Isolation and characterization of the glyceraldehyde-3-phosphate dehydrogenase gene of Aspergillus nidulans

49

Gene, 69 (1988) 49-57 Elsevier GEN 02533

Isolation and characterization Aspergillus nidulans (Recombinant cloning;

DNA;

gene libraries;

of the glyceraldehyde-3-phosphate

heterologous

hybridization;

gene amplification;

dehydrogenase

intron;

nucleotide

gene of

sequencing;

cDNA

phage 1 vector)

Peter J. Punt, Maria A. Dingemanse,

Brigit J.M. Jacobs-Meijsing,

Peter H. Pouwels and Cees A.M.J.J. van

den Hondel TN0 Medical Biological Laboratory, Rijswijk (The Netherlands) Received

6 February

Accepted

2 May 1988

Received

by publisher

1988 18 May 1988

SUMMARY

The isolation and characterization of the highly expressed glyceraldehyde-3-phosphate dehydrogenase (GPD)-coding gene (gpdA) of Aspergillus nidulans is described. The gene was isolated from an A. nidulans 13 gene library with a Saccharomyces cerevisiae GPD-coding gene as a probe. Unlike many other eukaryotes, A. nidulans contains only one GPD-coding gene. At the amino acid level, homology with other GPD enzymes is extensive. The A. nidulans gene contains seven introns, one of which is positioned in the 5’-untranslated part of the gene. The major transcription start point is found at 172 bp upstream from the start codon. Polyadenylation occurs at several sites about 200 bp downstream from the stop codon. Comparison of 5’ and 3’ flanking sequences with flanking sequences of other highly expressed (glycolytic) genes shows several regions of similar sequence.

Glyceraldehyde-3-phosphate dehydrogenase (GPD; EC 1.2.1.12) plays a central role in glycolysis and gluconeogenesis. In glycolysis it converts glyceraldehyde-3-phosphate into biphosphoglycerate, in gluconeogenesis it catalyses the reverse reaction. Much is known about the structure of the enzyme in

different organisms (Harris and Waters, 1976; Skariynski et al., 1987). Also, the nucleotide sequence of the GPD-coding genes of many prokaryotes and eukaryotes has been determined (e.g., Holland and Holland, 1980; Stone et al., 1985a; Tso et al., 1985b; Michels et al., 1986; Yarbrough et al., 1987). Comparison of the structure of the different GPD enzymes and of the nucleotide sequence of

Correspondence 10: Dr. P.J. Punt, TN0

gene coding

INTRODUCTION

ratory,

P.O.

Box 45,

2280 AA

Medical

Rijswijk

Biological

(The

Labo-

Netherlands)

Tel. (015) 138777.

nucleotide(s); gene;

for GPD;

kb, 1000 bp; mc, multiple

pgk, A. nidulans phosphoglycerate

SDS, sodium

dodecyl

0.15 M NaCl + 0.015 M Na, Abbreviations:

bp, base

glyceraldehyde-3-phosphate

pair(s);

ds, double

dehydrogenase;

stranded;

GPD,

&pdA, A. nidulans

gene coding merase-coding state.

037X-l 119/88~$03.50 0 1988 Elsewer

Science Publishers

B.V. (Biomedical

Division)

for GPD;

sulfate; citrate

copies;

ss, single stranded;

SSC,

pH 7.6; tdh, S. cerevisiae

fpiA, A. nidulans triosephosphate

gene; wt, wild type;

nt,

kinase-coding

iso-

[ 1,designates plasmid-carrier

50

their genes among

shows

different

Yarbrough

a high degree

species (Fothergill-Gilmore,

1986;

et al., 1987).

In several species multiple up to several present

of conservation

hundreds)

in the genome

(in higher eukaryotes

GPD-coding

genes

are

(Tso et al., 1985a; Michels

et al., 1986; Yarbrough et al., 1987). In some cases presumably only one of these genes is transcriptionally

the A. nidulans gpdA gene and the amdS

selection

marker.

