Isolation and developmentally regulated expression of an Aspergillus nidulans phenol oxidase-encoding gene, ivoB

Gene, 98 (1991) 69-76

69

Elsevier

GENE

03892

Isolation and developmentally regulated expression of an Aspevgiflus nidufans phenol oxidase-encoding gene, ivoB (Recombinant scriptional

DNA;

tilamentous

fungi; pigmentation

genes;

fungal sporulation;

genetics

of fungal differentiation:

tran-

regulation)

C.E. Birse* and A.J. Clutterbuck Department qf Genetics, Glasgow University, Glasgow Gil SJS Scotland (U.K.) Received by R.W. Davies: 2 April 1990 Revised: 10 June 1990 Accepted: 11 September 1990

SUMMARY

Ivory (ivn) mutants of Aspergillus nidulans lack conidiophore pigmentation. We have cloned ivoB which codes for a conidiophore-specific phenol oxidase (AHTase) via the adjacent selectable ureD gene. Gene-library transformants of a ureD4 strain proved defective for the vector, but we recovered both ureD and ivoB from a 2 library of transformant DNA. The ivoB transcription unit was localized to a SalI-XbaI 3-kb fragment and its 5’ end was located by hybridization with an oligodeoxyribonucleotide corresponding to the N-terminal polypeptide sequence of AHTase. Expression of the ivoB 1.4-kb mRNA corresponded temporally with AHTase in conidiating cultures, and the levels of both mRNA and AHTase in leaky MA mutants implied transcriptional control by brlA. A second developmentally regulated locus of unknown function adjacent to ivoB was also transcriptionally dependent on brlA, but was expressed 4 h later.

INTRODUCTION

The upper parts of the conidiophore which bears asexual spores or conidia in the lilamentous fungus A. nidulans is normally grey-brown in colour. ‘Ivory’ mutants have lost this pigmentation and we have shown that the ivoB gene

Correspondence to: Dr. A.J. Clutterbuck, University, Gll Glasgow Glasgow Tel. (44-41)3305114; Fax (44-41)3305994. * Present

address:

Department

of

Department of Genetics, Scotland (U.K.) 5JS

Microbiology

and

Irvine,

Abbreviations: A., Aspergillus; AHT, AHTase, AHT oxidase; Ap, ampicillin;

N-acetyl-6-hydroxytryptophan; bp, base pair(s); brlA, gene en-

coding developmental

regulator;

glycol-bis(/l-aminoethyl

ether)

ethidium

bromide:

I19~91,‘$03

50 C

92717

per min; EGTA,

N,N,N’,N’-tetraacetic

iroB, gene encoding

1000 bp; nt, nucleotide(s);

037X-I

cpm, counts

CA

Molecular

Genetics, University of California, Tel. (714)836-5265; Fax (714)856-8598.

AHTase;

Science

Publishers

ethylene

acid; kb, kilobase

oligo, oligodeoxyribonucleotide;

199 I Elsevier

(U.S.A.)

B.V

EtdBr, or

wt, wild type.

(Biomedical

Division)

codes for AHTase, a developmentally regulated phenol oxidase (EC 1.14.18.1) produced only during conidiophore differentiation (Birse and Clutterbuck, 1990; Clutterbuck, 1990b). The substrate of this enzyme has been identified as AHT (McCorkindale et al., 1983). It accumulates in ivoB mutants and is dependent on ivoA for its biosynthesis. Developmental regulation of ivoB is apparent from its expression in various morphological mutants (Clutterbuck, 1969; 1977; 1990b; 1991) in particular those at the hrlA locus; null mutants at this locus produce elongated conidiophore stalks which are unable to develop vesicles or subsequent structures (see Fig. 5). These mutant bristles are unpigmented and do not produce AHTase but all ‘leaky’ brl.4 mutants synthesize the enzyme, and the more morphologically developed mutants are heavily pigmented. The degree of pigmentation may also reflect control of the ivoA gene which is involved in synthesis of the substrate. The brlA gene has been cloned (Johnstone et al., 1985; Boylan et al., 1987) and sequenced (Adams et al., 1988).

