Primary structure of human salivary α-amylase gene

Gene. 41 (1986) 299-304

299

Elsevier GENE

1514

Primary

structure

(Recombinant

Takahiro

of human

DNA;

genomic

salivary library;

a-amylase flanking

Nishide a, Yusuke Nakamura”,

gene

sequences;

Sl mapping;

exons;

Mitsuru Emi”, Tatsuo Yamamoto”,

introns)

Michio

Ogawa b, Takesada

Mori b and Kenichi Matsuhara a* I2Institute forMolecular atld Cellular Biology. Osaka University, Yamada-oka, Suita 565, Tel. (06)877-5244. and h Second Departtnettt cf Surgery, Osaka University Medical School, Fukushima-ku. Osaka 5.53 [Japan) Tel. (06)451-0051 (Received

August

18th, 1985)

(Revision

received

October

(Accepted

October

19th, 1985)

25th, 1985)

SUMMARY

A recombinant clone which covers the human salivary r-amylase gene in a single insert has been isolated from a human genomic DNA library using a human salivary r-amylase cDNA as a probe. Restriction mapping and nucleotide (nt) sequence analysis revealed that this gene is approx. 10 kb long and is separated into eleven exons by ten introns. Its 5’-flanking region has some sequence homology with that of mouse salivary x-amylase gene [Schibler

et al., J. Mol. Biol. 155 (1982) 247-2661.

INTRODUCTION

Human r-amylases, which hydrolyze a- 1,4 glycosidic bonds, occur in two major isoenzymic forms, viz. salivary and pancreatic, which differ in M, (Matsuura et al., 1978; Stiefel and Keller, 1973), isoelectric point (Matsuura et al., 1978), and antigenie properties (Boehm-Truitt et al., 1978). Correspondingly, their mRNAs differ in size and nt sequence (Nakamura et al., 1984). Thus, human salivary and pancreatic z-amylases are produced by expression of different genes that act in a tissuespecific fashion. The molecular mechanism acting in

* To whom correspondence

and

reprint

requests

should

be

addressed. Abbreviations:

aa, amino

base(s) or 1000 bp; cDNA, nucleotide(s);

acid(s);

PA, polyacrylamide;

[2-cthancsulfonic DNA polymerase

bp, base

DNA complementary

pair(s);

kb, kilo-

to mRNA;

Pipes, piperazine-N,

acid]; Pollk, Klenow (large) fragment

ofB. coli

I.

037X-l 119!86/$03.50

0

1986 Elsevier

Science

nt,

N’-bis

Publishers

the tissue-specific expression of r-amylase genes has been studied in rodents by Schibler et al. (1983) and Young et al. (1981). They showed that, in mouse, salivary and liver amylases are encoded by a single gene whose transcription is controlled by two different promoters.

Human

liver, on the other hand, does

not produce amylase. In this connection it is of interest to examine what control mechanism acts in the tissue-specific expression of human x-amylase genes. x-Amylase gene(s) are also expressed ectopitally in some lung and ovarian cancers (Light and Ball, 1973; Zakowski et al., 1984). The molecular mechanism of this gene activation also poses an interesting problem. To elucidate these problems, we have begun studies with human amylase genes, and reported nt sequences of cDNAs for salivary and pancreatic amylases (Nakamura et al., 1984). In this communication, we report the isolation and characterization of genomic clones of the human salivary z-amylase gene.

B.V. (Biomedical

Division)

300 MATERIALSANDMETHODS

S 1 nuclease digestion was performed as described by Berk and Sharp (1977). The products of S 1 digestion

(a) Enzymes

were electrophoresed

and reagents

in a 6 “/, PA gel containing

7M

urea and autoradiographed. Restriction

endonucleases

were purchased

Takara

Shuzo

(Kyoto,

Japan),

Japan),

BRL (Maryland,

USA)

Toyobo

from

(Osaka,

and New England

Biolabs (Massachusetts, USA). PolIk was from Takara Shuzo, and Sl nuclease was from Sankyo (Tokyo, kinase

Japan).

RESULTS AND DISCUSSION

(a) Screening

of human genomic DNA library and

isolation of ChHaA2

T4 ligase and T4 polynucleotide

were generous

and Dr. 0. Chisaka,

gifts from Dr. T. Tsurimoto respectively.