The strain

contains

about

nine

copies of the gpdA gene (J. Dekker, in preparation). Plasmid pFLl-33 containing the S. cerevisiae GPDcoding

gene

(gap63/tdh2;

1980) was obtained

Holland

and

Holland,

from Dr. L.E. Edens.

(b) Gene libraries

active, the other copies being pseudogenes

(Hanauer

and Mandel,

1984; Fort et al., 1985). In

several other cases GPD tiple genes pressed

contains

which

(McAlister

is synthesized

sometimes

from mul-

are differentially

and Holland,

ex-

1985; Tso et al.,

1985b). In S. cerevisiue and rat muscle up to 5y0 of the total amount of cellular protein consists of GPD (Krebs et al., 1953; Piechaczyk et al., 1984). This implies that the expression signals of the GPDcoding gene(s) are strong, as was demonstrated by Edens et al. (1984). In our research on gene expression and gene regulation in filamentous fungi the expression signals of A. nidulans genes are being analysed (Van Gorcom et al., 1986). Both structural and functional features of these expression signals are under research. In this paper the isolation of the A. nidulans gpdA gene is described. The complete nucleotide sequence of the gene and its 5’ and 3’ flanking regions was determined. Furthermore, the nucleotide sequence of the messenger RNA was determined using cDNA clones and poly(A) + RNA as templates.

An A. nidulans 1Charon provided

AND

METHODS

(a) Strains and plasmids Escherichiu coli K-12 JM109 (Yanisch-Perron et al., 1985) was used for the construction and propagation of vector molecules. A. niduluns strain FGSC4 (Glasgow wild-type; Clutterbuck, 1986) was used for the construction of the A. niduluns cDNA library. Poly(A) + RNA from A. nidufans strain MH 1277[pAN45-lA] 1 was used as a template for mRNA sequence determination. This strain was obtained by transformation of A. nidufuns MH1277 (bti 1, amdS320, amdI18, amdA7, niiA4; Hynes et al., 1983) with plasmid pAN45-lA, which

Dr.

W.E.

4a gene library was kindly Timberlake

(Orr

and

Timberlake, 1982). A partial cDNA library was constructed using poly(A) + RNA isolated from a culture ofA. niduluns FGSC4 cultivated in minimal medium (Pontecorvo et al., 1953) with 2”$ galactose as a carbon source. The ds cDNA was prepared and cloned as described by Teeri et al. (1987). (c) DNA/RNA

manipulations

A. nidulans poly(A)‘RNA was isolated as described by Teeri et al. (1987). Primer extension experiments were performed as described for firststrand cDNA synthesis (Teeri et al., 1987). Heterologous hybridization experiments were carried out at 56°C with final washes in 3 x SSC, 0.1% SDS, 0.1% Na . pyrophosphate at 56’ C, as described by Van Hartingsveldt et al. (1987). All other DNA/RNA manipulations were carried out as described in Maniatis et al. (1982).

RESULTS

MATERIALS

by

AND DISCUSSION

(a) Isolation of the Aspergillus

nidulans gpdA gene

The A. nidulans gpdA gene was isolated from an A. nidulans FGSC4 1 library by heterologous hybridization with a DNA fragment containing one of the S. cerevisiue GPD-coding genes (gap63/tdh2; Holland and Holland, 1980) as a probe. From 25000 A clones screened, five positive clones were obtained. Restriction enzyme analysis revealed that the inserts in these clones had a 4.0-kb BglII-Hind111 fragment in common. Southern-blot analysis showed that only this fragment hybridized with the S. cerevisiae probe under heterologous hybridization conditions (results not shown), suggesting that a complete A. nidzdans gpd gene is located on the fragment.

51

Two lines of evidence confirmed nucleotide quence

sequence of a part

Fig. 2. The major part of the nucleotide

that the complete

A. nidufuns gpdA gene had been isolated. and predicted

First, the

amino

of the BgZII-Hind111

the gpdA mRNA complete

acid sefragment

revealed a clear similarity with S. cerevisiue tdh2 and other gpd sequences (for details see RESULTS AND

cDNA

sequence

was also determined,

of

using

in-

clones and poly(A) + RNA as tem-

plates.