70

The implied BRLA protein includes a basic region including two putative zinc-finger motifs essential for its function (Adams et al., 1990). The regulatory role of h&t is supported by misscheduled expression of this gene in vegetative cultures which led to premature development of conidial structures and transcription of a number of conidiation-specific genes including ivoB (Adams et al., 1988). With the aim of understanding the role of h&t in controlling conidiophore development, we have cloned the ivoB gene and confirmed that its transcription is developmentally regulated and dependent on hrlA (Btirse, 1989).

RESULTS

AND

DISCUSSION

(a) Cloning the iv&l gene

The ivoB gene maps very close to ureD (~2) (Clutterbuck, 1990a), a position which we confirmed in a cross (see Pontecorvo et al., 1953; Clutterbuck, 1974, for standard Aspergillus techniques) between strains GO286 and G841 (Table I, footnote a, for genotypes) which gave no recombinants between the two loci among 178 fully analysed hybrid progeny and 2000 further progeny plated selectively for Ure + by growth on a urea nitrogen source (Darlington et al., 1965; Mackay and Pateman, 1983). To clone ureD and the adjacent ivoB region, an urgB2

TABLE

I

AHTase

levels in surface

Mutant,’

strains

cultures

of conidiation AHTase

mutants

ureD4 strain (CEB033) was transformed with the pILJ16 gene library (Johnstone et al., 1985; transformation method adapted from Tilburn et al., 1983) and plated on regeneration medium lacking arginine (the selective marker in pIJL16) and containing urea as sole nitrogen source. One strongly growing colony was obtained and shown to be Arg + Ure + Among progeny of a cross between this transformant and an urgB2 ivoB 197 strain (AJC454.21; Table I. footnote a) a11Arg ’ progeny were also Ivo +, implying that the urgB ’ gene bank plasmid was integrated close to ivoB. Five Arg Ivo ’ progeny (out of a total of 62) can be explained as arising by excision of the plasmid during meiosis. DNA from the transformant failed to transform E. co/i to Ap resistance and restriction analysis revealed an apparently deleted 2.15-kb pUC vector sequence. Genomic DNA from the transformant was used to construct a 2 library in the replacement vector 1, EMBL3: approx. 15 000 plaques were blotted and probed with pUC8 which identified six positive clones. All six clones were also probed with pILJ 16 (Fig. I) and shown to contain the 2.15-kb pUC8 fragment and all except i 5 included the argB gene ( 1.9-kb and 0.8-kb Sal1 fragments). The presence of urgB + meant that these i clones could be used without further manipulation to transform trrgB3 ivoB63 (AJC9.4) and urgB2 ureD4 (CEB033) strains to test for ivoB and ureD. Transformation with i. clones 3 and J gave approx. 50”,, Ivo + colonies with AJC9.4 while clones 1, 2 and 6 gave a similar frequency of Ure + colonies with CEB033 indicating the presence of the two gents on different /Is.

h acti\?ty

(b) Subcloning, ectopic expression and restriction mapping

wt hrlA42

d ivoB (CEBO80)

9.7

the ivoB gene

2.4

Fig. 1 shows that clones 3,4 and 5 contained a 5-kb &r/l fragment not present in clones 1, 2 or 6. This fragment vva5 subcloned into the unique SnzuI site of pILJl6 to give pCEB 122 and shown to transform AJC9.4 to Ivo ’ at high frequency. We demonstrated that the 5-kb insert in pCEB122 contained the whole iwB gene, including expression signals, by transferring it to a vector (pCEB404) containing an incomplete argB gene to force integration at mgB. Of the transformants of AJC9.4 with this construction (pCEB2 18; Fig. 2) 14”,, were Ivo + and were accepted as evidence of ectopic expression of ivoB + Southern blots of transformant DNA probed with pUC8 confirmed the presence of plasmid in three Ivo ’ transformants while five Ivo transformants, assumed to have arisen by gene conversion ol argB without integration, contained no plasmid. One transformant (CEB075) demonstrating ectopic expression was included in the Northern-blot analysis (section c, below) and shown to produce normal i,oB mRNA (Fig. 3B).

