GPlW, 104 (1991) 177-3x2 4~’ 1991 Elsevier

GENE

Science

Publishers

B.V. All rights reserved.

0378-l 119,91 $03.50

277

04051

Structure of the entire human muscle phosphofructokinase-encoding (Recombinant

Tomoyuki

DNA;

exon-intron

boundary;

alternative

splicing;

Yamasaki”,

tissue-specific

Hiromu Nakajima”, Norio Kono”, Kikuko Hotta”, Kuwajima a, Tamio Noguchi b, Takehiko Tanaka’ and Seiichiro Tarui a

gene: a two-promoter system gene expression:

Kazuya Yamada”,

glycogenosis)

Enyu lmai “, Masamichi

” Second Depurtrnent ofh~terncd Medicirle. Ostrk~~University Medical School, Fukushinm-ku, Osuka 553 (Japm), and” Departrmwt cfNutritiotr crud Ph~~siological Chernistqs, Osukr! Uni\wsitjb Medical School. Suitu 565 (Japan) Tel. (Sl-06)877-5 I I I, est. 5435 Received by H. Yoshikawa: I3 August Revised: 25 December 1990 Accepted: 5 January 1991

1990

SUMMARY

WC have recently shown that three types (A, B, and C) of mRNA species are transcribed from a single gene encoding human muscle phosphofructokinase (hPFK-M) through alternative splicing [Nakajima et al., Biochem. Biophys. Res. Commun. 166 (1990) 637-6411. To determine its complete structure and elucidate the mechanism of alternative RNA splicing, we isolated the hPFK-M gene, which spans about 30 kb, and contains 24 exons. Transcription start points were observed for both exon 1 and cxon 2 by Sl nucleasc protection assay and primer extension. Motifs of an Spl-binding site were observed in the upstream region of exon 1 (promoter I). A TATA-box-like sequence and a CAAT-box-like sequence were identified in the upstream region of exon Z (p romoter 2). Reporter assay revealed that the promoter 1 region was functional both in I1cl.a cells and myoblastic clonal cells, and that the promoter 2 region was active only in myoblastic cells. Motifs of M-CAT knoun as a muscle-specific enhancer, were observed in the promoter 2 region. These results indicated that the hPFK-iZI gene contains at least two promoter regions, facilitating the expression of the heterogeneous gene transcripts in a cell-type-specific manner.

INTRODIJCl‘ION

Phosphofructokinase (PFK I. ATP : r>-fructose-6-phosphate-1-phosphotransferase; EC 2.7.1.11) is a key enzyme in glycolysis (Uyeda, 1979). Deficiency of this enzyme in human muscle, known as glycogcnosis type VII (Brown and Brown, 1968) was first reported by Tarui et al. (1965). C‘orr~.~/~~c/,o,,denc,e fo; Dr. H. Nakajima. Second Department Medicine. Osaka University Medical School, l-I-50 Fukushima-ku,

Osaka,

553 (Japan)

Tel. (81.Oh)45l-005l,

of Internal Fukushirna, ext. 1211;

Fax (El-Oh)453-0125. Abbreviations: bp, base pair(s); CAT, Cm acctyltransferaae; url; gene encoding CAT; Cm. chloramphenicol; hPFK, human phosphofructokinasc;

hPFK-M,

human

gene

encoding

1000 bp; -I., liver type; -M, muscle deoxyribonucleotide:

start point(s).

kb, kilobase

or

oligo, oligo-

-P, platelet type; Pipes. 1,4-piperxzine-diethanesul-

fonic acid; PolIk, Klenow (large) fragment ss, single stranded;

PFK-M;

type: nt, nuclcotide(s);

TLC, thin-layer

of E. coli DNA polqmerase

chromatography;

IF/J, transcription

I;