By comparison

mRNA

sequences

of the

the presence

genomic

and

of five introns

the

could

of

be established in the 5’ part of the transcribed region of the A. niduluns gpdA gene (Fig. 3). Comparison of

several copies of the putative gpdA gene in A. nidulans resulted in an increased GPD enzyme activity. The

the genomic sequence with that of the cDNA clones revealed the presence of two additional introns in the

DISCUSSION,

section b). Second,

level of GPD enzyme

introduction

activity appeared

3’ part of the transcribed

to correlate

region (results not shown).

with the number of gene copies (J. Dekker, in preparation). This indicates that the BglII-Hind111 frag-

As can be seen in Fig. 1, a small part of the mRNA

ment contains

donor

a functional

sequence

copy of the gpdA gene

together with its expression signals. The 4.0-kb BglII-Hind111 fragment of one of the A clones was subcloned into a pBR322 derivative, containing a polylinker with a unique BglII and Hind111 site, resulting in plasmid pAN5-22 (Fig. 1). Southern-blot analysis of genomic A. nidulans DNA digested with BglII + Hind111 showed that a single band of 4.0 kb hybridized with pAN5-22 under stringent conditions (results not shown), indicating that the A. nidulans genome, unlike S. cerevisiae and many other eukaryotic genomes, contains only one GPD-coding gene.

The sequence strategy used to determine the nucleotide sequence of the gpa!A gene and its flanking regions is given in Fig. 1. The sequence is shown in

1 St

ss

intron

in the corre-

H

P l,Y_

YII

ss

E St

SS

6

H

SC

-

-

01 kb

Fig. 1. Nucleotide termination

methods

1987) as templates. represents

sequencing

restriction

Nucleotide

for the A. nidulans &pdA gene. Nucleotide

(Sanger sequences

et al., 1977) ds-DNA were analysed

the map of the 4.0-kb BglII-Hind111 regions by narrow

enzyme sites are indicated

the position,

length and direction

clones, those marked

pAN5-22.

strategy

with ss-DNA

the ?I’- and 3’-noncoding

cDNA

LlllIIP

SC

ss

No obvious

sites were found

sponding part of the nucleotide sequence. The features ofthe introns are described in RESULTS AND DISCUSSION, sectionc. The similarity between the predicted amino acid sequence of the GPD polypeptide of A. nidufans (Fig. 2) and the sequences of Nicotiana tabacum (cytosolic) (Shih et al., 1986), Drosophila melanogaster (Tso et al., 1985b), chicken (Stone et al., 1985a), man (Tso et al., 1985a), rat (Tso et al., 1985a), S. cerevisiue (Holland and Holland, 1980) and E. coli (Branlant and Branlant, 1985) is 65-70%. Between the A. nidulans GPD polypeptide and those of Bacillus stearothermophilus (Walker et al., 1980a) and Thermus aquaticus (Walker et al., 1980b) the similarity is 50-55%. In parts of the GPD polypeptide known to be essential for enzymatic activity (Harris and Waters, 1976) similarity is almost 100%. Such relatively high percentages of

(b) Structure of the gpdA gene and GPD enzyme

69

was not investigated.

or acceptor

using UWGCG

insert in pAN5-22.

bars. The introns

as follows: B,BamHI;

of the nucleotide

with ‘m’ sequences

obtained

analysis

data were obtained

1985) and poly(A)+ programs

using dideoxy

by hatched

(Devereux

determined.

from poly(A)+

bars numbered

H,HindIII;

Arrows

marked

with roman

with ‘c’ represent

et al.,

et al., 1984). The top line by open wide bars, numbers.

