Rat aldolaseA messenger RNA: the nucleotide sequence and multiple mRNA species with different 5′-terminal regions

Gene. 39 (1985) 17-24

17

Elsevicr GENE

1407

Rat aldolaseA messenger RNA: the nucleotide sequence and multiple mRNA species with different 5’ -terminal regions (Recombinant

DNA;

cDNA

clones;

isozyme;

primer extension;

skeletal muscle)

Keiichiro Joh, Tsunehiro Mukai, Hitomi Yatsuki, and Katsuji Hori* Department

of Biochemistry,

(Received

April Sth, 1985)

(Revision

received

(Accepted

Saga Medical

School, Nabeshima,

Saga

840-01

[JapanJ Tel. (0952)

31-651 I

July 2nd. 1985)

July 3rd, 1985)

SUMMARY

The nucleotide sequence of aldolase clones and a cDNA synthesized

A mRNA in rat skeletal muscle was determined by primer extension. The sequence is composed

cDNA

using recombinant of 1343 nucleotides

(nt) except for the poly(A) tail. Based on the sequence analysis we have deduced an open reading frame with 363 amino acids (aa) (A4, 39 134). The sequence suggests several nt polymorphisms in the mRNA population, one of which causes an aa change. The determined sequence of rat aldolase A mRNA was compared with the published ones of rabbit aldolase A or rat aldolase B mRNAs. The homology between rat and rabbit aldolase A mRNA sequences is greater than that between rat aldolase A and B mRNA sequences. Multiple aldolase A mRNAs having different M,.s were detected in the various tissues, and appeared to be expressed in a tissue-specific manner. Further analysis suggests that differences in mRNA length are due to differences in the 5 ’ -noncoding terminal region.

INTRODUCTION It is well known that the multiple molecular of enzyme, “isozyme” (Markert and Moller,

* To whom

correspondence

and reprint

requests

forms 1959),

should

be

addressed. Abbreviations: cneglycol Hepes, kilobases

EGTA, ethyl-

ether)-N,A’flfl’-tetraacetic

acid;

4-(2-hydroxyethyl)-I-piperazineethanesulfonic or 1000 bp; nt, nucleotide(s);

poly(A) + RNA, sulfate;

aa, amino acid(s); bp, base pair(s);

bi@aminoethyl

polyadenylated

RNA;

acid; kb,

PA, polyacrylamide; SDS,

sodium

SSC, 0.15 M NaCI, 0.015 M Na. citrate,

0378-l 119~85.‘$03.30 0

1985 Elsevicr

Science

dodecyl

pH 7-8.

Publishers

exhibit tissue- or cell-specific patterns. That is,the isozyme patterns are different in different tissues and characteristic for the stages of differentiation in each tissue. Fructose 1,6-diphosphate aldolase (EC 4.1.2.13) is a glycolytic enzyme that catalyzes reversibly cleavage of fructose 1,6-diphosphate to glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. Three isozymic forms of the aldolase have been reported in vertebrates (Horecker et al., 1972): aldolase A (muscle type), aldolase B (liver type), and aldolase C (brain type). The isozymes are different in their catalytic, electrophoretic, and antigenic properties and have been studied extensively with respect

IX

to the tissue

distribution

development

and

1972; Lebherz Because

and the changes

carcinogenesis

and Rutter,

of their

during

(Horecker

et al.,

1969).

characteristic

expressions,

it

alcohol (24 : 1) followed by LiCl

chloroform-isoamyl

precipitation, as usual. The poly(A) + RNA was isolated by oligo(dT) cellulose chromatography. Enrich-

seems that rat aldolase isozymes are an ideal material

ment of aldolase A mRNA successive centrifugations

for studying

a regulatory

gradients

sion during

development,

cinogenesis.

Furthermore,

mechanism

of gene expres-

differentiation the aldolase

offer a useful evolutionary

and carisozyme

will

model for understanding

the process

by which closely related genes and their

regulatory

mechanisms

have

diverged.

