Cloning and sequence analysis of porcine myoglobin cDNA

137

C;ene, 40 (19XS) 137-140 Elsevicr GENE

1452

Cloning and sequence analysis of porcine myoglobin cDNA (Hybrid-arrested

translation;

nucleotide

sequencing;

human

and seal myoglobin

genes;

introns)

Eiko Akaboshi

(Received

March

(Revision

received

(Accepted

3rd. 1985) June 21st. 1985)

July 3rd. 1985)

SCJMMARY

Porcine myoglobin cDNA clones have been isolated from a cDNA library prepared from enriched heartmyoglobin mRNA. Sequence analysis revealed 59 nucieotides (nt) in the 5’-untranslated, 462 nt in the amino acid (aa)-coding, and 590 nt in the 3’-untranslated regions. The myoglobin cDNA showed a high C t C content (60%). When the nt sequence of the porcine myoglobin cDNA is compared with those of seal and human myoglobin cDNAs deduced from the corresponding genomic myoglobin genes [Blanchetot et al., Nature 301 (1983) 732-734; Weller et al., EMBO J. 3 (1984) 439-446; Akaboshi, Gene 33 (1985) 241-2491, a high degree of homology is observed in the 5’-untranslated region and in parts of the 3’-untranslated region, as well as in the coding region.

INTRODIJCTION

EXPERIMENTAL

Myoglobin is a heme protein contained in cardiac and skeletal muscles. The 3’-untranslated cDNA correspondent to seal myoglobin mRNA (Wood ct al., 1982) and seal and human genomic myoglobin genes (Blanchetot et al., 1983; Weller et al., 1984) were cloned and sequenced. In addition, Akaboshi (1985) cloned human myoglobin gene by using porcine cDNA as a probe. In this report, the nt sequence of the porcine myoglobin cDNA was determined and compared with those of the seal and human genomic myoglobin cDNAs.

(a) mRNA

Abhrcviations: tu mRNrZ;

aa. amino acid(s); ds. double-stranded;

reading

frame;

dodecyl

sulfate;

p, plasmid;

cDNA,

DNA complementary

nt, nucleotide(s);

PA, polyacryiamide;

ORF,

037X-i I IY:X5,$03.30

0

1985 Elsevier

Science

Publishers

DISCUSSION

isolation

Total nucleic acid was isolated from fresh pig hearts obtained in a slaughterhouse by the procedure of Lomedico and Saunders (1976), and poly(.A)+ RNA was prepared by two cycles of oligo(dT)cellulose (Type 7, P-L Bioch~micals) column chromatography. Poly(A) + RNA was fractionated on 5-202, sucrose gradients in a buffer containing 0.05 M Tris. HCl (pH 7.4), 1 mM EDTA and 0.25:,; SDS. (b) Cloning methods

open

SDS, sodium

ss. single-stranded.

AND

Single-stranded cDNA was synthesized at 42°C from 1 ng of the mRNA as described by Maniatis

138

et al. (1982). After alkaline hydrolysis, synthesized, digested with nuclease selected on a BioGel A-l.5 The resulting (Roychoudhury pBR322

m column.

ds cDNA was tailed with poly(dC) and Wu, 1980), annealed with

that had been digested

with poly(dG),

ds cDNA was Sl and size-

and transformed

strain HB 101 as described

by PstI and tailed into Escherichiu coli

by Morrison

(1979). The

screened (Hanahan transformants were and Meselson, 1980) using ‘*P-labeled cDNA synthesized from the mRNA

or j2P-labeled

ments of the myoglobin

cDNA.

DNA

frag-

ly, with poly(A)+ RNA and the enriched mRNA.

globin mRNA

Poly(A) + RNA was separated by two successive 5-20% sucrose gradient centrifugations. In vitro translation products of fractionated poly(A) + RNA are shown in Fig. 1. Of the incorporated 35S, 0.82, and 8% were associated with myoglobin, respective-

185

28s

1

1

2345676

1

Fig. I. Fractionation

and enrichment

of myoglobin

assayed

by in vitro translation.

Total poly(A)

fractions

separated

sucrose gradient

in the rabbit

on a i-X”,,

rcticulocyte

were separated

lysate

on an SDS-12.5

1973); 2.5 ~1 of each sample RNA fractions sucrose

grltdient:

of myoplobiil.

taken

system.

products and Favre,

per lane. Lanes l-8:

from the top to the bottom

lane m: total poly(A)’

as

wera translated

Translation

“,) PA gel (Lacmmli was loaded

mRNA

’ RNA and RNA

of 3 5-20”,,

RNA. Arrow,

position

myo-

1000 bacterial

were

picked

transformants

to a cDNA

myoglobin

in

mRNA,

up and

probe and 48

assayed

by

hybrid-arrested translation as described (Paterson et al., 1977). Only when total poly(A) + RNA was hybridized