SC, ScaI; Ss, SsfI; St, StuI. Arrows

RNA. All other sequences

chain-

RNA (Johanningmeier

The coding region of the gpdA gene is indicated

are indicated

Bg, BglII; E,EcoRI;

sequence

sequence

(Chen and Seeburg,

sequences

were obtained

Relevant indicate

obtained

from

from subclones

of

52

-712

-532

-352

-172

369

45Y

549 ggagctaattatgtttagcteacgtcgtcgtagCCCGGGCACCATTGA .. . intron V ]A Y M L K Y

D

S

Q

H

G

Q

F

K

G

T

I

E

639 GACCTACGACGAGCCTCTTATTGTCAACGGCAAGAAGATCCGCTTCCACACCGAGCGTGACCCCGCCMCATCCCCTGGGGCCAGGACGG TYDEGLIVNGKKIRFHTERDPANIPWGQDG 729 TGCTGAATACATTGTCGAGTCCACCGGTGTCACTACCGCTTGTCAT AEYIVESTGVFTTQ EKASAHLKGGAKKVVI I319 CTCTGCCCCATCTGCTCATGCCCCTATGTTCGTCATGGGTGTC~C~~CGAGACCTAC~G~GCACATTCAGGTCCTCTCC~CGCTTC SAPSADAPHFVHCVNNETYKKDIQVLSNAS 909 ~TGCACCACCAACTGCCTTGCCCCTCTCGCC~GGTCATC~CGAC~CTTCCGTATCATCGAGGGTCTGATGACCACCGTCCACTCCTA CTTNCLAPLAKV INDNFGI IEGLHTTVHSY 999 CACTGCTACCCACAAGGTCCTCGACGGCCMGGCATCATCCCCTCCTCCAC TATQKVVDGPSAKDWRGGRTAATNIIPSST 1089 TGGTGCTCCCAAGCCTGTCGGCAAGGTCATTCCTTCGCTC~TGGC~GCTCACCGGCATGGCGATGCGTGTTCCCACCTCC~CGTCTC GAAKAVGKVIPSLNGKLTGMAMRVPTSNVS 1179 CG~GTTGACCTGACCGTCCGCACCGAGAAGGCTGTTACCTACGACCAGATC~GGATGCCGTC~G~GGCTTCTGAG~CGAGCTC~ VVDLTVRTEKAVTYDQIKDAVKKASENELK 1269 GGgtastgtgaatgtgcCtttgctgttggacaCcttcgact~actagttgntttagGCATCCTTGGCTACACCGAGGACGACATCGTCTC . Incran vI........."" JILGYTEDDIVS GI"“" 1359 TACCGACCTCAACGGTGACACCCGCTCTTCCATCTTCGATGCT~GGCGGCTATTGCCCTClZACTCCAACTTCATCAAGCTCGTTTCCTG TDLNGDTRSSIFDAKAGIALNSNFIKLVSW 1449 GTACCACMCGAGTGGGGTTACTCCCGCCGTGTTGTTGACCTCATCAgta~gtc=t=~g~~~g~tgga~c~ttttgtg~gttg~t=~~t= inCron "II.. Y D N E W G Y S R R V V D L I S[""". 1539 gcacccagCCTACATCTCCAGGTTGATGCCCAATAGgaaacagg~cgg~agccaatggccaggagctccttgtaaaa~aat~~t~~ttg "'.."I Y I S K V D A Q * 1629

Fig.2. Nucleotide sequence of the gpdA gene ofA.

nidulans and the predicted amino acid sequence. Coding regions are indicatedin

upper-case letters, all other sequencesinlower-case letters. Below the coding regions,the

the standard by roman

one-letter

numbers

code. Nucleotides

are numbered

(see Fig, l), are indicated

sites( + 1778, + 178 1,

by dotted

box (-616 underlining.

region (between

to -593)

underlining.

to the transcription

The major transcription

predicted

amino acid sequence

isgiven using

start point (+ 1). The introns,

numbered

start point ( + 1) and the polyadenylation

+ 1784) are indicated by asterisks. The putative TATA box (-52 to -47) is overlined. The C + T-rich region (-47

to -1) is overlinedwith a dashed line.The putative the 3’noncoding

with reference

showing

polyadenylation

+ 1640 and + 1730) are indicated

a clear similarity

to a sequence

signal ( + 1760 to + 1766) is underlined. by pairs of convergent

upstream

arrows. The sequence

The inverted upstream

repeats

in

of the TATA

from the TATA box of the A. nidulans pgk gene is indicated

by

53

GA

similarity

RNA

DNA T

G

C

A

T

between

different

homologous

tides have also been found

CwtmC

zymes

(Pichersky

et al.,

Forthergill-Gilmore,

polypep-

for other glycolytic 1984; Tani

et al.,

en1985;

1986).