2.4

hrlA I MA6

21.6

MA 7

73.6

hrlA I4

11.2

MA42

26.4 64.0

uhaA I

100.4

rnedA I5

I’ Strains

were grown

as top-layer

cultures

for 26 h at 37’C

on agar

dishes (Birse and Clutterbuck, 1990; Clutterbuck, 1972). All A. niduluns strains were derived from Glasgow stocks which carry the WA 1 mutation. The wt and single morphological nutritional

marker

(pcrhaA 1; hrlA42 crhoA6

ivoB 197), AJC9.4

(pcrbcrri I; mgB2; [argB + ureD

CEBOXO

brlA42

(pabaA

ureD4),

’ I), CEB075

(pnbaA

mutant

strains all carried the additional

strains were: GO286 (hiA 1; urrD4), G841 uaY9 iwB63), AJC454.21 (rihoA1 >,A2 hiAl; argB2; hiA I. Other

I;

argB2;

1; argB2;

brlA42

CEB041 (pabaA

(pabaA

1; [argB + ivoB

brlA42

ureD4

ieoB63),

1; urgB2;

’ 1; brlA42

AivoB-[cc@

Clutterbuck (1973; 1990b) for explanation of symbols. h 100x.%4 4,0 per min per culture. AHTase was extracted as described by Birse and Clutterbuck (1990). Background CEBO80 and brlA 1 is due to nonspecific phenol oxidases.

CEB033

brlA42 ureD4 ivoB63),

+I).

See

and assayed activity

in

71

A 12345678

kb -2.7

Fig.

I, Southern-blot

analysis of six I clones from the Arg + Ure + transformant

gel. (Panel B) Southern size markers; techniques

8, pILJl6

digested

were employed

et al.. 1982).

argB

ivoB

pUC8/18 E

with SalI. No hybridization

(Maniatis

s

S

SM

to the upper part of the blot was seen. Standard

XCH

E

II pCEB122

I

II II EBGB

pCEB218

2

1

I

K

K

i

b

a

c

1"

pCEB208

1

pCEB509

i

'ti

X

X pCEB512

-

pCEB604

---;

Fig 2. Linearized

shaded EcoRI;

fragment of plasmids

Single line, pUC8 or pUC18; in pCEB122.

hybridizing

XhaI. Methods.

blackened

Restriction

Plasmid

and phage

ivoB

box, argB; open box, oligo is

(see Fig. 3) hybridizing sites: B, BarnHI;

K, KpnI; M, SmaI;

H, WindIII;

the putative

with the N-terminal-derived

a, b, c, transcripts

and its subclones. G, BglII;

incorporating

to

C, C[aI; E,

P, PsfI; S, SalI;

DNA were isolated

X,

using standard

procedures (Maniatis et al., 1982). A. nidulans DNA and RNA were extracted from mycelium frozen in liquid nitrogen and ground in a mortar. DNA isolation

used a scaled-up

Broda

Gel-isolated

(1985).

version of the procedure

DNA

were purified by the Geneclean

fragments

procedure

for plasmid

(BiolOl

of Raeder

and

construction

Inc., P.O. Box 22284,

La Jolla, CA 92038-2284). pCEB122 was constructed by blunt-end ligation of the Klenow-tilled 5-kb Sal1 fragment from A4 into the SnzaI site

religation. digestion

were obtained

Similarly

pCEB604

and religation.