(c) cDNA cloning strategy

clones

from the enriched

by hybridization

made from the enriched positive

mes-

and cloned in E. co/i as described

b. Initially,

were screened

mid

m

Thus about tenfold purified myoglobin

sage was obtained. cDNA was synthesized section

myoglobin

with the DNA of the recombinant

pMG293

(Figs. 2 and

3; nt 686-l

plas-

11l), the

translation of myoglobin mRNA was partially arrested. The sequence of the PstI insert of pMG293 revealed poly(A) at the 3’ end, but no coding region for myoglobin. Then an additional 1500 transformants were screened by hybridization to the &I insert of pMG293; clone pMG387 (Fig. 2) contained complementary DNA since a band corresponding to myoglobin disappeared in hybrid-arrested translation (not shown). The sequence of the P.~tl insert of pMG387 is shown in Fig. 3 (nt 468-l 103). As expected, the pMG387 clone contained the region coding for aa 13 l-l 53 corresponding to the C-terminal aa, and part of the 3’-untranslated region. Next, to isolate the remaining coding sequence, the primer extension method was used. First, using Hue111 digested fragments (nt 522-562, 563-568 and 569-619) of a DdeI fragment (Fig. 3. nt 522-619 in pMG387) as primer, I constructed a cDNA library as described in section b and screened 200 colonies by hybridization to a 5 ’ -end-labeled Hp~l I I-.&&J1 (Fig. 3, nt 477-521: upstream region of the primer) and found 16 positive colonies. The nt sequence of a PstI insert of a clone with the largest insert, pM E 12 (Fig. 2) was determined (Fig. 3, nl 244-619). This clone contained 152 nt overlapping pMG387 along with 224 nt upstream, but was not long enough to cover the entire aa sequence of porcine myoglobin reported by Rousseaux et al. (1976). Therefore, the primer extension was repeated using pME12. A Sau3A digest (Fig. 3, nt 33 l-355 and 356-409) of a HueIII-PstI fragment (Fig. 3, nt 331-409) was used as a primer and a P.stI-Hue111 (Fig. 3, nt 244-330; this PstI site was created by dG-dC tailing) as a probe. I screened 400 colonies and selected eight positive clones, and obtained a clone pME1612 (Fig. 2) containing 59 nt upstream in addition to

400

200

600

1000

800

pMC293

pl?G387

c-e_

, +--_--_---_-_____-_____

< --___+~_---_pPEl2

-____,c____t___sc_-______I f---_--__

pMEl612

t_------

pM6G

--_----_+

I

Fig. 2. Strategy

by dashed sequcncin~

I

and nucleotide

sequencing.

sites used in primer extensions

The coding area is boxed. pMG293 indicated

4 ?

+_----_-------_-_-_

for cloning

Only the restriction

didcoxy

___--

G------

contains

The top line represents

I1 1 I-nt segment of the porcine mpoglobin

a

are shown below. The PstI site in parentheses

poly(A),,

at the 3’ end. pMB6 was constructed

arrows

were determined

by the technique

(Sanger

ct al., 19X0). Isono’s programs

of Maxam

and Gilbert

was generated

from pMEI2

and pME1612.

(1980). The sotid arrows

(1984) were used for computer

analysis

cDNA.

by the cloning procedure. The sequences

indicate

the results of

of nt sequences.

300 nt coding region (Fig. 3, nt l-359). Finally, I constructed a clone pMB6 (Fig. 2 and Fig. 3, nt 46-619) covering the entire aa sequence by ’ ligating a PstI-SphI fragment (Fig. 3, nt 46-342) from pME1612 to the SphI-XVII fragment (Fig. 3, nt 343-619, oligo(G),,, and a PstI-XWII pBR322 fra~ent) from pMEl2. (d) Primary structure of the myoglohin

message

Fig. 3 shows the nt sequence of myoglobin cDNA and its deduced aa sequence. One ORF of 462 nt corresponds to 153 aa plus initiation codon. The deduced aa sequence agrees with that from protein sequencing (Rousseaux et al., 1976). Since the initiation codon ATG immediately precedes the N-terminal codon, the first 59 nt are the 5’-untranslated region. This 59 nt corresponds to those spanning 12 through 70 in the human and seal myoglobin cDNAs (79-8 1% homology) (Fig. 3). Since the first 11 nt of the human and seal myogiobin cDNAs are completeiy identical, the porcine myoglobin cDNA also Fig. 3. The nt sequence comparison cDNAs

of the porcine

of this sequence

in the 5’-untranslated

quence of the strand

and coding

corresponding

from the 5’ to the 3’ direction. above the coding sequence.

myoglobin

cDNA

and

with the human and seal myoglobin regions.

to the mRNA The aa sequence

The aa and nt sequence

The nt se-

shown

above

and at the right of each

rcfcrs to the initiating product.