Codon usage in the A. niduluns gpdA gene is clearly biased, with a preference for a pyrimidine in the third position.

Of all codons

that position

79% have a pyrimidine

(C:55%,

T:24%,

and when a choice between is allowed, chosen. highly

in 93%

G:20%,

in

A:2%),

a purine or a pyrimidine

of the cases

a pyrimidine

is

This bias is similar to that found for other expressed genes in lilamentous fungi

(Kinnaird

and

1983 ; Clements

Fincham,

and

Roberts, 1986; May et al., 1987) but clearly different from that in highly expressed genes of S. cerevisiae (Bennetzen and Hall, 1982).

an

(c) Introns Of the seven introns detected by sequence analysis (Fig. 1) introns II to VII interrupt the coding region of the gene, intron I is part of the 5’-noncoding region of the gene. The nucleotide sequence of the exon-intron boundaries of all introns of the gpdA

from A. niduluns MH1277[pAN45-lA]

and poly(A)‘RNA dicated

by ‘RNA’) primed

1 (in-

with an oligodeoxynucleotide

(CTCAATGGTGCCCTTGAACTGACCGTGC)

primer

complemen-

tary to a part of the coding region 3’ of intron V. In the sequence ladder

the exon-intron

indicated

boundaries

by arrowheads

ladder obtained is indicated

numbers.

lation start codon

arrowheads.

read from bottom

RNA sequence

polarity,

as CAT in the figure). In the RNA sequence structure point(s) 6

of the RNA. of aspecific

To determine

bands)

Poly(A)’

primer extension

containing

gpdA gene, was used for primer and ‘mc’ lanes extension Fig. 3. Sequence

analysis and primer extension

the transcription

start point and the position of introns.

A, T, C show the products quencing

reactions

of dideoxy

with pAN5-22

analysis to locate Lanes G,

chain-termination

DNA (indicated

se-

by ‘DNA’)

are shown.

products

multiple

extension

start

as a con-

experiments

longer exposure.

to additional The rightmost

used as size markers

were

primer used for copies

(wt) and

(mc) of the

reactions.

In the ‘wt’

from these RNA preparations

In both cases one major band was observed.

wt bands (identical products

the transcription

in the RNA sequence

RNA from A. nidulans FGSC4

MH1277[pAN45-lA]l,

to the

thus ATG is read

several bands across

out with the same oligodeoxynucleotide

sequencing.

(note that

due to stops caused by secondary

(which were obscured

sequence carried

probably

of the trans-

by an asterisk

to top, is complementary

and has the opposite

all four lanes appear,

of these introns

The position

(ATG) is indicated

are

In the sequence

with poly(A) + RNA the position

by numbered

the sequence,

of the first five introns

and roman

Minor

mc bands) can only be seen after lane A shows sequence for the primer extension

reaction products.

54

gene fit (with one or two mismatches) to the consensus sequence for fungal introns (Ballance, 1986).

region has been reported

The size of the introns

eukaryotes,

Comparison

varies from 50 to 120 bp.

of the position

GPD-coding

of introns

genes (Fig. 4) shows that intron V of

the A. nidulans gene coincides chicken

in different

GPD-coding

Furthermore,

gene

(Stone

chicken

et al., well

in

nt upstream

known. These results do not support the hypothesis that introns originally mediated exon assembly and thus are present in homologous genes at corresponding positions at boundaries of regions encoding structural domains. Such a hypothesis was proposed on the basis of analysis of different triosephosphate isomerase genes (Straus and Gilbert, 1985; Gilbert et al., 1986) and the chicken GPD-coding gene (Stone et al., 1985b). The presence of very small exons in the A. nidulans gpdA gene (too small to be structural domains) is not consistent either with this hypothesis, although introns very close to each other may have resulted from duplication and movement of early introns (Gilbert et al., 1986). The presence of an intron outside of the coding

