The human liver and reticulocyte cytochrome b5 mRNAs are products from a single gene

Vol. 178, No. 1, 1991 July 15, 1991

BIOCHEMICAL

The Human

Liver

mRNAs

are

Sara Department

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 38-44

and Reticulocyte Products

J.

Giordano

of

Biochemistry

from

Ohio

W. Steggles Pathology

Universities

of Medicine

Rootstown, 3,

b,

Gene

and Molecular

College

May

a Single

and Alan

Northeastern

Received

Cytochrome

Ohio

44272

1991

Using a combination of standard cDNA library screening techniques and the Polymerase Chain Reaction (PCR) we have isolated and sequenced DNA fragments corresponding to the human reticulocyte cytochrome b, mRNA. The reticulocyte specific sequence codes for amino acids 97 and 98 only, with a TAA stop codon. The reticulocyte specific 3'non-translated sequence has 15 new nucleotides then utilizes the liver mRNA sequence from amino acid 97 onward. This indicates that the reticulocyte specific exon has 24 base pairs (bp). In addition, we have isolated sequences that are derived from a transcribed cytochrome b, pseudogene. This transcript contains multiple mutations which prevent the synthesis of any functional protein. 0 1991Academic Press, Inc. Cytochrome in

two forms.

Met in

b, (b,)

as 1,

One with

found

the

liver

between

the

is

in

and

other are

b) the

98th

aa for

all

species

aa l-97

are

always

the

same in

two forms

transcriptional human

and

of b, could

processing rabbit,

post-translational

etc.

(1).

a) the

end and serve

(1)

processing

0006-291X/91 $1.50 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

protein

that

counting

the

other

The

extra

with

36 aa in

the

liver

except

is

the

two forms. from

the

one mRNA.

It

erythrocyte of

38

the

bS's liver

different,

tissue

has

and i.e.,

suggested

specific

possible

could b,,

b, occur

domain,

This

is

initial

differences

binding

bovine

exists

134 aa found

essential

as a membrane

arise

of

(aa), the

tissues

C terminal

the

acids

reticulocyte,

at the

that

amphipathic

98 amino

the

two forms

a small

postthat

the

be derived

by

but

this

would

Vol.

178, No. 1, 1991

entail

the

of

protein.

the

tubulin the

BIOCHEMICAL

addition

(2). liver

one

chicken,

the

same coding

alteration

However,

for

bovine

reticulocyte and

sequence

the

(3)

with

the

liver

a real

reported

specific

b,

is which

in

for of

possibility. analyzed.

mRNA's

only

end

processing

been

reticulocyte

There

form

C-terminal

has been

mRNA has

differing

regions. membrane

b, is b,

the

post-translational

reticulocyte

liver

mitochondrial

MATERIALS

one new aa to

novel

the

RESEARCH COMMUNICATIONS

least

rare

non-translated

identity

at

This

b, to

Only

of

AND BIOPHYSICAL

the

also has

have length

the

exactly of

one only

In

the

their

report

3' of

58% amino

a

acid

(4).

AND METHODS

The human reticulocyte XZAP cDNA library was a generous gift from Dr. J. Prchal (Birmingham, AL). Human total RNA was isolated from reticulocytes (5) obtained from blood taken from normal individuals. Blood was drawn following NEOUCOM IRB approved procedures. A total of 11.4~10~ plaques were screened using standard procedures (6) and Stratagene (La Jolla, CA) protocols. Fourteen plaques were purified corresponding to four independent clones containing inserts of 0.52, 0.54, 1.9, and 3.9 kb, respectively. The inserts were isolated in Bluescript KS- according to the manufacturer's instructions (Stratagene). DNA sequencing was carried out using the chain termination method with Sequenase (USB, Cleveland, OH) and (r35S dATP (NEN, Boston, MA). Single stranded cDNA was prepared from reticulocyte total RNA (6) using random primers and MLTV (BRL, Gaithersburg, MD), and used for PCR without further purification. For the amplification of human reticulocyte cDNA, oligonucleotide primers A, B, and C derived from the liver and reticulocyte b, sequences (8 and Fig. 1) were obtained from National Biosciences (Hamel, MN) and used without further purification. The PCR amplification conditions were 30 cycles of 1.5 min at 94O, 1 min at 60°, 2.25 min at 72O with a final 7 min step at 72O. The initial PCR amplification used one tenth volume of the cDNA synthesis reaction. When samples were subjected to a second set of 60 cycles of amplification, they were purified through UltraFree columns (Millipore, Bedford, MA). PCR products were analyzed by 4% Nusieve agarose gel electrowere subcloned into phoresis and selected bands of interest Bluescript KS- (7).