was partially fragments

et al., 1985). pCEB208

into the Sal1 site ofpUC18,

and pCEB512

diagrams

ivoB insert. The segment pCEB208

blotting

+ EcoRI

and probing

(c) Identification of the ivoB transcript Northern analysis was used to localise the ivoB transcription unit within subclone pCEB208. Northerns from a time series of a hrL49 culture probed with pCEB208 identified three transcripts (Fig. 3A): transcript h is expressed constitutively, while expression of transcripts a and c is apparent after about 20 and 24 h growth, respectively. An identical result was obtained for a series of wt cultures by M. Stark (unpublished).

of pILJ 16 (Johnstone

sequence.

DNA electrophoretic,

;-

1 kb -

Scale

0.9”” agarosc

'ti KBXS

pCEB503

(Panel A) EtdBr-stained

To facilitate further analysis, the 5-kb Sal1 fragment from 14 was cloned into the Sal1 site in the multiple cloning region of pUC18 giving pCEB208 and this was mapped as shown in Fig. 2. Three subclones of pCEB208 were also constructed (pCEB503, pCEB509 and pCEB512; Fig. 2) for use as probes in the localization of the ivoB transcript within this 5-kb region.

insert

MXP

CEB041 (see Table I for genotype).

pUC and argB sequences). Lanes: l-6, I clones 1-6, digested with Sal]; 7, i,‘HindIII

blot probed with pILJ 16 (identifying

incorporated

and subclones

from this by appropriate was obtained

For gene-library

the same 5-kb

pCEB503,

from pCEB122

construction

pCEB509

digestions

and

by X&z1

genomic

DNA

digested with Sau3A followed by selection of 14-kb to 22-kb

on a 5-242,

ligated into IEMBL3

NaCl gradient.

Appropriate

digested with EcoRI + BarnHI,

DNA fragments

were

then packaged

with

a Packagene system. The primary gene library contained approx. 330 A. nidulans genome equivalents of insert DNA. E. coli strain DH1 (F ~, recA 1, endA 1, gyrA96, fhil, hsdR 17, supE44) was used for plasmid propagation and for attempts to rescue transforming plasmids from A. niduluns (Johnstone

et al., 1985). E. coli transformation

the CaCl,/RbCI

method

(Maniatis

followed a modification

et al., 1982) as detailed

of

by Johnstone

et al. (1985). The IEMBL3 vector, phage-packaging kit and the permissive [NM538 (supF, hsdR)] and restrictive hosts [NM539 (supF, hsdR, PZrou3)] were obtained

from Promega

(Madison.

WI).

72

16

20

24

28

32

a

a

b

b

C

C

brlA9 Fig. 3. Developmental identity of transcripts mutants

grown

regulation

of ivoB transcription.

a, b and c is discussed

for 26 h. CEB075

c; see Table 1 for genotypes.

and CEBOSO are, respectively,

For RNA isolation.

Total RNA was extracted

according

Gels were clectrophoresed

at 100 V for 7 h. Northern

High Wycomb,

Northern

blots of total RNA from surface cultures

of various

strains

probed

in section c. (Panel A) A brlA9 strain grown for the stated time before harvesting.

to Timberlake

strains

the ectopic

were grown

expression

transformant,

as liquid top-layers

(1986) and fractionated blotting was carried

on 1.2% agarose

and the ivoB deletion

over agar (Clutterbuck, gels containing

out on nylon membranes

with pCEB2OX. The

(Panel B) Developmental strain described

in section

1972; Birse and Clutterbuck.

2.2 M formaldehyde

following the manufacturer’s

(Maniatis instructions

1990).

et al., 1982). (Amersham,

U.K.).