(Ter) refers to the terminator

is displayed

pentanucleotidc,

is displayed

the 5’.untranslated

numbers

nucleotides

are

line, respectively.

Met not associated

AATAA,

codon

for poly(A) addition

and coding

UAA.

sequence

A signal

is underlined.

regions,only human

which differ from porcine

(Ini)

with the final protein In

and seal

are indicated.

140

may

have

eleven

more

heart poly(A)+ (522-l

RNA (not shown).

111) consist

a possible

polyadenylation

of pig

The last 590 nt

of 3’-untranslated signal,

sequence

and

AATAA,

at

tcriophagc L.omcdico.

Maniatis,T.,

tant for the functions

of the mRNA.

package

protern

T4, I. DNA

pxkagm#

I’. I. and Saunders.

well with those of the human and /or seal myoglobin cDNAs (not shown). The very highly conserved to be impor-

and

for

storing

sequence

data.

and Nucl.

of the hc:td 01 bat-

events.

J. Mol. Biol. X0

([V’i) i:~-ylw

nt 1091. This region also can be aligned reasonably

could be inferred

program

DNA/RNA

Acids Res 13 (19X3)101-I 11. I.ucrn~nli.U.K. and Favre. Iv.: Maturation

mRNA:

region (nt 610-680)

.A computer

retrteving

( 1122 nt) coincides blot analysis

K.:

Isono,

nt in its 5’-untranslated

region. This predicted length with the result from Northern

cell-free

polypeptide. A Laboratory Maxam,

i~i‘pancrc:ittc

of an i~~sulin-imntunorc;~ctive

Nucl. Acids Res. 3 (1976) 3X1-391.

Fritsch,

Spring

G.F.: Prcparatnm

translation

L.F. and Sambrook,

Manual.

Harbor,

J.. Molecular

Cold Spring Harbor

Cloning.

I.aboratory,

Cold

NY, 1982.

A.M. and Gilbert,

with base-specific

W.: Sequencing

chemical

cleavages.

end-labclcd

Methods

DNA

Enzymoi. 65

( 1980) 499-560. Morrison,

D.A.: TraIlsfornlation

bactcrtal

i”tC:KNf)\it’LEDGEhlENTS

ceils

3’6-33

Thanks are due to Dr. K. Matsubara for his hospitality, discussions and encouragement, Dr. S.M. Heywood for mRNA purification and Mr. T. Wakabayashi for his advice in the synthesis of cDNA. I am grateful to Dr. K. Isono for supplying computer programs and Mr. Y. Katsuki for help with computer analysis. I thank Dr. Y. Sakoyama for reading the nlanuscript.

Paterson,

B.M., Roberts,

cell-free

translation.

Rousseaux.

acid

ungulate

sequence

33

.A.. Wtlson, V., Wood, D. and Jeffreys.

myoglobin

gene: an unus~~ail~ long &bin

A.J.: The seal

gcnc. Nature

301

colony

Han, K.: Comparrson

and

Biochnn.

( 1977)

mqoglobin

Biophys.

Acta

with

ofthc othct

4.79 ( 1976)

55-62. of nucleotides

of DNA. Mcthodr

65 (1980) 43-62.

F., Coulson,

A.R.. Barr&l. B.G.. Smtth. A.J.H. and Rot.

in single-stranded

DNA sequencing.

zation

transfcrase-~3t;ii~z~~{

to the 3’ tcrmim

bacteriophage

as an aid to

J. Mol. Biol. l-13 (1980)

A.J.. Wilson. V. and Blanchctot.

of the human

myoglobin

Wood,

D.. Blanchetot.

of seal

m~o~lobin

gene.

EMBO

161-17X. A.: OrganJ. 3

A. and Jeffreys,

A.J.: Molecular

mRNA.

Acids

7133-7144

(1983) 732-734. Hanahan.

gcnc

(I’lX3)

439-446.

(19X5) 241-249. Blanchetot,

M.

of pig heart

myoglobins.

Weller, P.. Jetfreys. gene. Gene

Structural

mRNA hybrid-arrested

R~~~~l~oudhur~. R. and Wu, R.: Terminal

rapid my&bin

by DNA

Proc. Nutl. ,Acad. Sci. USA 74

_I., Dautrevaux,

amino

B.A.: Cloning

of the human

6S (1979)

4370-1374

Sanger,

E.: Cloning

ot.~[)l~lp~t~llt

Enzqmol.

B.E. and KuB’, EL.:

identiticatr~~n and mapping

Enzymol.

Akaboshi.

Methods

t.

addition

REFERENCES

and prcscrvation

by freezing.

D. and

Meselson,

density.

Gene

if)

M.: Plasmid

(1980)63-67.

screening

at high Communicated

by H. Yoshikaua

Nucl.

Res.

cloning

lit (1982)