For

types in a species and genes of same type

in different

species.

cDNA clones and its nucleotide sequences have been reported for rat aldolase B (Tsutsumi et al., 1984a), rabbit aldolase A (Tolan et al., 1984) and human aldolase B (Rottmann et al., 1984; Paolella et al., 1984) mRNAs. Here, we describe the cloning and characterization of cDNA for aldolase A mRNA from rat skeletal muscle and also describe an analysis of the mRNAs with a distinct molecular size from the various tissues.

MATERIALS

AND MEI’HODS

(a) Materials Avian myeloblastosis virus reverse transcriptase was purchased from Life Science Inc. and nuclease Sl was from Boehringer Mannheim. Restriction enzymes and other enzymes were from Bethesda Research Laboratories Ltd., New England Biolabs, or Takara Shuzo Co., Ltd. Radioisotope-labeled compounds were obtained from Amersham. (b) Purification

rev./min 26°C

w/v, sucrose in 807” formamide,

pH 7.6 and 3 mM EDTA)

in a Beckman if necessary.

estimated

by two density at 37000

SW40 Ti rotor for 48 h at

The extent of enrichment

to be 3-4 fold by in vitro translation

was assay.

these

purposes, it is necessary to clarify the structural relationship of the relevant genes, that is, genes of different

(5-20%,

3 mM Hepes,

was performed on sucrose

(c) Construction Construction

and isolation of cDNA clones of cDNA

clones

using

a pBR322

vector and colony hybridization were performed as described previously (Tsutsumi et al., 1984a) with slight modifications. Depending upon the probe DNA used, one of two conditions with different stringency were chosen for the hybridization experiments as follows. Hybridization was carried out in a solution containing 1 x Denhardt’s solution (Denhardt, 1966), 50 mM Na. phosphate, pH 6.5, 200 pg/ml denatured salmon sperm DNA, 0.1% SDS and 6 x SSC at 55°C for aldolase B cDNA as a probe or in the same solution except for 4 x SSC at 65’ C for aldolase A cDNA as a probe. After hybridization, the filter was rinsed with 2 x SSC, 0.1 y0 SDS at room temperature and then washed twice with the same solution at 42’ C for B probe or 65 ’ C for A probe. (d) Primer extension The DNA fragment and poly(A)+ RNA were hybridized as described with some modifications (Maniatis et al., 1982). The hybrids were used to synthesize cDNA with reverse transcriptase as described (Tsutsumi et al., 1984a).

of poly(A)+RNA (e) Cleavage of mRNA with RNase H

Skeletal muscle of male Wistar rats was pulverized in a Waring blender filled with liquid nitrogen. The powder was suspended at 1 g/20 ml in 50 mM Tris . HCI buffer, pH 9.0, containing 0.3 M KCl, 10 mM MgC12, 5 mM EGTA, and 1 y0 SDS and mixed with an equal volume of phenol-chloroform-isoamyl alcohol (25 : 24 : 1 v/v), and the total RNA was extracted. The RNA was further extracted with phenol-chloroform-isoamyl alcohol and then with

Poly(A) + RNA and cDNA were hybridized as in section d. The resulting hybrid was digested with RNase H at 37°C for 30 min in 50 ~1 of 10 mM Tris HCl pH 7.6, 0.13 M NH,Cl, 10 mM Mg acetate, 5% sucrose and 2 units of RNase H (P.L. Biochemicals). After phenol extraction and ethanol precipitation the digests were analyzed by blothybridization using an appropriate 5’ or 3’ probe.

19

(f) Nucleotide Labeling

sequencing

and sequencing

of DNA by the method

of Maxam and Gilbert was performed according to a protocol (Maxam and Gilbert, 1980; Maniatis et al., 1982). For DNA chain terminator cloning

sequencing

method,

vector.

Sequencing

scribed (Messing,

M13mp8

by the dideoxy was used as a

was performed

as de-

1983) with some modifications.

