of i s o c y t o c h r o m e

repeats

has

site,

in

P-450,

at

TGACTC -685

and

proteins.

Studies

and

sites

their

spacers

this

are

of

that

short

herne,

hemoprotein

implicated -772

C gene

containing

laboratory

regulates

sequence seen

by

this

and

the gene

in

general

may

provide

to

identify

underway

binding in

is

elsewhere

sequence

This

flanking

and

growth

P1-450

to

systems(14).

the

-134

human

cytochrome

a heine r e g u l a t o r y

transcription(4,16]. aminoacid

sequence the

core

i n v o l v e d in the

repeats(15).

prosthetic

detected

in

at

s e q u e n c e TTTATATCAGAATGATCTTTCACA(-339 to -315)

sequence in yeast

direct

of

sites

figuring

CTTT-CACA(-322

i m m u n o g l o b u l i n and

a l s o c o n t a i n s the be

regions

sequence

regulatory

TGTCCT

regulatory

opposite

is

other

The

hormone

genes(12,13].

can

few

kP-450eS.

the

5'flanking

r e g i o n of c y t o c h r o m e P-450 genes.

ACKNOWLEDGEMENTS T h i s work was c a r r i e d out i n t h e Genetic E n g i n e e r i n g Unit s u p p o r t e d b y t h e D e p a r t m e n t of S c i e n c e and T e c h n o l o g y , New D e l h i . The t e c h n i c a l a s s i s t a n c e of M s . P . G . V a t s a l a i s a c k n o w l e d g e d .

REFERENCES 1.

Ryan,D., Thomas,P.E., Koreniowski,D. J.Biol. Chem. 254, 1365-1374.

and Levin,W. ( 1 9 7 9 )

2. Vlasuk,G.P., Ryan,D.E., Thomas,P.E., (1982) Biochemistry. 21, 6288-6292. 3. Adesnik,M.

and Atchison,M.

(1986) Crit. Rev.Biochem.

4. Ravishankar,H. and Padmanaban,G. J.Biol. Chem. 260, 1588-1592. 5. Benton,W.D.

and Davis,R.W.

6. Maniatis,T.,

Fritsch,E.F.

Levin,W.

and

19, 247-305.

(1985]

(1977) Science,

and Sambrook,J. 262

196, 180-182. (1982)

Walz,Jr.F.G.

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Manual,

Cold S p r i n g Harbour L a b o r a t o r y ,

7. S a n g e r , F . , Coulson,A.R., Barrek,B.G., (1980) J . M o l . Biol. 143, 161-178.

Smith,A.J.H.

8. M i z u k a m i , Y . , S o g a w a , K . , S u w a , Y . , Muramatsu,M. (1983) P r o c . N a t l . A c a d . S c i . USA. 80, 3958-3962.

and

and

Roe,B.A.

Fujii-Kuriyama,Y.

9. Suwa,Y., M i z u k a m i , Y . , Sogawa,K. and F u j i i - K u r i y a m a , Y (1985) J . B i o l . Chem. 260, 7980-7984. 10. A r n d t , K . and F i n k , G . R . (1986) Proc. N a t l . A c a d . S c i . U S A . 83, 8516-8520. 11. Nawa,K., Nakamura. T, Kumatori,A., J.Biol. Chem.261, 16883-16888.

Noda,C.

and Ichihara,A.

12. J o n e s , P . B . C . , D u r i n , L . K . , G a l a e z z i , D . R . and W h i t l o c k , J R , J . P . Proc. N a t l . A c a d . S c i . U S A . 83, 2802-2806. 13. H i g a s h i , Y . , Y o s h i o k a , H . , Yamane,M., Gotoh,O. (1986) P r o c . N a t l . A c a d . S c i . USA. 83, 2841-2845. 14.

Renz,M., N e u b e r g , M . , K u r z , C . , EMBO.J. 4, 3711-3716.

15.

Guarente,L.

and Mason,

Bravo,R.

and

and M u l l e r , R .

(1986)

[1986)

Fujii-Kuriyama,Y.

(1985)

(1983) Cell 32, 1279-1286.

16. S a t h y a b h a m a , S . , S s t h i a g n a n a S e e l a n , R . B i o c h e m i s t r y , 25, 4508-45t2.

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