Using cDNA

5’-end-labeled insert

section b) as a human genomic library. eleven positive recombinant One recombinant phage, bridizing with 5’ and 3’ METHODS,

(b) Library screening The human genomic DNA library was vided by Dr. R.M. Lawn (Lawn et al., combinant phage plaques were screened cedure of Benton and Davis (1977) using labeled human salivary a-amylase cDNA plasmid pHSA7 (Nakamura et al., 1984) (c) Genomic mation

DNA

and cDNA

kindly pro1978). Reby the prothe 5’-32Pinsert from as a probe.

beteroduplex

for-

Homologous regions in the cloned human salivary x-amylase genomic DNA fragments and the cDNA plasmid were mapped by the formamide procedure of Westmoreland et al. (1969) as described by Davis et al. (1971). (d) Poly(A)+

human

of pHSA7

(see

salivary

xc-amylase

MATERIALS

AND

probe, we screened the From 3 x lo5 plaques, phages were obtained. named ChHxA2, hyend probes made from

pHSAlO0 (see legend to Fig. 1) was taken as carrying a full-size gene. That this clone carries the salivary type gene was shown by its hybridization with the 5’-non-coding sequence of human salivary a-amylase cDNA which differs from the 5’-noncoding sequence of pancreatic r-amylase cDNA (Nakamura et al., 1984). Electron microscopic examination of heteroduplexes formed from the ChH xA2 DNA and cDNA from pH SA 100 revealed that the human salivary z-amylase gene contains at least nine exons (Fig. 1). (b) Restriction mapping and DNA sequencing analysis of ChHaA2

RNA preparation The ChHxA2

Human salivary gland was frozen in liquid nitrogen immediately after resection in surgical operations and stored at -70°C. Cytoplasmic RNA was isolated by a modified phenol-chloroform method (Lomedico and Saunders, 1976; Nakamura et al., 1984). Poly(A)+ RNA was obtained by chromatography on oligo(dT)-cellulose (Krystosek et al., 1975). (e) Sl nuclease mapping The 5 ‘-end-labeled DNA restriction fragment (30 pmol) was denatured and hybridized with human salivary poly(A) + RNA (2 pg) in SOo/, formamide, 40 mM Pipes (pH 6.4) 0.4 M NaCl at 47’C for 3 h after heating at 85 a C for 10 min. After hybridization,

DNA covering

a 13-kb region was

digested with EcoRI or HindIII, and the fragments were subcloned into pBR322 for fine restriction mapping and DNA sequencing. The resulting subclones, restriction map and the sequencing strategy are shown in Fig. 2. The nt sequence is shown in Fig. 3. The predicted aa sequence of 5 11 residues is shown below the nt sequence. The nt sequence of the aa-coding region, along with the 5 ’ - and 3 ‘-flanking regions, agrees completely with that of human salivary x-amylase cDNA obtained from different sources. Comparison of the genomic sequence with that of human salivary I-amylase cDNA has allowed us unequivocally to determine exon-intron junctions. The human salivary xc-amylase gene is about 10 kb long and is separated into eleven exons by ten introns. The eleven exons vary in size from 100 to

Fig. 1. Heteroduplex ChHaA2

analysis

1984). (B) An interpretive AND

METHODS,

A:Charon

s

1

z-amylase

ChHaA2

drawing ofthe heteroduplex

sectionc.

Numbers

1 through

gene. (A) An electron

is a recombinant

a-amylase

structure.

as constructed

The heterodupiex

8 represent

Two small exons, as revealed

cDNA,

of a heteroduplex

studies

loops.

formed by annealing

a full-size human

from pHSAI5

salivary

and pHSA7

analysis was performed

single-stranded

by nt sequencing

micrograph

phage isolate carrying

as described

pBR and i represent

r-amylase

(Nakamura

arms

of pBR322

1 kb

I

I

/

:, I

, 512

2.2

, I

S 702

2.5kb

5601’

1.4!&

4 5320

,

1

kb

1 1.9kb

I

I

s90

.5410

0.85kb

,

Fig. 2. Restriction the locations

constructed nuclease

map, subclones

and borders

I

fragments mapping.

P, PsfI; C, C/al;

strategy

for the EcoRI insert in ChHaA2.

from results

in Fig. 3. Each arrow

indicates

The numbered the direction

of Maxam and Gilbert (1980) and Sanger et al. (1980). Open bars represent

(Sl2-S702).

independently

and sequencing

of exons as deduced

by the procedure

7.2kb

3.55kb

s 511

restriction

and

are not seen with this technique.

I

, I

.EH

determined

et al.,

in MATERIALS

Pmbs

'\ '\

pHSA100.

carries full size human salivary

4A, respectively.

-

c

of the human salivary

DNA with DNA of plasmid

gene. Plasmid pHSAlO0

301

(B)

(A)

The restriction

by Southern

E, EcoRI;

S, Sau3A;

blotting

map of the region spanning procedures

Hi, HincII;

(not shown).

Av, AvaII; H, HindIII;

Sa, SacI. Only those sites that are relevant

to mapping

exon 1 through

black boxes (1-11) represent and region of the sequence

the subcloned

4 where subclones

S 1 probe is the restriction

fragment

was

used as a probe

in S 1

A, AccI; B, BglII; T, TaqI; Ha, HapII; are shown.