The discrepant), bet% ecn the activities in erl throcytes and muscle of these patients prompted investigaticms on isozymes of PFK. Three isozymes have been found in humans, viz., muscle-type (PFK-M). liver-type (PFK-I_), and platelet-type (PFK-P) PFK (Vera. 1982). The genes for these isozymes, PFK-M, -L, and -P, have been mapped on chromosomes 1. 21. and 10, respectively (Vera, 19x1). Poorman ct ai. (1984) demonstrated that the N-terminal half of the rabbit enzyme was homologous to the other half and that each half of the enzyme was also homologous to PFK of Btrcillus .st~~N~(~therrnophil~~.r.They suggested that gent duplication had occurred during the evolution of the PFK gene. The genomic structure of rabbit PFK-A4 gene was reported by Lee et al. (1987), but the promoter and 5’-untranslated region of the gene were not determined. We have reported the complete nt sequence of hPFK-M cDNA (Nakajima et al., 1987). Valdez et al. (1989) reported part of the genomic sequence of hPFK-M, suggesting the c.xistence of a splice junction within the 5’-untranslated region

278

of PFK-M mRNA. Recently, we identified three types of hPFK-M mRNA with different 5’-untranslated regions and named them types A, B, and C (Nakajima et al., 199Oa). Types A and B mRNAs share a common sequence in part of their 5’4mtranslated region. The putative intron sequence reported by Valdez et al. (1989) is retained in type B mRNA. The sequence of the 5’-untranslated region of type C mRNA is different from those of type A and B mRNAs, and essentially identical to that of a cDNA cloned from a placenta library (Sharma et al., 1989). We considered that two promoter systems and alternative splicing could bc responsible for these heterogeneous transcripts from a single gene. Recently, Sharma et al. (1990) reported the existence of another type of alternative splicing within the coding region ofthis gene. However, the whole structure and promoter region of the hPFK-M gene remained to be determined. To investigate the complete genomic structure and the mechanisms of expression of these multiple mRNA species for hPFK-M, we cloned and analyzed the hPFK-M gcnc.

EXPERIMENTAL

AND DISCUSSION

(a) Structure of the human PFK-M gene Thirteen positive clones were isolated from library LIO03 with hPFK-M cDNA as a probe. Eleven of them had identical inserts of 17 kb long, and the other two clones (RPFKMGl and ;IPFKMG3) had 3 kb longer inserts. One (2PFKMG16) of five positive clones isolated from library LIO 18 overlapped APFKMG 1 and -3 by 0.8 kb. The entire gene for hPFK-M was included in I,PFKMG 1, -3

III

I

III

HBB

H

EEE

II KH

B

I

Ill

E

BEE

and - 16. The cxon-intron organization of the hPb’K-M gcnc is sho\vn in Fig. 1. The hPFK-M gene has 24 cxons dispersed over a Icngth of about 30 kb. Exon I corresponds to the 5’-untranslated region oftype C mRNA. The donor site of intron I begins with GC instead of the commonI\ used GT dinucleotide (Mount, 1982). Other examples of the GC dinuclcotidc in the 5’-end of the intron have been listed by Shapiro and Senapathy (19X7). The scqucncc around the donor site of this intron, ‘AAG/gcaag ’ is common in these cxccptional

cases.

Exon 2 corresponds

to the 5’-untrans-

lated region of types A and B mRNAs. and the sequence ot intron 2 is identical to the 89-bp inserted sequence oftypc B mRNA. Thus, in types A and B mRNAs. intron 2 is altcrnativeI\ spliced by being rcmovcd and retained. rcspcctivcly. The remaining downstream cxons arc common to the three types of mRNAs. The exon-intron organization in cxons 3-23 is essentiali> the same as reported for the rabbit gene (Lee et al., 1987). t;xon 3 contains 8 nt of the 5’untranslated region sequence and the LranslaCion start codon (ATG). The last cxon contains the stop codon (TAA). the 3’-untranslated region scqucncc and the polyadenctation signal (AATAAA). In introns 2-33. the nt scqucnces of the boundaries arc a11 consistent \vith the conscnsus sequence (Mount, I982 ; Shapiro and Senapathq, 1987). (b) Identification of the tsp of exons 1 and 2 The results of primer extension and Sl mapping, performed for identification of the tsp of the hPFK-M gene are shown in Fig. 2. One of the primers. HP1 l/RM7. was an antisense-primer common to all three types of mRNA. The other primer, HPClOj20. was specific to type C mRNA. The products of 150. 138. and 1 15 nt extended from the

