DEVELOPMENTAL

BIOLOGY

148, 117-128

(19%)

Developmental Expression of Genes Involved in Conidiation and Amino Acid Biosynthesis in Neurospora crassa

Accepted

August

2, 1991

The levels of transcripts for Neurospora c~usso genes concerned with cellular and metabolic functions changed dramatically at different stages of asexual development. Transcripts for some conidiation-related (con) genes were present at high levels in conidiating cultures and in dormant conidia, but were absent or reduced during mycelial growth. Levels of some CMZ transcripts increased transiently during conidial germination, while others disappeared. Transcripts for amino acid biosynthetic enzymes, ribosomal proteins, cytochrome oxidase subunits, histones, and other polypeptides important for cell growth were detected in newly formed conidia and were present at reduced levels in dormant conidia. Levels of these transcripts increased upon germination of wild-type conidia in minimal medium, reaching their highest levels during this stage or during the early phase of exponential growth. The increased transcription of amino acid biosynthetic genes observed during germination in minimal medium was not dependent on a functional cpc-1 gene. However, cpc-1, which encodes a DNA binding protein presumed to function as a transcriptional activator, was essential for increased expression of amino acid biosynthetic genes when amino acid starvation was imposed during germination c 1991 Academic Press, IX. or at any subsequent stage of mycelial growth.

INTRODUCTION Neurospora crassa is a haploid, filamentous ascomycete that undergoes complex developmental and morphological changes during its asexual and sexual reproductive cycles. Mutations at more than 180 loci have been described which visibly affect events in these cycles (Perkins et ab, 1982). Wild-type strains of N. crassa produce abundant, multinucleate asexual spores (macroconidia) on aerial hyphae in response to environmental challenges including desiccation and nutrient deprivation. The process of conidium formation is completed in less than 24 hr in synchronized cultures (Berlin and Yanofsky, 1985a; Springer and Yanofsky, 1989), although several days are required for conidia to mature fully (Schmit and Brody, 1976). Conidia are activated for growth by hydration, and, if provided with appropriate nutrients, they swell and produce multinucleate filamentous hyphae. Hyphal branches generally anastomose on contact to form an intertwined mycelium. The asexual reproductive cycle is completed upon differentiation of new conidia from the cells of aerial hyphae. Investigations into the regulatory mechanisms responsible for conidiation have been initiated (Berlin and Yanofsky,

1 Current, address: Depart,ment of Chemical and Biological Sciences, Oregon Graduate Institute of Science and Technology, 19600 NW von Neumann Drive, Beaverton, OR 97006-1999. ’ To whom correspondence should be addressed.

1985a,b; Roberts et ah, 1988; Roberts and Yanofsky, 1989). Regulation of enzyme synthesis in N. crassa has been studied (e.g., Davis, 1986). Alterations in gene expression have been noted in response to changes in the availability of sources of carbon (McNally and Free, 1988), nitrogen (Fu and Marzluf, 1988; Fu et ah, 1989a; Fu and Marzluf, 1990; Fu et al, 1990), phosphorus (Mann et aZ., 1989; Kang and Metzenberg, 1990), sulfur (Fu et al, 198913;Ketter and Marzluf, 1988), and amino acids (Paluh et ah, 1988; Ebbole et al., 1991). The levels of many amino acid biosynthetic enzymes increase upon starvation for single amino acids (Carsiotis et aZ., 1974; Barthelmess, 1982). This increase, a regulatory phenomenon also observed in Saccharmyces cerevisiae and Aspergillus nidulans (Hinnebusch, 1986), is called crosspathway control in N. crassa and general control in S cerevisiae. Mutational studies have identified a locus, cpc-1, that mediates N. crassa cross-pathway control (Barthelmess, 1982; Paluh et al., 1990). The cpc-1 gene has been cloned; it encodes a DNA binding protein, CPCl, that has been characterized in some detail (Paluh et aZ., 1988; Ebbole et al., 1991; Paluh and Yanofsky, 1991). By analogy with the extensive studies on the related phenomenon in yeast, it is thought that CPCl is a transcriptional activator of genes under its control. In view of the complex asexual cycle of Neurospora, it was desirable to characterize gene expression throughout the cycle before proceeding with mechanistic studies 117

0012-1606/91 $3.00 Copyright All rights

0 1991 by Academic Press, Inc. of reproduction in any form reserved.