RESULTS

AND

From library,

DISCUSSION the

screening

4 independent

of

the

recombinant

human phage

39

reticulocyte were

purified.

XZAP The

cDNA two

Vol.

BIOCHEMICAL

178, No. 1, 1991

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ATG Met

GCA Ala

GAG Glu

CAG Gln

TCG Ser

GAC Asp

GAG Glu

GCC Ala

GTG Val

AAG Lys

TAC Tyr

TAC Tyr

ACC Thr

39 13

CTA Leu

GAG Glu

GAG Glu

ATT Ile

CAG Gln

AAG Lys

CAC His

AAC Asn

CAC His

AGC

Ser

AAG Lys

AGC Ser

ACC Thr

78 26

TGG Trp

CTG Leu

ATC Ile

CTG Leu

CAC His

CAC His

AAG Lys

GTG Val

TAC Tyr

GAT Asp

TTG Leu

ACC Thr

AAA Lys

117 39

TTT Phe

CTG Leu

GAA Glu

GAG Glu

CAT His

CCT Pro

GGT Gly

GGG Gly

GAA Glu

GAA Glu

GTT Val

TTA Leu

AGG Arg

156 52

GAA Glu

CAA Gln

GCT Ala

GGA Gly

GGT Gly

GAC Asp

GCT Ala

ACT Thr

GAG Glu

AAC Asn

TTT Phe

GAG Glu

GAT Asp

195 65

GTC Val

GGG Gly

CAC! TCT His Ser

ACA Thr

GAT Asp

GCC Ala

AGG

Arg

GAA Glu

ATG Met

TCC Ser

AAA Lys

ACA Thr

234 78

TTC Phe

ATC Ile

ATT Ile

GGG Gly

CTC Leu

CAT His

CCA Pro

GAT Asp

GAC Asp

AGA Arg

CCA Pro

AAG Lys

273 91

TTA Leu

AAC Asn

AAG Lys

CCT Pro

GAG Glu G CCA Pro

GAA Glu

CCT Pro

TAA xxx

AGGCGGTGTTTCAAGGAAA

316 98

CTCTTATCACTACTATTGATTCTAGTTCCAGTTGGTGGACCAACTGGGTGA

367

TCCCTGCCATCTCTGCAGTGGCCGTCGCCTTGATGTATCGCCTATACATGG

418

CAGAGGACTGAACACCTCCTCAGAAGTCAGCACAGGAAGAGCCTGCTTTGG

469

ACACGGGAGAAAAGAAGCCATTGCTAACTACTTCAACTGACAGAAACCTTC

520

ACTTGAAAACAATGATTTTAATATATCTCTTTCTTTTTCTTCCGACAT'$AG

571

AAACAAAACAAAAAGAACTGTCCTTTCTGCGCTCAAATTTTTCGAGTGTGC

622

CTTTTTATTCATCTACTTTATTTTGATGTTTCCTTAATGTGTAATTTACTT

674

ATTATAAGCATGATCTTTTAAAAATATATTTGGCTTTTAAAGT-

725 740

Fiqure 1. cytochrome underlined, polymorphism

The complete cONA. b, the alternative at nucleotide

nucleotide sequence The reticulocyte polyA site triple 288 indicated.

of

human

reticulocyte

specific

is

sequence

underlined,

and

the

The PCR primer A was AAAACAGCTGGCTCGCGGCGAACCG and based on nucleotides -1 to -19 from the human liver b, sequence Primers B and C were (8) AAAACAGCTCTTCCTGCGCTGACTTCTandCACCGCCTTTAAGGTCwerecomplementary to nucleotides 459+439 and 306-+289 of the reticulocyte

smallest for liver

(A,B;

524 and 540 bp, respectively)

analysis. b,

from

additional sequence

DNA sequencing the

showed that 3'

polyA

between

b, for

a recognizable

(Fig. sequence

1, single was

the reticulocyte

clone tail

but

sequence

underline).

and the break 40

of The 5' point

the

chosen

A corresponded

to

contained

aa 96 and aa 97.

now part WINA.

sequence.

were initially

was GAACCTTAAAGGCGGTGTTTCAAG corresponding

liver

sequence

to

24 bp inserted

stop-non-translated the

aa 86

b,

This to

an extra

glu-pro-

The remainder 3'

non-translated

end of clone between

of

the

A was not 5' sequence

Vol.