I

I’

I

I

E

HH

H

B

~1 I KH

B

I H

5kb

I

I Fig. 1. Structure RrrruHI,

and

restriction map ofthe hPFK-M gene. Boxes indicate exons and lines in bet\vecn specify introns. Restriction

and KprlI are indicated

as E, H. B, and K, respectively.

Library L1003 was constructed from human genomic partially digested with Sau3AI and cloned in iEMBL3. using PolIk (Takara to type B mRNA)

Shuzo. Kyoto, Japan). Thirteen probe, were isolated

Two libraries,

prepared

site\ ofE~R1.

by Dr. R. Sakaki

Hiudlll.

were xrecned.

DNA partially digested with EcoRI and cloned m iCharon4A, and LIOlX from genomlc Labeling ofthe probes was performed by the multi-prime method (Feinberg and Vogelstein,

clones. detected

from approx.

LIO03 and LIOl8. originally

with a “P-labeled

1 x IO” clones of library

1.1018 with a probe ofthe D&I-PvuII fragment ofcDN.4 [nt \+erc determined by the didcoxq chain-termination method Biochemical Corp., Clcvcland, OH).

full-length UFK-M

cDNA (Nnkajima

L1003. Five clones were isolated

DNA 19X3)

et al., 1990b, corresponding

from approx.

2 x IO” clones of library

13 1 to 232. numbered from the adenine residue ofthe start codon (ATG)]. The nt sequcnccs (Sangcr ct al.. 1977) using modified T7 DNA polymerase (Sequenasc’h’. United States

279 TABLE

I

Nucleotide

sequences

Location

of exon-intron

Sequence

boundaries”

of exon-intron Exon”

I

.4GACTAAAAz

Exon 2

CCTGACTGAG

Exon

junctions 5’

Intron

gcaaga

,_,,_,

3’

Exon

..,,,,,,

Intron

1

gtggct

.Intron

2

tgtcttaaattctag

AGTGGATCAT

gtaagg

.Intron

3

tgtccctcctttcag

GTATGAATGC

gttggt

.Intron

3

taatgtgtcacacag

GGTTATCAAG

gtatgt

.Intron

5

ctacctcttccaaag

GGAGGCACGG

gtaaga

.Intron

6

..gcttctcattgtcag

GTAAGATCAC

gtaagg

.Intron

7

..gtctatctcttgcag

CCACCAGAGG

gtaaga

.Intron

8

tgactctcatctcag

ATACCTGGCC

gtactt

.Intron

9

tttgttctcaaccag

ACAAGGACCC

gttcgt

.Intron

10

tgttggtcccttcag

CTGGTGGTTA

gtaagt

.Intron

I1

tctgggctcctgcag

GGCAGCAGGA

gtaagt

.Intron

I2

gtgagg

.Intron

gtactg

x5

Exon 3

GATGCCCAAG IS’1

Exon 4

TGTCCATGAG ??7

Exon 5

GCTTCAGCTG 417

Exon 6

CAGAAAGCAG 5’)?

Exon 7

CTGCCCAGAG h3ii

Exon 8

GCCACTGTGG 747

Exon 9 Exon Exon

ACTCAGCGAG YJ1

IO

C.4TCAAGAAT ‘)3h

II

Cr\GAATTCTG I MT?