EcoRI or Hind111

are not overlapped

R, RsnI; Sp, SphI;

302

Fig. 3. Primary

structure

of the human

salivary

shown. A few errors in the cDNA sequence box (Goldberg,

1979) and the CAT box (Benoist

signal in the 3’.flanking Sequences

region is underlined.

for exon and 5’- and 3’-flanking

nt are numbered

starting

r-amylase

previously

gene. The nt sequence

published

(Nakamura

et al., 1980; Efstratiadis

The putative

mRNA

of the sense strand

et al., 1980) in the 5’-flanking

start points and polyadenylation

regions are given in capital letters, and those for introns

from the start codon;

aa are numbered

23 1 bp and the ten introns range from 94 to 2400 bp. The sequences around the 20 splice junctions conform with the GT---AG rule (Sharp, 1981) and further flanking sequences show general agreement with the favoured nt frequencies reported by Mount (1982) and Breathnach and Chambon (1981). Schibler et al. (1982) have observed by the electron microscopic RNA/DNA hybrid analysis that mouse salivary a-amylase gene also consists of eleven

and the deduced

et al., 1984) have been corrected.

aa scqucncc

arc

The Goldberg-Hogness

region are boxed. The poly(.4)

sites are indicated

by arrowheads.

are given in lower-case

letters. The

from the start codon.

exons.

Among

those

they

have

identified

three

splicing junctions, viz. donor and acceptor sites of the second exon and donor site of the sixth exon. These junctions agree perfectly well with ours, showing that the site of interruptions in the x-amylase coding region is highly conserved between mouse and man. On the other hand, none of the introns are similar in size or sequence between these two mammalian species.

(c) Mapping the transcription To determine

start points

the transcription

start point(s),

we

prepared a 5’-end-labeled 230-bp AvaII-HincII fragment containing the upper portion of exon 1 (Fig. 2). This DNA was hybridized poly(A) + RNA,

followed

with the human by digestion

clease and electrophoresis. monstrate

that

the

ly(A) + RNA synthesis gene can be mapped -222 _ -223. Another

major

The results start

in human at positions

salivary

with S 1 nu(Fig. 4) de-

points salivary

for

po-

r-amylase

-214 _ -217 and

minor start point is found at

around -298. In view of the general observation that an A residue is preferred for transcription initiation (Corden and Chambon, 1980) the A at -215 is the most likely candidate for the major start point for human salivary a-amylase poly(A) + RNA synthesis. These conclusions are supported by primer extension analysis (not shown). Fig. 4 also demonstrates the presence of a minor start point at around -298. This may reflect a sequence that is infrequently recognized by RNA polymerase II. Alternatively, it may indicate the presence of another functional, closely related amylase gene(s) having different transcription start point(s). In this connection, our recent observation (unpublished) suggests that human genome carries at least three, possibly more x-amylase genes that are very closely related to the salivary amylase gene reported in this paper. In the 5’-flanking region, a Goldberg-Hogness box (Goldberg, 1979) and a CAT box (Benoist et al., 1980; Efstratiadis et al., 1980) are found at positions -248 N -243 and -330 _ -315. The human sequence around the CAT box has high homology with that of mouse salivary a-amylase gene (Fig. 5), suggesting that it plays an important role in tissuespecific expression of the r-amylase gene in the salivary gland.

Fig. 4. S 1 nuclease man salivary was hybridized electrophoresed

with

We thank Dr. Tomoko Ogawa, Department of Biology, Osaka University for her guidance in preparation of specimens and operation of the electron microscope. This work was supported by a Scientific Research Grant from Ministry of Education, Science and Culture Japan.

marker

of the 5’ termini

Human

salivary

with AvaII-Hind111 fragment, with various

represent

mapping

mRNA.

in Fig. 2, treated length ACKNOWLEDGEMENTS

protection

r-amylase

concentrations

chemically

(shown

an Sl probe shown of S 1 nuclease

degraded

DNA

in the right four lanes).

respectively,

samples treated

Lane 4 shows a sample

treated

of mRNA.

Negative

flanking

regions

putative

start points are indicated

along

numbers

with exons

and

as a chain

Lanes

1, 2, 3,

with 2 u, 0.5 u,

I u of S 1.

similarly

addition

of hu-

poly(A) + RNA

as lane 3, but without indicate

from the start by arrowheads.

the nt of 5’codon.

The

304

Human Sali

vary

Mouse Sali vary

AAGAACAATGTTTTTCTTAGATGCTAATAAATTGTCCTGCTCAGGTTAGAGCAGCCA

Mouse Pant reds

ACTGTACCTTAAATATTTACTCATGAGCATTTACTTTGGAAATGTACTTTTTGTAGAAATATAAATAGGCGCTAGAGAGAAAGAACACTG

Mouse Liver

M

TGATGAATTATGTTGCAATAGAGGAGGTTATAAAGCAGCAGCCAAGGGAAGGCAGTGGTTTCCAAAGGAACCACAGGGTGGTGGTGCCCAG

Mouse Liver

m

TAGAAGAGGTTGTTGTGTATTGAGAAAGCAAAGTGATGAATTATGTTGCAATAGAGGAGGTTATAAAGCAGCAGCCAAGGGAAGGCAGTGC -80

Fig. 5. Comparison sequences. Human

of the sequences

The mouse sequences

and mouse salivary

-40

around

cap sites of cc-amylase genes. Sequences

are taken from Schibler et al. (1982). The sequences

r-amylase

genes have a highly conserved

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