Fig. 1. Restriction quencing RESULTS

maps

strategy.

of rat

Aldolase

shown as a solid bar (coding

(a) Aldolase A cDNA clones from rat skeletal muscle poly(A) + RNA

body method and electrophoresis on a SDS-PA gel (not shown). A possible aa sequence deduced from the nt sequence of one of the clones, pRAAM 1 (Fig. l), was found to be highly homologous to that of rabbit aldolase A reported by Lai et al. (1974). Therefore, we concluded that pRAAM1 was a rat aldolase A cDNA clone. Two clones, pRAAM40 and 83, were obtained by colony hybridization using pRAAM1 as a probe (Fig. 1). Their inserts were 1.2 kb long excluding the poly(A) sequence and covered 90% of the mRNA. The third clone, pRAAM103, whose cDNA insert was 0.55 kb long and covered

73% (48 nt) of the

5’-noncoding region (66 nt) and part of the coding region of the mRNA, was obtained by primer extension (Fig. 1). (b) Nucleotide

sequence of aldolase A mRNA

Most of the nt sequence of aldolase A mRNA in rat skeletal muscle was determined by sequencing

A cDNAs

and

of rat skeletal

se-

muscle is

region) and open bars (noncoding

regions)

at the top. The cDNA

positions

relative

to the mRNA.

inserts

are depicted

The restriction

in the

sites indicated

are: PslI (Ps), Pvull (Pv), Hinfl (H), TuqI (T) and Hue111 (7). of Hue111 sites in the pRAAM40

The mapping

been done yet. The sequencing

Poly(A) +RNA from rat skeletal muscle was enriched for aldolase A mRNA and used to construct cDNA clones as described in MATERIALS AND METHODS, sections b and c, respectively. The cDNA clones were screened by colony hybridization using rat aldolase B cDNA (pRAB3031; Tsutsumi et al., 1984a) as a probe at a low stringency. Several hybridization-positive clones were obtained and identified as an aldolase A cDNA clone by hybrid selection and translation experiment as described (Maniatis et al., 1982) followed by the double-anti-

aldolase

A mRNA

arrows.

The cDNA

fragment)

and

pRAAM103 pRAAM83 box and

fragments

probe

(see

arrow

DNA (ZzP-labeled

under

fragment)

section

as a filled and a hatched broken

insert

is indicated

used as a primer

(PsrI-HinfI

RMIJLTS,

a)

(Hid-HaelI for

are

cloning

shown

box, respectively.

pRAAM103

has not

by horizontal

indicate

portion

synthesis,

respectively.

of the mRNA

and the direction

This DNA fragment

of

under An open

a primer

at the 5’..4luI end) used for sequencing

5’-terminal experiment

strategy

ofthe

of cDNA

was also used for the

showsn in Fig. 6.

the cDNA inserts of pRAAM1, 83 and 103 (Fig. 1). To determine the nt sequence corresponding to the 5’ terminus of the mRNA, which appears to be missing in pRAAM103, a cDNA was synthesized using the primer shown in Fig. 1 and sequenced. Fig. 2 shows the nt sequence of mRNA for rat skeletal muscle aldolase A and the aa sequence deduced from the nt sequence. The mRNA has a chain length of 1343 nt except for the poly(A) tail and encodes 363 aa for aldolase A. Several microheterogeneities were observed among the cDNA inserts of pRAAM40, 83 and 103 in their restriction sites and nt sequences, as shown in Figs. 1 and 2. The variation in the codon at position 90 causes an aa change, GCC (pRAAM83) for alanine and GGC (pRAAM103) for glycine. There is no discrepancy between pRAAM1 and 83 so far as determined. (c) Comparison of rat aldolase A with rabbit aldolase A and rat aldolase B The aa sequence of rat skeletal muscle aldolase A deduced from the nt sequence of the mRNA is in