Exon 12

TGTCCAGGTG

..gtgctcccccctcag

ACCAAAGATG

13.

ttgtacttcctacag

GAGCTTCATG

.Intron

14.

cttcctcctgtatag

AGTGGTTCGC

gtatgg

.Intron

I5

..ggcttatccccacag

ATAGAGGAAG

gtaagt

.Intron

I6

tcactgatcaactag

GACTCTACCC

gtgagt

.Intron

II

ctctcttcttcttag

GCTTACACAG

gtgaga

.Intron

18.

ccattgtccttgcag

ACCTGTGACC

gtagct

.Intron

19

ctctttcattttcag

GCAAATGTTG

gtacct

Intron

20

ttctccacctggcag

GAATGAAAAG

gtaggg

Intron

2I

tgtttctttctccag

GGTGGGAGCC

gtaggt

.Intron

22

catccctcattgcag

GGCGGATCTT

gtgagt

.Intron

23

ctcattcctctgtag

GCATCGAATC

!llll\‘.Y’.“n~ag C‘C‘LL‘LLCC

G

II?:

Exon 13

TGAGAGGCCG IIYI

Exon 14

GGTATCTAAG 1341

Exon 15

CAAGGGGCAG IJI:

Exon 16

GGACTAAAAG Ii00

Exon 17

GGGCTTTGAG Ihii

Exon IX

TATCTGCACA

,x,x Exon 19

AGACCTGCAG

IXX0 Exon 20

TGGTGTTAAG

IW2 Exon 21

C.4TGCAGCAG XY?

Exon 22

T.4CCGTAATG ?lW

Exon 23

Consensus

CAGATTTTGA

I’ The sequence ’ Numbers residue ’ Mount

C’4G .\

sequence’

gt;agt

data in this table will appear

above the sequences

of the translation

.._._.._._..........

in the GenBank

nt sequence

of the 3’-end of exons correspond

start codon

(ATG)

data bank under

to nt numbers

as nt 1. Since intron 2 was retained

of cDNA

accession

Nos. M59719-M59741.

[type C (exon 1). type A (exon 2)], taking the first adenine

in type B mRNA,

the nt number

of the 3’ end of exon 2 was -9X.

(1986).

HP1 l/RM7 primer (Fig. 2A), indicated identical nt in the genomic sequence to the signals of 83, 71, and 48 nt protected with AccI-Hi& probe (type B specific) in Sl mapping (Fig. 2C). The product of 61 nt in Fig. 2A was shorter than that of 150 nt by 89 nt, corresponding to the length of intron 2. These results indicated that types A

and B mRNA were transcribed from the same rsp located 3 1 nt upstream from the 3’ end of exon 2 as the major one. In type B mRNA, there would be some other minor ts~, such as the one located within intron 2. The products of 43, 54, and 81 nt from the HPC10/20 primer (Fig. 2B) were identical to the products of 72, 83,

3x0

A

B 12

gested that transcription

C

M

123

M

P12

of type C mRNA

was initiated

M

(c) Analysis of the mechanism of tissue-specific of the hPFK-M gene

83nt--c

-

72 nt -; *

54nt-L -w

61 nt -se

-

-m

-:

es-*;

43 nt _

kig. 7. Identification

on denaturing

aqlamidc)

arc shown.

polyacrylamidc

Lane M shows

for M 13mpIX primer

in section (h) are indicated extension labeled

using

PolIk,

and

denatured

primer

7.5,X)

after

Primer

annealing

was hybridized mM EDTA

~$35 performed

KC%3 mM

MgCI,/IO

D/50(1 btM each offour

the

at 60’C

at 37’C rnM

deoxyribonucleotide

rnyloblastosis

virus reverse transcriptase MA) for

20 ~8 of human ofthe

oligo,

muscle r.y oftype

RNA

C mRNA.

bj -14 polynuclcotide hybridized

ml

nctinomycin

triphosphatc;? (DuPont’NEN

umts ofavian Research

Pro-

analysis

using

kidney

total RNA control)

for

Oligo HPC-10 (5’~CTTTT.4GTCT) (5’-AGGAGAGCT4AGACTAAA-

analysis fragment

kinasc.

the extension

(lane 3, non-apecitic

the prlmcr

(HPC

using total RN.4 of human muscle

(lane I) and rat liver (Ianc 2, nonspecific 2. ThcAccI-Hir~fl

HCI pH

Hc‘l pll 7.5,40 mM

75 peg per

AGA), and \\as 3’-end labeled lvith Pollk to gencratc lO~30). (Panel C) SI nuclcase