The same blot was reprobed with pILJI6 to show the UrgB transcript (Birse, 1989) showing that transcript a is slightly larger than argB mRNA, 1.3 kb (Upshall et al., 1986) and that in all samples the constitutive transcript b is found at a very similar level to that of argB mRNA. As urgB transcription shows little variation during vegetative growth and conidiation (Yelton et al., 1983) transcript b could be treated as a gel-loading control showing that there is no increase in mRNA loading in the later time points which have increased levels of transcripts a and c (Fig. 3). In further blots (Birse, 1989) subclones of pCEB208 were used as probes. Plasmid pCEB509 hybridised with only the two developmentally regulated transcripts a and c, while pCEB5 12 identified only transcript c. Similarly, pCEB503 hybridiscd only with transcripts a and b, giving the order of the three transcription units as b-a-c, in the orientation of Fig. 2. It was concluded that the most likely candidate for the iwB mRNA was transcript a: both a and c arc temporally regulated, but it is probable that only transcript a (1.4 kb) is large enough to code for AHTase: 48-50 kDa, even allowing for possible glycosylation (Birse and Clutterbuck, 1990). To settle the identity of the ivoB transcript, a deletion mutant was constructed by gene replacement (Miller et al., 1985). A pCEB 122 subclone (pCEB604; Fig. 2). deleted for the central XhuI fragment was transformed into the urgB2 MA42 strain CEB033. Among the Arg’ transformants a small proportion had an Ivo phenotype. The deleted status of one such transformant (CEBO80) was confirmed by Southern blots of genomic DNA probed with pCEB208 (Birse. 1989). This mutant was fully viable and we detected

no abnormality in the brlA42 background in which it was induced other than the ‘ivory’ phenotype. It failed to complement in a heterokaryon with ivoB63 but complemented satisfactorily with ivoA and ygA controls. The mutant was assumed to have arisen by gene conversion of the chromosomal copy of ivoB by the deleted plasmid version during integration. The resulting chromosome would have two deleted copies flanking the argB plasmid. Northern analysis (Fig. 3B) showed that the deletion strain (CEBOSO) possesses transcripts b and c, but has lost transcript a. which is therefore confirmed as the ivoB mRNA. (d) Identification of the 5’ end of the translated region of ivoB We have obtained the sequence of the N terminal of the AHTase polypeptide (Birse and Clutterbuck, 1990). Choosing regions of minimal codon ambiguity and taking into account the limited codon bias in A. niduluns (Gurr et al., 1987) two oligos were constructed and hybridised to a variety of subclones (Figs. 2 and 4). A 20-mer derived from AHTase residues 6-12 gave only nonspecific hybridisation, but a 35-mer derived from residues 14-25 identified the 1. I-kb region between the lefthand X&I site and the KpnI (shaded in pCEB122, Fig. 2) as containing the 5’ end of the ivoB gene (Fig. 4). This confirms that we have identified the structural gent for AHTase, and agrees with the location of the gene as determined from Northern blots. Since the Northern-blot analysis indicated that the ivoB transcription unit extends through the PstI site at the left of the shaded box in Fig. 2, but not beyond the X&I site to the right of the box, it is

73

I

kb

2

B

3

phology 4

Fig.

4.Identification with

of the 5’ end of the translated ivol3 sequence by ‘*P-end-labelled oligo 5’-TTCCTCACCC-

the

CAAGGGAGTGGGGGCTGCTCGTCGA Scientific and Engineering Biochemistry minal peptide

Department, sequence

rose gel. (Panel XbaI,BamHI Lane

M, markers

Council oligo synthesis

Glasgow

University,

B) Southern

and HindIII, (phage

constructed

Research of AHTase.

(Panel

blot. Lanes: respectively;

in leaky brlA mutants

suggests

second, individuality of each brfA mutant protein in terms of its specificity for the promoters of ivoB, genes concerned with morphogenesis, and probably also ivoA (Clutterbuck, 1990b).