ala GCC

Glu GAA

Gly GGC

Ala GCA

Ala GCU

Gin CAG

Gly GGA

Val GUG

Cys UGU

Met Val GUG

Gly GGG

Glu GAA

Gly GGU

Pro CCA

Tie AUU

Val GuU

Arg ccc

Ile AUC

Asp GAU

Leu CUG

Val Thr GIJC ACA

Asn AAU

Leu CUG

Leu CUC

Trp uGG

Thr ACU

Tie AUC

Gfy GGC

Gly GGC

Lys AAG

Ile AUC

Leu CUU

Thr ACA

His CAU

Asn AAU

Gln CAG

Gln CAG

Lys AAG

Rsn AAC

Arq CGU

Leu CUG

Asp GAC

Ala GCC

Tyr UAU

Ile Lys AIJIJAAG

Val GUG

Ile AUU

~I Asp GAC

Asn AAC

Cjy GGA

Ala Ser UCU

Cys UCU

Thr ACA

Lys AAG

Glu GAG

Lys AAG

Arq CGA

Leu CUG

AAG

UUG

Phe Ser ~~Ala

leu CUG

Ala GCC

Pro CCU

Thr KU

Ser AGC

Glu GAA

Gln CAG

Gly GGG

Gly GGC

Glu GAG

lie AUC

Lys AAG

Ala GCU

Cys AAG

Arg CGC

G!y GGC

Glu GAG

Ser Leu IJCU CUC

Glu GAG

Trp Ala IJGG GCC

Thr Val Pro GUC: KU

Thr XC

Val GUA

Ala GCC

I.ys Pro AAG CCA

Ala GCC

~Val GUC

Tyr UAU

Tyr UAC

P-.-1 Ala Ser GCC AGU

320 Leu Lys CIJG AAG

Ala GCA

Ala GCA

Leu CUG

290 Pro CCC

350 Ala GCC

Leu CUG

Fro CCC

Pro CCU

Met AUG

Gln CAG

Arg CGU

Val GUG

260

Asn AK

230 Pro CCC

Cys

200 Arg

-~ Lys UGC

Ala GCC

170 Val Leu GULJ CUG

CGC

Gly CGA

140 Asp GAU

Lys AAG

Val Pro GIJG Ccc

Gin CAG

Arq AGG

Gnc U~IJ Gee

Val GUA

Gly GGU

110 Lys AAG

Asp GAU

Leu

Asn AAU

Lys AAG

Val GUA

Leu Tyr CUG UAC (A)

Asn AAC

Gee

Thr ACA

80 His Glu CAC GAG

Glu GAG

Ala Pro GCIJ CCG

Asp

Glu GAG

50 Asn Thr AAC ACC

Glu GAG

Val GUA

Ala

Ile AUU

20 His Arg CAC CGA

Ala GCU

Leu Phe ClJIJUUC

Thr ACC

Ile AUC

AGCCCACUGCChAUAAACAGC~JAUUUAAGGGGnnAAnnnAAA----------

Ser AGU

Gly GGG

Asp GAC

Gly GGC

Gly GGG

Ser UCC

Glu GAG

Lys AAG

-Ser Lcu Ala CUC, GCU Gly GGC

30 Leu Ala CUG GCU

ala GCC

Thr KU

Gln CAA

240 Thr ACC

210 Ala GCA

360 Asn AAC

330 Arg CGA

Val 1l.c Lys AUC AAG

His CAU

Ala GCC

Lys AAG

Pro CCC

Ala GCU

Ile AUU

Gly GGA

Ala GCC

363 Tyr UAC

Lcu Ala ClJG GCC

Arg CGA

Gly GGG

tcr UAA

Asn AAC

Ala WC

GLn CAG

Glu GAG

Glu GAG

Leu CUG

Leu CUC

His CAC

Glu GAA

Ser UCG

Gly GGG

Ser KC

Val GUG

Ile AUU

Ser UCU

Glu GAG

Gln CAA

Ala GCU

Ala GCA

Ile Ala AU11 GCC

Asp GAC

Ala Cys GCrJ UGU

ala GCC

GAA

Glu

Glu GAA

Ser AGU

Pro CCU

Pro ccc

Asp GAC

Lys RAG

Arq CGU

Ser AGC

Pro CCG

340 Gly GGA

310 Leu CUA

280 Ser KC

250 Met AUG

220 His CAU

Glu GAG

Gly GGCJ

Cys LIGC

Arq CGC

Lys AAG

Lys AAG

Lys AAG

Ile AUC

Ala GCA

Ile Val GUC

{Le~clld o*ipage21,

Tyr []A('