Heat-

for 1 h. Then

total RNA (lane 1). human

\~a\ arrnealcd with oligo HPC-20

,.YJ~ ofckon

1 (5’-ATGGT-

IIPI

I 11.(Panel B) Primer extension

(lane 2) and 1:‘. WI; ribosomal analysis

muscle (ianc 1) and

HPI VRM7 was 3’-end-

in 50 mM Tris

dithiothreitol,

to

(Panel A) Primer

with RNA in 20 mM Tris

ducts.

Boston.

The signals referred

RNA ofhuman

control).

pal}-

of the sequencing

KM7 (i’-ATGACCCATGAAGAGCACCAT).

mM NaCI:I

reaction

size rnarkcrs

from nt 6284-6290.

analysis usmg 20 pg oftotal

after elec-

gel (7 M urea 7”,,

by the arrows with numbers.

rat liver (lane 2, nonspcclfic GCTCTT)

nt-

oftsp for cxons I and 2. Autoradlograrns

trophorcsis ladder

* 48

control)

for dctcrmination

ofi,GMPFKlh

Samples

ofthe

was 5’-end-labeled

of 20 ~cg of total

in 40 mM PipcsXJ.4 M NaCI: 1 mM EDTA:SO”,,

1X c‘ for 16 h. DIgestion by Sl nuclease wah performed as described (Sambrook

(4500 units,ml; et al., 19%)

expression

The hPFK-A4 gene seems to be a single copy per onchaploid genomc, judging by Southern-blot analysis (Sharma et al., 1990), and exons 1 and 2 both contained specific tsp as described above, so the existence of two alternate promoters in the hPFK-A4 gene is very likely. As shown in Fig. 3. t\vo Spl-binding sites are found in the upstream region of exon 1 (promoter I), and TATA-box-like and

81 M--t

83~

at

these positions. The distinct signal observed at 130 nt and 105 nt in Fig. 2A were considered to be artifacts because ability to form stem structures around these positions was shown by computerized sequence analysis.

RNA

were

formamide Tukara

at

Shuzo)

and 110 nt, respectively, from the HP1 l/RM7 primer (Fig. 2A). The product of 72 nt from HP1 I/RM7 was also detected as a major product when total RNA from kidney was used in the assay. In kidney, type C mRNA is the main type expressed (Nakajima ct al., 1990~). These results sug-

CAAT-box-like sequences are found upstream from exon 2 (promoter 2). The TATA box is generally thought to play an important role in precise initiation of RNA transcription (Breathnach and Chambon, 198 1). The heterogeneous ts11 of exon 2 may be explained by the fact that the location and sequence of the TATA box of this gene arc not typical. To confirm the promoter activity of these two regions, we pcrformed a reporter assay using a synthetic Curt gene. As shown in Fig. 4, promoter 1 was active in HcLa cells in which only type C mRNA was detected by the polymerasc chain reaction (data not shown), but not in the C2Cl2 mouse myoblast cell line, whereas promoter 2 was active in both cell lines. These observations were made using hcterologous cell lines, but the results indicate that promotcr 2 would facilitate muscle-specific cxprcssion of this gene. Muscle-specific enhancer, ‘M-CAT’-like scquenccs were also found in the promoter 2 region. In addition, in recent work (Nakajima et al., 199Oc), we f(>und that type A and B mRNAs arc cxprcssed almost exclusivciy in muscle, whereas type C mRNA is expressed not only in muscle but also in most other tissues cxamincd. Recently, 1.i et al. (1990) reported a 5’4lanking region and two altcrnativc noncoding cxons of the rabbit PFK-M gene and concluded that multiple transcription initiation by ;I single house-keeping promoter Lvas responsible for the heterogeneous expression of the mRNA. Stauffer et al. (1990) reported that one promoter and subscqucnt two noncoding cxons active in the human aldolase A-encoding gene arc not active in the rodent aldolase A-encoding gents. Thus, thcrc may bc a difference in the expression mechanisms of the human and rabbit Pb’K-A4 gents. Hcrc, \ve report the structure and function of hPFK-M gene. with special reference to the tissue specificity of two promoter regions. These results contribute not only to clucidation of the molecular defect in hPFK-M deficiency (Tarui et al., 1965; Nakajima et al., 199Ob). but also to resolution of regulation of the muscle glycolytic pathway at the gene level.