178, No. 1, 1991

and

the

coding

junction. been

sequence

Analysis

attached

new polyA

at

gave

aa 72 did

sequencing

species

have

bovine

reticulocyte

In

to

we obtained

were

purified

by

(ca l/10)

A and

C,

and

ca 350 bp.

gave Subcloning

nucleotide

organization

of

the

Clone

Pl

is

the

analysis

to

be less

(this

paper,

exon

ends

at

aa 96,

junction.

The

presence

or absence

value

in

because

the

in

restriction

analysis

enzyme

of this of

b, gene 41

A 5 /bl primers

product

of

gave

the

product

confirmed

at

96.

The

codon

a

overall

b, cDNA was confirmed.

found not

think

that

this

is

both

sequences

in

shown). of

one CCA. be

The CCA seems

6 human If

a CCA/GT

HpaII site in

A and B

with

this

analysis

restriction the

primers column.

We do not

would

cDNA,

and

5 were CCG, only there

b, cDNA,

aa l-98,

we have

the

only

and most

homogenous

specific

(data

for

5' and 3' sequence

using

of

P2 is CCG.

then

reticulocyte

reticulocyte

amplification

and P2)

A.

intron-exon

rabbit

UltraFree

to

DNA clones

8, 9, lo),

an

on the

sequencing

because

of genomic

24 bp

exception.

an apparently

reticulocyte

artifact

common,

an

coding

as clone

the

human

based

a second

(Pl

CCA and clone

a PCR generated

of the

5' end

the

reticulocyte

be the

corresponding

two clones

site

specific

expect

organized

for to

in

The

but

that

would

through

polymorphism

1).

of the

resemble

indicated

rest

and

sequence

Fig.

position

The PCR products

rise

had

Analysis

not

primers

was used

tail

alternative

reticulocyte

still

the

passage

polyA

sequence,

one

could

b, cDNA (8).

the

relative

a similarly

isolate

3'

in one exon of 24 bp coding

oligonucleotide

liver

aliquot

data

By analogy,

to

order

same

is contained

acids.

to

The

at

sequence

of the

aa 72.

an intron-exon

underline,

an unrecognizable at

the

b, mRNA (9).

breakpoint The

that

was different liver

RESEARCH COMMUNICATIONS

resemble

(triple

the

amino

but

for

not

B showed

in

junction.

other

clone

was present the

specific

aa 86 did

site

started

sequence

AND BIOPHYSICAL

a new position

B again

information

two

of

described

clone

Again

at

adenylation

previously of

BIOCHEMICAL

human

recognizes

b, cDNAs

the

previous

or

a CCG/GT CCGG,

the

may be of some future genomic

DNA.

BIOCHEMICAL

Vol. 178, No. 1, 1991

The chicken

difference is not

between

surprising

and possess

endoplasmic

the

of

finding

type

not

Northern

analysis

hybridizing

data

and

Chicken

(3). reticulum

RESEARCH COMMUNICATIONS

that

obtained

erythrocytes

from

are nucleated

as do hepatocytes.

membrane

binding

the

Therefore,

domain,

i.e.,

a liver

be unexpected.

signal

studied

our

a b, with-a

b5, would

AND BIOPHYSICAL

had either

of

human

of

ca 720bp,

little

or

reticulocyte

RNA showed

suggesting

that

transcribed

no

the

only

one

individual

pseudogenes

(data

not

shown). Clones represent could the

the

have same

from

C and

clone

liver

been 5'

D

or

partially

terminal A.

3900

(1900,

bp)

reticulocyte

However,

both

mRNA's

as clone C and

clearly

forms

processed sequence

were

D

of (9).

B (Fig.

had the

GGGGCTGTGTG;LGCTGGGk!TGGCTCGC$G$$W&CCGAG

too

b,,

however,

Both 2),

long

to they

C and D had but

differed

same 3' sequence.