Ala WII

Asn AAC

Thr AC(

Tyr UA[I

Pro CCIJ

Leu Ala CIIC GCC

Ser UCU

Gly GGU

190 Ile Leu AUU CUC

130 Leu CUG 160 Ala Ser ucc

100 Lys AAG

70 Asn Pro AAU CCC

40 Ala Lys GCC AAG

10 Glu Gln GAG CAG

CChGAGCUGAlJClJAAGGCUGCUCCAUCGACA

Ser AGC

Thr ACU

Ceu CUG

Ile AUC

~Asp GAC

Gly GGA

L,eu Thr CUG ACC

Val Glu G[JG GAG

His CAU

Gly GGG

Val GUU

Asp GAU

Thr ACU

Ala GCA

T,eu Gln CIIG CAG

Ser AGU

Tyr ~- His 1,~s Phe Ser Asn AAA lJUU IJCC AAU

Tyr Gly [JAI1GGC

Ser UCU

Gln CAG

Ala Val Tyr GClJ GIJC UAC

Ile AUU

Val GUA

Thr ACU

180 Asn Gly AAlJ GGC

300 Ser UCC

Str UCU

Thr ACU

Ser UCC

--~~~ Phc Pro Gin IJUC CCC CAA

Leu CUG

Glu GAG

Pro CCA

Ile tily Glu RUIJ GGG GAG

Thr ACC

Pro CCC

Leu CUG

Asp GAC

His Tyr UAC

150 Val T,eu Lys GIJC CUA AAG

270 Phe Leu UIJC CUG

Cys UGC

Lcu CUC

Gln CAG

Cys mxi

Arg CGC

Ala GCA

1 Ser Pro 111s Pro CCC CAC CCA

60 _.__ Gln Leu CAA CUG 90 (Ala) Asp Gly Arg GAU GGC CGU (C) ~_~~ -120 Asn Gly Glu AAU GGC GAG

Ile AUC

Phe UUC

Thr AYU

Thr ACU

Ala GCU

Val GUA

Gln CAG

Arg CGC

Thr ACC

~.. Asp GAU

Phc Ilr lJUC AK

Tyr UAC

Ltu UUG

Val GUC

His CAU

Lys AAG

Cys UGC

Trp uGG

Gly GGA

~-Ala GCA

-Phe Tyr UlJC UAC

Lys AAG

NNNNGCIJGCUGACCAGGCUCIJGCGGCUUCllIJUCAC!JGCACCAc:AGcAAAGCGCUGCCACCGGCACC

Met AUG

21

agreement

with that of rabbit

from the mRNA for

12 aa,

homology

M

aldolase A deduced

indicating (97%).

remarkably

There

high

sequence

S

H

M

28S+

is also a high sequence

homology between the noncoding regions, especially between the 3’ regions, although they are considerably

B

sequence (Tolan et al., 1984) except

23S-,

different in length (Fig. 3). This size differ-

ence seems to be caused sequence and/or seen in Fig. 3.

by insertions

deletions

in the rabbit

in the rat sequence,

18s-, 16S+

as

Comparison of the sequence of rat aldolase A with those of rat aldolase B (Tsutsumi et al., 1984a) shows that many aa substitutions occurred between these enzymes (Fig. 2). The homology between the aa sequences was 70%. The 3’-noncoding regions of the mRNAs for rat aldolases A and B are quite different in length (182 nt for aldolase A and 384 nt for aldolase B) and have no significant sequence