9,

*

nidulans

e

~~~~~~~aster

Fig. 4. Position

was determined

*

of the introns

et al., 1987) and D.

sequences of the 3’ end of the gpdA mRNA by analysis

out of eight cDNA

clones

analysed,

In five

the poly(A)

found in one of the cDNA clones. A putative polyadenylation signal (AAUACA) was found 11-17 bp upstream from the poly(A) track. This sequence is related to a sequence (AAUAAA) found at comparable sites in many other eukaryotic messengers (Proudfoot and Brownlee, 1976). The 3’-noncoding sequence of the messenger shows stretches of dyad symmetry, as indicated in Fig. 2. Comparable results were obtained for other A. nidulans genes (Clements and Roberts, 1986; Ward and Turner, 1986). These sequences might be functional in 3’ processing and polyadenylation of the precursor of the messenger RNA (Platt, 1986).

4f

I 7

10

14

17

25

27

31’

227

334

*__‘-:I: in the GPD-coding

genes ofA. niduluns (this report),

chicken (Stone et al., 1985a), C. elegans (Yarbrough

melanogaster(Tso et al., 1985b). The location of the introns in the genes is indicated

A. nidulans gene. The total number region of the GPD-coding

of gpdA cDNA.

track started at nt position + 207 downstream from the stop codon (UAG), in two clones at nt position + 204 and in one clone at + 201 (Fig. 2). Thus, some heterogeneity was observed at the site of polyadenylation. The length of the poly(A) track is not known, but is at least 70 nt since this length was

*

L

et al., 1985b) coding

I

I Chicken

(Stone

(Tso et al., outside the

1985a). as

from the start codon (ATG) (Stone et al., 1985a; Tso et al., 1985b). None of the other introns in the A. nidulans gene coincides with an intron found in one of the other GPD-coding genes of which the mRNA and/or genomic nucleotide sequence is

4 21 ‘IpI

chicken

region have been observed.

The sequence

at IO-20

the

D. melanogaster genes, introns

(d) 3’-Noncoding

an intron

as

including

1985a) and GPD-coding

a

contains

the

et al., 1988). In several genes of higher

D. melanogaster GPD-coding gene the 5’ part of the gene corresponding to the untranslated region of the mRNA

in

with intron III of the

(Saloheimo

for one other fungal gene

genes.

of codons

in each gene is also given. Asterisks

indicate

the position

by the codon numbers

of the

of an intron in the 5’.noncodmg

55

(e) 5’-Noncoding

sequences

Analysis messenger

The transcription

start point(s)

transcription

(corresponding mRNA)

upstream

(ATG)

(Fig. 3).

start point is localized

to a leader

region

Some

minor

sites

172 bp

(ACAAUGG) role

start codon were

in the

eukaryotes

found

sensus

major

AAAAUGG

region of about

*****

*

of a repetitive

the

is thought

efficiency

of translation

ACCUG

1986) in

AUG

and

initiation

resembles

in higher the

genes

of

in

the coneukaryotes

consensus

glycolytic

codon

to play an important

1986) closely

(Cigan and Donahue,

50 bp was observed. This sequence is preceded by a putative TATA element (TATTTT). Similar sequences have been observed upstream from other Aspergillus and N. crussu genes (Ballance, 1986). In the highly expressed A. nidulans oliC (Ward and Turner, 1986) and N. crussa am (Kinnaird and Fincham, 1983) genes the C + T-rich region is particularly long. It has been suggested that the length of the C + T-rich region between the TATA box and the major transcription site in A. niduluns is related to the level of transcription (Ballance, 1986).

(A)

which

sequence

(Kozak,

C + T-rich

1986). around

(Kozak,

between 140 and 170 bp in front of the ATG codon. In the sequence immediately upstream from the site a prominent