281 tgcttcactgccagaatcccqcccccatccctccctccccactgcaggcttgagaagtggcctcacagacacgcccttcctcttgacactttca . . . .. . . .. . (a) attccaaatagctagcttttctgtactttctctgagaaaggtaatgtaaatgtaaaagaagtttttgaggctattctatagaagatgtca taattcaatgttcctcaaaggcagggactatatttatttttttaaacctctcggcacctaatgcaaaatctagcacacagactcccaaga aatgtttgcttaaagacggatagagggccacaggggcaggttttattagcagttgctgaagatgttggaatgcaaggtgtctcaggcagt ctggactaggaaaaagttctgtgttgaaaacaagagaagcaacgaaataatttattattcccattaaataaagatgctgatggctctcga gatagatatgaggaccccaggactctggggagtgaaggaactaagccgctgccatatctgagctgagtacttagggggaggaggaagagg

aggagaaaggcaagcaggagqaqqc~qacttcttgtcagcatctgttagtggaggttgggaagcctctcctccttccccctccctctttg .. . .. . .. . (b) 1 7 1 cctccacctggctcctccccatqttcqtccatcacccctcccccctttcccaaqqacaatctqcaaqaaaqcaqcaqcqqaqqaqaqcta Exon aqactaaaaq~gcaagaggggccattgagtga----------(3000 1ntron

1

bp)---------ctttttctcctccctttctcatttctattc .... .. 1

agtcaacttctcttttccctgaccttagtttattccccaaataggttccaatcqgggtgggagggatcagggaggaattaggaccttagt ... .. .

CC)

Cd) r AccI agtcttgqgcttgattacatqacatttcagcttgtcagtctacaagggtgtggctttcctctggaagaagtccaaagctctcaqgctgca . .. .. . .. . . . (e) (f) 1 7 v aagctca~actt~~tataat~~~a~a~cct~act~a~/gtggctctagccagtctaattqccgttcctttagctagtggcatcttgattc Exon 2 1ntron 2

rHinfI

ctgctqtgtcttaactgaccattqtcttaaattctag/aqt~~atcATGACCCATG~GAGCACCATGCAGCC~CCCTGGGGATTGG Exon 3 CAAAGCCATTGCTGTCTTAACCTCTGGTGGAGATGCCCAA/gtaaggaggagggqacaaaaaacatggctggtgggactttt Fig. 3. Nuclcotide

sequence

of the 5’-flanking

indicate

splice sites. Upper-case

binding

site

match

with the consensus and Chambon,

of these elements. Restriction

sequence); sequence

The

(c) M-CAT

GGFCAATCT;

of the consensus

box (617 match Breathnach

fspobtained

by primer

extension

sequence

with the consensus

and Chambon,

and S 1 nuclease

are noted. The sequence

the 5’-end of intron 3. Exons arc underlined

region. Double

198 1;(f) M-CAT box (617 match with the consensus

sites used for St mapping

Nos. M59719

gene including

the protein-coding

[lo/10 match with the sequence complementary

match with the consensus Breathnach

region of the hPFK-M

letters in exon 3 indicate

underlines

indicate

“GGGCG~~$ TA sequence

with thin lines. Slashes

motifs of regulatory

CATTCCT;

Nikovits

protection

sequence

TATA:A;\;

The nt above the closed circles match the consensus

assays

are shown by downward

data in this figure will appear

in the GenBank

(a) Splsite (9jlO

et al., 1986); (d) CAAT box (619

1981); (e) TATA box (5/7 match with the consensus sequence).

elements:

(Briggs et al., 1986)]; (b) Spl-binding

arrowheads

nt sequence

sequences

above the sequence.

data bank under accession

and M59720.