Fig. 4. RNA various cellulose agarose

filters

of aldolaseA

Poly(A)‘RNAs

after

mRNAs

expressed

were blotted

electrophoresis

gels (Maniatis

translated

homology. On the other hand, the 5’-noncoding regions are similar in length; yet no significant sequence homology was observed.

blot-analysis

rat tissues.

on

pRAAM83

[32P]cDNA

insert

pg), brain (B, 5 pg), heart

(H, 5 pg) and spleen (S,I5pg).M,

region

: NNNNGCUGCUGACCAGGCUCUGCGGCUUCUUUCACUGCACCAC AGGAAAGCGCUGCCACCGGCACC . . . _.. . .._. . . . . . . ..-. .._..._e... ..-....--.. ACUACUGCUGACCAG CUCUGCGGCUCCUACAGCCUCGCCGCAAGGAACU UGCUACCAGCACC Rabbit:

AUG---

Rat

3.noncoding : --UAA --UAA

Rabbit:

.._

..

.-..._..

..

..

.-

AUG---

region

CCAGAGCUGAUCUAAGGCUGCUCCAUCGACACUCCAGGCCCCUGCCU

.

(Mukai

muscle (M, 0.25

markersarerRNAs.

5 noncoding

Rat

?a

with a nick-

as described

RNAs are from skeletal

in

nitro-

formaldehyde-l

et al., 1982) and hybridized

et al., 1984). Poly(A)+

onto

..--.._--..

ACCCA

. . ._

.. ..

GCGGAGGUGUUCUAAGGCCGCCCCCUCAACACUCCACUCCAGGCUCCAGCACCGGCCCCCCUACACACACA

CUUGCUAUUGAAGAGGG GCCUU CAGGCUC UUUCCCAUCACUCUUGCUGCCCUCGUG . . . . . .-........ . . .. .-...m _..._.. _..._..._ _...__.. CUCGCUCUUGAAGAGGGAGCCUUCUUGGGGGUUCCAGGCUGUGUUUCCCACCACUCUUGCCUCCCUCGUGACUUUGG

UGUGCAGUGUUGUCUGUGAAUGCUAAAUCUGCCAUC CCUUCCAGCCCACUGCCAAUAAACAGCUAUUUAAGGGG . ... . . ..-...-. . . . . - . . . . . . . . _.._..._....._..._..._............._ UGUGUGGUAUUGUCUGUGUACGCUAUAACU CCAUCACUUUCCAGCCCACUGCCAAUAAACAGCUAUUUAAGGGGG Fig. 3. Homologous and rabbit

mRNAs

sequences

and 221 nt, respectively.

aa sequence indicate

of aldolase

ofthe pRAAM83

A mRNA

indicate

aldolase

The 3’ sequences identical

from rat skeletal

in the mRNA sequence of the primer

region is also underlined.

mRNAs

aldolase

A are shown

previously

are from the pRAAM103

used for the primer extension

was determined

are unknown

and 103 inserts are shown. Those nt in parentheses

the HhaI site, which is a 3’ terminus

The 5’-noncoding

AAA--

sequences

of the rat

are composed

of 182

nt.

muscle. The sequence

aa in rabbit B reported

A mRNAs.

of the rat and rabbit

The initial four nt of the 5’ terminus

from those of rat aldolase

insert; the nt at the same positions signal in the 3’-noncoding

of rat and rabbit

A is also shown. The different

those that are different

the sequences

two sequences

and 103, and by primer extension. of rat aldolase

regions

of 66 and 62 nt, respectively.

Dots between

Fig. 2. Nucleotide sequence pRAAM83

of the noncoding

are composed

AAAA--

(Tsutsumi

from the cDNA

and are indicated

inserts

of

by N. The deduced

above the rat sequence.