118

DEVELOPMENTALBIOLOGY vOLUME148.1991

on gene expression regulation during conidiation and amino acid starvation. Here we have systematically examined changes in mRNA levels for a variety of cloned genes during the asexual reproductive cycle and in response to amino acid starvation. Our findings on transcript levels for genes involved in amino acid biosynthesis, conidiation, respiration, ribosome assembly, and DNA replication extend the observations of others (Berlin and Yanofsky, 1985b; Brambl et al., 1987; Hoang-Van et al., 1989; Paluh et al., 1988) and provide a basis for investigations on the mechanisms of regulation of these genes in this organism.

threitol), phenol (6 ml), chloroform (6 ml) and 10% SDS (1.2 ml) for one minute. The disruptive force was generated using a jigsaw modified to hold small tubes (Miller, 1972). The preparations were then mixed for an additional lo-20 min using a tube rotator. The beads were allowed to settle and the liquid phase was decanted into centrifuge tubes; the glass beads were washed with 7 ml of extraction buffer and the washes and homogenates combined. Aqueous and organic phases were separated by centrifugation at 8000~for 10 min; the aqueous phase was removed and extracted twice: once with 15 ml phenol:chloroform (l:l), and once with chloroform. The nucleic acids in the aqueous phase, mainly RNA, were precipitated with ethanol and dissolved in water. METHODS Smaller RNA samples were obtained using 2-ml Growth of N. crassa screw-cap Eppendorf tubes. Acid-washed glass beads (1 g), extraction buffer (580 pl), phenol (420 pl), chloroform N. crassa stocks were obtained from the following sources: wild-type N. crassa strain 74-OR23-1VA from D. (420 pl), and 10% SDS (84 ~1) were placed in each tube; Perkins, Stanford University; FGSC strain 893 (arg-1p), 50 mg (wet weight) of N. crassa mycelium, frozen in Fungal Genetics Stock Center, University of Kansas liquid nitrogen, was added to the tube. The tubes were Medical Center; MPlOl (Acpc-l::trpC-hph) from M. Pla- capped and nucleic acids were extracted by homogenizmann, Stanford University. Stocks carrying the cpc-1 ing with the disruptor for 45 set at room temperature, mixing the homogenate for an additional 5 min, and alleles CD15 and CD86 were obtained from I. Barthelmess, Universitat Hannover, Federal Republic of Ger- then homogenizing an additional 10 set with the disruptor. Tubes were centrifuged in a Sorvall Microspin 24 many. Cultures were maintained by standard procedures centrifuge for 30 set to separate the aqueous and or(Davis and deserres, 1970). Macroconidia from cultures ganic phases. The aqueous phase was removed, exgrown for 2-3 weeks on 250 ml minimal agar (1X Vogel’s tracted once with 0.8 ml phenol:chloroform, and once minimal medium/2% sucrose/2% agar) in 2 liter Fern- with chloroform. Nucleic acids were precipitated from bath flasks were harvested in a dormant state using the the aqueous phase and dissolved in water. Poly(A) RNA was prepared from total RNA by two hydrophobic solvent Soltrol 170 (Bonnen and Brambl, 1983). Conidia were filtered through cheesecloth and cycles of oligo(dT)-cellulose chromatography essencollected by centrifugation. Germinating and mycelial tially as described (Sachs et aZ.,1986). Hybridization of cultures were obtained by resuspending harvested coni- RNA to oligo(dT)-cellulose was accomplished by continuous mixing of RNA in binding buffer with oligo(dT)dia in growth medium by rapid vortexing and inoculatcellulose in a closed tube through repeated, gentle invering liquid media with the resuspended conidia (final sions using a tube rotator; the oligo(dT)-cellulose with concentration of 2-5 X 106/ml). Cultures were grown at bound RNA was pelleted by centrifugation and the hy32°C on a rotary shaker for 0.25-48 hr. Germ tubes first bridization solution removed. The resin was resusemerged approximately 1.5 hr after inoculation; 95% of pended in wash buffer and transferred to a chromatogthe conidia germinated within 5 hr of inoculation. Cells raphy column for the subsequent washing and elution were collected by vacuum filtration onto Whatman 541 steps. filter paper and frozen in liquid nitrogen. Samples from cultures induced to conidiate were obtained as described by Berlin and Yanofsky (1985a). Northern Blot Analyses Preparation of RNA Total RNA was prepared from N. crassa by a modification of the method of Kurtz and Lindquist (1984). Cells (0.5-l g wet weight) were frozen in liquid nitrogen and immediately disrupted by shaking with acid-washed, 0.5-mm-diameter glass beads (20 g) in sealed tubes with extraction buffer (6.8 ml; extraction buffer contains 100 mM Tris-HCl, pH 7.5,lOO mM LiCl, and 20 mM dithio-