4.2 3.4

hybridization

and pigmentation

that morphological development and pigmentation arc independently regulated by brlA. The series of brlA mutants can be explained by the superimposition of two factors: first, increasing leakiness of the mutants in this series and

pCEB208

4, pCEB509

i DNA digested

the

on the basis ofthe N-ter-

A)EtdBr-stained 1-3.

by

facility at the 0.9% agadigested

with

digested with KpnI.

with Hind111 + EcoRI).

perhaps most likely that the 5’ end is to the right and that the transcribed sequence extends out of the box leftwards in the orientation of Fig. 2. (e) Expression of ivoB in brfA mutants Bristle mutants can be arranged on morphological criteria in a series of increasing leakiness (Fig. 5 and Clutterbuck, 1969; 1990b). For the alleles used here, the series consists of brL4 1 and brlA 14 (null phenotype, also lacking conidiophore pigmentation), and then pigmented mutants in order of increasing morphological development: hrIA6, -7 and -42. Fig. 3B includes Northern blots of 26-h surface cultures of strains carrying br/A alleles. Parallel cultures were analysed for AHTase levels (Table I). In Fig. 3B it can be seen that transcript b, which was previously shown to be constitutively expressed in the hrlA9 strain, is present in all strains tested. In the h/A mutants, transcripts a and c appear to be similarly regulated: they are present in brlA7, brlA9, brlA 14 (faintly) and brlA42, but absent from brlA 1. Transcript a levels, moreover, correspond well with AHTase levels (Table I). We therefore conclude that a functional br/A gene is required for transcription or processing of ivoB mRNA and also transcript c. The incomplete correlation between mor-

(f) Expression of iv& in medA and abaA mutants The medA 15 mutant morphology (Fig. 5C) is interpretable as prolongation of the metula stage to give three or four layers before phialides and conidia are formed (Clutterbuck, 1969). The medA15 mutant has maximum ivoB mRNA and AHTase (Fig. 3B; Table I), although it is only lightly pigmented; we have shown that this is due to low levels of the AHTase substrate (Clutterbuck, 1990b) and can postulate that in nzedA ivoB is fully responsive to b&i activation, but both ivoA and the morphogenetic promoters can only respond partially. The morphology of the ubuA 1 mutant (Fig. 5B) suggests that it is derived by repetition of the phialide stage. This mutant gave good enzyme activity in 26-h cultures (Table I) but the mRNA level (Fig. 3B) was very low. Mature ahA colonies also have low AHTase activity (Clutterbuck, 1990b) so the enzyme level in ubuA 1 at 26 h may represent accumulation of a protein whose synthesis is now declining. It is therefore possible that ivoB is transcribed in the conidiophore vesicle and metulae, but only at a lower level, or not at all, in the phialides which represent the predominant cell type in abaA. It seems unlikely that the ahA gene plays a vital role in either the rise or decline of AHTase levels, although Mirabito et al. (1989) have demonstrated that it can induce synthesis of a number of developmental mRNAs in misscheduled expression experiments. The highest AHTase levels are found in brlA mutants when ubuA is apparently not expressed (no ubuA mRNA is found in brlA42 developing cultures; R. Prade, unpublished) and the decline appears to have begun, although it is not complete. in mutants defective for ubuA. We suggest that ivoB is induced by low levels of hIA product, but that the full level developed in ubuA and the wt may repress ivoB again. This may also explain why the leakiest bristle mutant, brlA42 has lower AHTase and ivoB mRNA than brlA7. (g) An additional bvfA-regulated gene The finding of a second developmentally regulated gene responsible for transcript c adjacent to iwB is intriguing. It may imply that one or more conidiation-specific enhancers lie in this region, and it would be interesting to analyse

adjacent DNA clones for further developmentally regulated genes. Curiously enough, Miller et al. (1987) concluded that many mRNAs expressed exclusively in the spores were derived from gene clusters, but they contrasted this distribution with both vegetative and conidiophore-expressed genes which are not clustered. On the other hand, in the one case which has been analysed in some depth (SpoCl), the cluster of spore-expressed genes contained one conidiophore-