Bars on the aa

et al., 1984a). Two discrepancies

between

under codons No. 83 and 90 are from the pRAAM83 insert. GCGC experiment

in the 5’.noncoding

described

region indicates

in Fig. 6. The polyadenylation

22

(d) Aldolase A mRNAs regions

with different 5’-terminal

reported

(Mukai

for the mRNA Aldolase

A mRNAs

sues were analyzed

expressed

smaller

in various

by blot-hybridization

The aldolase A mRNA slightly

than

(Fig. 4).

from skeletal

those

from

rat tis-

muscle

other

was

tissues

as

A cDNA

3'

Tsutsumi

et al.,

to be approx.

1.55 kb

from skeletal muscle and 1.60- 1.65

kb for those from other tissues. We performed

an experiment

analogous

to that

described by Hagenbtichle et al. (1981) to locate the sequence that is responsible for the size difference. The aldolase A mRNA was hybridized with the

portion

mRNA

by digestion

RNA fragments

1

RNaseH

ization

with RNase

H. The 5 ’ and 3 ’

were then analyzed

with appropriate

by blot-hybrid-

5’- and 3’-specific

probes

prepared from the cDNA clones (Fig. 5). A size difference of approx. 50-100 nt was detected with the mRNA fragments bearing the 5’ end, but not with the 3’ fragments. For further analysis of the 5’

B a 1

1984;

220-bp HinfI fragment of pRAAM83 and cleaved into two fragments by removing the hybridized

fragment

w

5’

et al.,

1984b). The size was estimated

portion of the mRNA, cDNA was synthesized on the mRNA by primer extension using the Hhal-A/u1 cDNA fragment of pRAAM103 (Fig. 1) as a primer

b

and analyzed on a urea-PA gel (Fig. 6). Three major cDNA products, designated as I, II and III, whose sizes were estimated to be 110, 160 and 205 nt, respectively, were observed in the mRNAs from different tissues. The major cDNA product of the mRNA from skeletal muscle was cDNA I, whereas those of mRNAs from other tissues were cDNA II and III. The size difference between cDNA I and II or III was approx. 50-100 nt, which corresponded

12

2

-

1630

-

520

-

M

-

H

B

S

L - 1631

-

Fig. 5. Location (A) Schematic molecules

of size

220

in aldolase

of the procedure

into 5’ and 3’ fragments

fragment

-

difference

representation

J510 394 - 344 - 290

with RNase

used is the 220.bp ffinfl fragment

insert (see Fig. 1). (B) Blot-hybridization ments of aldolasc and

ascites

express heart,

a longer aldolase mRNAs

gels and blotted hybridized

onto GeenScreen

species

(bp) are Hbzfl-digested

(lane 1)

is known

on glyoxal,‘2”,, filters (NEN).

pBR322.

Fig. 6. Analysis

of cDNA

results).

minal region ofaldolase

agarose

DNA

Filters

ofpRAAM

is shown

products

synthesized

on the 5’-tcr-

A mRNA by primer extension.

in Fig.

I. Poly(A)’

RNAa

are from

A primer skeletal

were

muscle (M, 2 keg), heart muscle (H. 4 pg), brain (B, 4 pg), spleen

103

(S, 15 llg), and adult liver (L, IO pg). 0.08 pmol of the primer

probe or (b) 460-bp P.ul fragment

insert as a 3’-specific

primeL

to

like those of brain,

with either of(a) 1X0-bp PstI fragment cDNA

muscle

et al., 1984 and unpublished

were electrophorcsed

cDNA

of the 5’ and 3’ frag-

(lane 2), which

A mRNA

insert as a 5’-specific

of pRAAM83

H. The cDNA

of pRAAM83

from rat skeletal

AH60C

and spleen (Mukai

Cleaved

cDNA

A mRNAs

hepatoma

A mRNAs.

to cleave mRNA

probe. Size markers

DNA

was hybridized

products

with each

were subjected

gels (Maxam

and Gilbert.

poly(A)

to elcctrophoresis

’ RNA. The cDNA on 50’1,J urea:X”,, PA

1980). Size markers

are as in Fig. 5.