presence

one to several copies of which

(Ward and Turner, The sequence

of 56 nt in the

from the translation

the

have also been found in many other A. niduluns genes

were localized by sequence analysis of the gpdA messenger and by primer extension analysis. The major

showed

oligomer (CCAUCU),

of the @dA gene

sequence of the gpdA

of the 5’-noncoding

sequence S. cerevisiae

1987).

Comparison of the sequences between the putative TATA box and the major transcription start point of three A. nidulans glycolytic genes (gp&t, this paper; pgk,

Clements

McKnight

et al.,

and

Roberts,

1986)

(Fig. 5A). Comparison the TATA

1986;

shows

and

a clear

of sequences

tpiA,

similarity

upstream

from

box of the gpdA gene and the pgk gene

shows a region of similar sequence around 500-600 nt upstream from the transcription start point (Fig. 5B).

*

****

**

**

**

A.nidulans

tpiA

+1 TTATTTTCGTCATTCCTCCTTCCCAACCTTCACTCTTCC..aGTTTCCAACT

A. nidulans

Egk

TTATTTA.

A. nidulans

pvdA

ATATTTT....

***

**

. TCCCTGGTCTCTCCCCACTAG..CTGTTCCTGCCcGTCCATCT CCTG..CTCTCCCCACCAG..CTGCTCT

-592 (B) A 2

nidulans

ppdA

TGGCGCTCTGAGGTGCAGTGGATG *

A 2

nidulans

a

*

*

*******

*

*

*

*

*

*

TGCTATTTTGAGGTGTAATGCATG

- 501 Fig. 5. Comparison of sequences of the 5’ flanking region of glycolytic genes of A. niduluns. (Part A) Comparison of the region around the transcription start point. The sequences of the gpdA (this paper), pgk (Clements and Roberts, 1986) and tpiA(McKnight et al., 1986) genes are aligned for maximal similarity by introducing gaps (indicated by dots). Nucleotides identical for all three genes are indicated by asterisks. The major transcription start point is indicated by an underlined lower-case letter. (Part B) Comparison of 5’ upstream sequences of the &p&4and pgk genes. The distance (in nt) from the transcription start point is given. Identical nucleotides are indicated by asterisks.

Clements,

(f) Conclusions

J.M. and Roberts,

signals

The dete~ination

of the nucleotide

sequence

of

the 5’ flanking region of the A. njd~~ans gpdA gene and the comparison of this sequence with that of other genes suggests the presence tory regions. Detailed

functional

gene fusions (Van Gorcom to correlate

functional

analysis

signals

using lacZ

and structural

has

Clutterbuck,

Glasgow

1986. Fungal

Devereux,

J., Haeberli,

of sequence

stock

list of Aspergilius

P. and Smithies,

analysis

plant thaumatin P., Marty,

by gpdA

Jeanteur,

demonstrated

in

express

a

gene from ~~~~~~~~

News Lett. 33 (1986) 59-69. programs

L.E., Born, J., Ledeboer,

controlled

number of Aspergirrus species (Van Gorcom et al., 1986; Punt et al., 1987; Mattern et al., 1987; Mullaney et al., 19SS), Penicilfium chvysogenum (Kolar et al., 1988), Trichoderma reesei (PenttilB: et al., 1987) and in other tilamentous fungi, showing that the expression signals of the A. nidulans gpdA gene are a useful tool in heterologous gene expression in tilamentous fungi.

(PGK)

0.: A comprehensive

set

for the VAX. Nucleic Acids

Res. 12 (1984) 387-395.

Fort,

been

A.J.:

strains

Visser, C. and Verrips,

features of the

and processing

kinase

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Holland,

We gratefully acknowledge the valuable advice and assistance with regard to the construction of the cDNA clones by J.K.C. Knowles and T.T. Teeri. The authors wish to thank R.F.M. van Gorcom, F. Bleichrodt and P. Crowley for critical reading of the manuscript.

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