expressed gene at its centre (Miller et al.. 1987; Aramayo et al., 1989). Transcript c is evidently produced later than the iv& mRNA and it remains high in the ah& 1 strain (Fig. 3B) where the ivoB mRNA level has declined. A 4 h interval between appearance of transcripts a and c is found in the wt (M.S. Stark, unpublished), but it is interesting that the same interval is seen in the h&I 9 strain (Fig. 3A) despite the

fact that the morphology

of the brlA9 mutant

is arrested

in

development. Therefore, while transcription of this gene depends on brlA expression, the timing of induction depends on a clock which is independent of morphological progression.

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G.A. and Clutterbuck,

a natural

Miller, B.L., Miller, K.Y. and Timberlake, ACKNOWLEDGE
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development

T.H., Deising, H. and Timberlake,

Aramayo,

(h) Conclusions (1) Our success in cloning the ivoB gene represents a combination of good and bad fortune; we were unable to recognize Ivo + -transformed colonies on protoplast regeneration medium and thus clone the gene directly, but ivoB is adjacent to the selectable marker ureD which could be cloned. We were unable to recover the integrated plasmid from transformants, but a ,I library of one transformant yielded ivoB and ureD clones without further chromosome walking, and the presence of argB in the ,J clones enabled us to transform A. nidulans test strains directly. (2) A 5-kb Sal1 fragment is capable ofgiving normal ivoB expression when integrated ectopically at the argB locus, and therefore includes the complete gene and expression signals. The 5’ end of the ivoB coding region has been localised to an O.8-kb XbaI-KpnI segment in the centre of the 5 kb analysed. The transcription unit extends at least 1.1 kb to the left and not more than 0.2 kb to the right of this segment. We conclude that transcription is probably leftwards. A deletion mutant constructed by gene replacement is fully viable but lacks conidiophore pigment, AHTase and the 1.4-kb ivoB mRNA. It does not complement ivoB63. (3) ivoB mRNA synthesis is developmentally regulated, the transcript appearing at about the same time as the AHTase enzyme encoded by ivoB. In leaky MA mutants mRNA and AHTase also run in parallel and imply that low levels of brfA activity stimulate ivoB transcription, but that full br/A activity may switch ivoB transcription off again. ivoB transcript and AHTase levels in medA and abuA mutants t’it with this hypothesis, and in the latter case imply that the wt abaA allele is not required for ivoB down-regulation. (4) A locus adjacent to ivoB gives a second developmentally regulated transcript of unknown function. This transcript is also dependent on brlA, but in both the wt and a hrlA9 mutant which has limited morphological development, the transcript is not seen until 4 h after the iwB mRNA, implying a timing system independent of morphol-

W.E.: brlA is necessary

T.H., Boylan, M.T. and Timberlake,

sufficient

J.: Molecular Laboratory,

Cold Spring

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ofthe SpoCl gene cluster ofAspergil1u.y rtiduluns. Mol. Cell.

Biol. 7 (1987) 427-434.

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P.M., Adams, T.H. and Timberlake,

sequentially

expressed

genes control

in Aspergillus development. Pontecorvo, Bufton,

G., Roper,

W.E.: Interactions

temporal

and spatial

of three

A.W.J.: The genetics

L.M., MacDonald,

York,

Interactions

of Aspergillus nidulans. Adv. Genet.

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U. and Broda, J., Scazzocchio,

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Upshall,

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by integration

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A., Gilbert,

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P.J., Wggleilskl,

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of Aspergillus nidulans. Mol. Gen. Genet. Yelton,

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1986, pp. 343-357.

Miller, K. and Timberlake,

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K.D. and

(1953) 141-238. Raeder,

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J.A., Hemmons,

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J.E.,

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E.R., regulation

Mullaney, of the

E.J.

and

Asperx;i/u.c

nidulans wpC gene. Proc. Natl. Acad. Sci. USA 80 (1983) 7576-7580.