23

to the differences between seen in Fig. 4 and 5.

the sizes of the mRNAs

between

cDNA

I (110 nt) and cDNAs

and 205 nt) is in agreement between

aldolase

A mRNAs

II, III (160

with the differences in skeletal muscle and

other tissues observed in Figs. 4 and 5. If this is the case, the difference between two aldolase A mRNA DISCUSSION

species is located

in the region upstream

from the

HhaI

site (3’ terminus of the primer DNA) in the 5’-noncoding sequence. When aldolase A mRNAs

We have isolated several cDNA clones corresponding to a nearly entire region of aldolase A mRNA from rat skeletal muscle. We observed that

in skeletal muscle and brain were blot-hybridized with the cDNA fragment upstream of the HhaI site

there were several microheterogeneities in the nt sequences among the cDNA clones analyzed (Fig.

in the 5’-noncoding sequence of pRAAM 103 insert, a hybridization band was observed in skeletal muscle

2). It is most likely that these heterogeneities

poly(A) + RNA but not in brain poly(A) + RNA (not

resulted

from a heterogeneous mRNA population arising from allelic variation, since the mRNA used for cDNA cloning was prepared from many rat individuals. The observed aa change (Fig. 2) does not affect the net charge of the protein and probably not the enzymatic properties. Such allelic variation or polymorphism may be ubiquitous in eukaryotic genomes, since electrophoretic variants which arose from allelic variations have been reported for many enzymes or proteins of vertebrates, including man, and insects such as Drosophila (Johnson, 1977; Koehn and Eanes, 1979; Kreitman, 1983). Aldolase A mRNA in skeletal muscle is slightly shorter than those in other tissues (Fig. 4). This difference is mainly in the 5’ portion of the mRNA, not merely in the length of the poly(A) tail because, as seen in Fig. 5, only 5’ fragments of the mRNAs show a size difference which agrees well with the difference between the intact mRNAs observed in Fig. 4. It is therefore concluded that at least two species of aldolase A mRNA, whose 5’ portions are of different lengths, are expressed in a tissue-specific manner; i.e., the shorter one is in skeletal muscle and the longer one is in other tissues. Furthermore, three cDNA products with different lengths were produced by primer extension (Fig. 6). Although we cannot exclude the possibility that these cDNA products were produced by multiple premature terminations of cDNA synthesis on an aldolase A mRNA, it is more likely that cDNA I was synthesized on the shorter mRNA abundant in skeletal muscle and cDNA II and III were on the longer one abundant in other tissues by the following reasons. First, cDNA I is a major product only with mRNA from skeletal muscle and a very minor one with mRNAs from other tissues. Second, the size difference

shown). This observation suggests that two aldolase A mRNA species differ not only in length but in the nt sequence of the 5’-noncoding region. Comparing the rat and rabbit aldolase A mRNAs, highly homologous sequences were found in both the 5’- and 3’-noncoding regions, as shown in Fig. 3. In contrast, no distinct homologous sequence was found in either noncoding region between rat aldolase A and B mRNAs. The divergence of aldolase A and B genes should have occurred before the appearance of vertebrates, i.e., prior to the divergence of the rat and rabbit aldolase A genes, because these isozymes have been found in all the vertebrates so far examined (Lebherz and Rutter, 1969). Therefore, the presence or absence of homologous sequences in the noncoding regions may be explained by the difference in time after divergence ofthe genes. Alternatively, it may also be possible that the common sequences in the noncoding regions of the aldolase A mRNAs carries a regulatory function specific for aldolase A gene expression.

ACKNOWLEDGEMENTS

an

We thank Dr. M. Ikehara for generously providing antibody against rat aldolase A and Dr. Y.

Nabeshima for valuable suggestions about the preparation of RNA from skeletal muscle. This investigation was supported in part by a Grant-in-aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.

24

Markert.

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