A

C

pPFK-M-AB-CAT

pPFK-M-C-CAT

I Spl-bIndIng

Site

Exon 1

CAAT-llke

Exon 2

Exon 3

TATA-llke

Fig.4. CAT activities Part A represents by inserting dPFKMGl6.

of transfected

cell lines. The activities

the L’(~I gene constructs

the HindIll-XhaI Autoradiograms

used for transfection.

of the two promoter The plasmid,

regions

were determined

pPFK-M-AB-CAT,

by reporter

was constructed

fragment of IPFKMG16, and pPFK-M-C-CAT was also constructed of TLC plateused for CATI analysis in C2C12 cells. a mouse myoblast

assay using GUIsynthetic

from pUCOCAT

(Takenaka

gent.

et aI.. 1980)

by inserting the BumHIXcoTl41 fragment of cell line (panel B), and in HeLa cells (panel C),

transfected by these plasmids, are shown. The amount of protein in cell extracts used for the analysis is indicated in parentheses under each lane. Two plasmids. pUC2CAT and pUCOCAT, were used as positive and negative controls, respectively, as described (Takenaka et al.. 1989). The cells were cultured

in Eagle’s minimum

of DNA was performed cell extract nonacctylated

essential

by the strontium

with 3.7 kBq [‘“C]Cm

medium

(Nissui

phosphate

(DuPont:NEN)

Cm by TLC in chloroform-methanol

Pharmaceutical

method

Co. Ltd. Tokyo,

Japan)

supplemented

(Brash et al., 1987). CAT activity was determined

in I60 mM Tris. HCI pH 7.8;550 mM acetyl-CoA, as described

(Gorman

et al.. 1982).

with IO”,, fetal calf serum. Transfection 48 h after transfectlon, by incubating each

at 37’C for 2 h. Then AcCm was separated

from

2x2 Mount.

ACKNOWLEDGEMENTS

S.hI:

.A cata~oguc

of splice

~~l~lcl~on

Nuclclc

scqucnctzs.

/\&is

Rea. 10 (lYX2) 459-472.

This work was supported in part by a grant for basic research from the Muscular Dystrophy Association of the United States, by a Grant-in-Aid for Scientific Research. and a Grant-in-Aid for Scientific Research on Priorit) Areas from the Ministry of Education, Science. and Culture of Japan. a Grant-in-Aid from the Ministry of Health and Welfare ofJapan, a grant from the Foundation for Research on Metabolic Disorders of the Yamanouchi Foundation, and a grant from the Uehara Memorial Foundation. We are grateful to Dr. Y. Nabeshima (Division of Molecular Genetics, Institute of Neuroscicncc, National Center of Neurology and Psychiatry, Japan) for providing the C2C12 cell line. We thank the Japanese Cancer Research Resources Bank for supplying two human gcnomic libraries and HeLa cells. We also thank Drs. K. Imamura. T. Hanafusa. and T. Tamaki for advice. Drs. Y. Yamada. T. Hamaguchi. T. Matsuda, and M. Takenaka for helpful discussion. and Mr. T. Tanaka for preparing oligos.

Naka.jima,

H., Noguchi.

Tarui,

S.: Cloning

1 ., Yamasaki, of human

T., Kono,

muscle

N., Tanaka,

H.. Yamasaki.

I‘arui, S.: Evidence

T.. Noguchi,

I . I‘anaka,

for alternative

in the human muscle phosphofrLlctokill~l~cgcl,c

culture:

Stable expression

gen gene in primary

ofthe simian virus 40 large-T-anti-

human bronchial

epithelial

cells. Mol. Cell. Biol.

5’ untranslated

region. Biochem. Biophys. Res. Commun.

Nakqjima.

H.,

Kono.

Kuwajima,

N.. Yamasakl.

M., Noguchl,

muscle

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