12 ATG

GC$

3 ZAG

4 CAG

5 TCG

6 GAC

7 $AG

8 GCC

9 GTG

10 AAG

11 12 TS;'P Tt$

13 ECC

14 CT9

15 GAG

16 GAA

17 ATT

18 CA-

19 AAG

20 CAC

21 AAT

22 CA2

23 AGC

24 AA$

25 AGC

26 ACC

27 TGG

34 GTG

35 TAC

36 GAT

37 TTG

38 ACC

39 AAA

40 TT;

41 CTG

42 G$A

43 GAG

44 CAT

45 CCT

46 GGT

47 GGG

48 $AA

49 GAA

50 GT$

51 TTA

52 AGG

53 GAA

54 ;AA

55 GCT

56 GGA

57 GA-

58 GA;

59 GCT

60 ACT

61 GA$

62 AAC

63 TTT

64 GAG

65 GAT

66 GTC

67 GGG

68 CAC

69 TCT

70 ACA

71 GAT

72 GCC

73 AG$

74 GAA

75 TT$

76 y'c

77 AAA

78 ;CA

79 T$$

80 ATC

81 ATT

82 GGG

83 G;;

84 -T-

85 CAT

86 CT$

87 GAT

88 GAC

89 AGA

90 -TA

91 AAG

92 TTA

93 A$:

94 AAG

95 CCT

96 TC$

97 GAA

98 ;TT

stop TTT

TTTTTTTTT Fiqure 2. The partial nucleotide sequence of the reticulocyte cytochrome b, pseudogene transcript. Only the 5' and 3' end sequences and the hybridizing region are shown. The numbers 1-98 correspond to the reticulocyte b, codons (Fig. 1). * represents a single bp change, - indicates a-1 bp deletibn, -and . indicates a 1 bp insertion. The 18bp deletion occurs between codons 27 and 34.

42

In

Vol. 178, No. 1, 1991

order

to

analyze

was isolated

clone

as a HinfI

Analysis

showed

pseudogene of the

BIOCHEMICAL

D in more

detail,

fragment,

subcloned

that

spanning

the

sequence

amino

pseudogene

acids

sequence,

The

represent

transcripts

The

data

pseudogene

deletions,

there

bp

(Fig.

it

is

different (9,13). are

a rare from As yet,

present

in

the

to obtain

2).

of

a

b,

the

rest

and used as sequencing clone

D, and clone

an 18bp

b, pseudogene.

deletion,

plus

43

the

C,

5 single

one

bp

authentic

bp

changes.

reticulocyte

1). of

a pseudogene

event. three

that

a reticulocyte

with

(Fig.

TTGGTGAAATCGTAC-

that

insertions,

sequence

and sequenced

In order

from

contains

The transcription but

l-98.

is a ca 87% homology

b, cDNA sequence

hybridizing

represented

2) indicates

derived

single

the

were synthesized

transcript 2

Overall,

(Fig.

RESEARCH COMMUNICATIONS

two oligonucleotides,

ACC and TTTGAGGATGTCGGGCAC, primers.

AND BIOPHYSICAL

The liver

we do not any individual's

has been

sequence

of

b, pseudogenes know

exactly

described

this

pseudogene

previously

how many

(11,12) is

isolated

b, pseudogenes

genome.

ACKNOWLEDGMENTS We thank Dr. Prchal for the human reticulocyte library. This work was supported by funds from the American Heart Association, Ohio Affiliate, The and the United Way of Stark County, Ohio. accession number for the reticulocyte b, and the pseudogene sequences are M60174 and M64714, respectively.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.

Kimura S, Abe K, and Sugita Y (1984) FEBS Letters, 169, 143146. Raybin D, and Flavin M (1977) Biochemistry, 16, 2189-2194. Arch. Biochem. Biophys., Zhang H, and Somerville C (1990) 280, 412-415. Lederer F, Ghrir R, Guiard B, Cartial S, and Ito A (1983) Eur. J. Biochem., 132, 95-102. Chomczynski, P, and Sacchi, N (1987) Anal. Biochem., 162, 156159. in Molecular Maniatis T, Fritsch EF, and Sambrook J (1982) Cold Spring Harbor. Cloning. A Laboratory Manual. BioTechniques, 3, 452-453. Struhl K (1985) Yoo M, and Steggles AW (1988) Biochem. Biophys. Res. COmmUn., 156, 576-600. 43

Vol.

9.

10. 11. 12. 13.

178,

No.

1, 1991

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Yoo M, and Steggles AW (1989) Biochem. Biophys Res. Commun., 163, 18-24. Miyata M, Nagata K, Yamajoe Y, and Kata R (1989) Pharmacol. Res., 21, 513-519. Sorge J, Gross E, West C, and Beutler E (1990) J. Clin. Investig., 86, 1137-1141. McCarrey JR, and Thomas K (1987) Nature, 326, 501-505. Yoo M, and Steggles AW In preparation.

44