Cloned Neurospora genes with diverse functions were used as probes (Table 1). In most cases, probes were prepared using gel purified fragments of Neurospora DNA, obtained by digestion of recombinant plasmids with appropriate restriction enzymes. Specific details are as follows. The arg-2 probe was either the PvuIINcoI fragment of plasmid pAR241 or a cDNA insert (Orbach et al., 1990). con-Z, con-g, and con-11 probes were

SACHS AND YANOFSKY

Neurospora Transcript

Function

COT-2

Conidiation related Conidiation related Conidiation related Conidiation related Conidiation related Conidiation related Arginine synthesis” Histidine synthesis* Tryptophan synthesis” Tryptophan synthetase Cross pathway control regulator Nitrogen regulator Cytochrome c oxidase subunit V Cytochrome c oxidase subunit VIII Histone H3d Histone H4 Ribosomal protein, large subunit Ribosomal protein, small subunit fl-Tubulin

con -6 COW8

co?-9 con-lo COY-11

urg-2 his-3 h-p-1 trp3

cpc-1

nit-2 cos-5 cox-x

HS H4 rpl-1 rps- 1 tub-2

119

Developmental Express&m in N. crassu

TABLE 1 crassa mRNA TRANSCRIPTS EXAMINED Transcript

size (kb)

0.95 0.57 1.05, 1.25 1.0, 1.25 0.82 1.0, 1.75 1.85 2.9 2.45, 2.7 2.67 1.9 4.5 0.89, 1.33 0.53 0.87, 1.0 0.75, 1.2 1.06 0.9, 1.2 1.95

Reference Berlin and Yanofsky, 19851, Berlin and Yanofsky, 1985b Roberts and Yanofsky, 1989 Berlin and Yanofsky, 19851, Roberts et al., 1988 Berlin and Yanofsky, 1985b Orbach et aZ., 1989 Legerton and Yanofsky, 1985 Schechtman and Yanofsky, 1983 Burns and Yanofsky, 1989 Paluh et ul., 1988 Stewart and Vollmer, 1986 Sachs et al, 1986 Sachs et al., 1986 Woudt et al., 1983 Woudt et al, 1983 J. Heckman, Indiana U., Bloomington, Kreader and Heckman, 1986 Orbach et aZ., 1986

IN

carbamoyl phosphate synthetase. a arg-2 encodes the small subunit of arginine-specific b his-8 encodes a multifunctional protein with histidinol dehydrogenase, phosphoribosyladenosine 5’-triphosphate pyrophosphohydrolase, and phosphoribosyl adenosine 5’-monophosphate cyclohydrolase activities. synthetase, phosphoribosyl-anthranilate isomerase, and indole-glycerol ’ trp-1 encodes one subunit of an aggregate which has anthranilate phosphate synthetase activities. d N crassa histone genes HA’ and H.4 are unique loci.

prepared from plasmids pCON2, pCON9, and pCONllA (Berlin and Yanofsky, 1985b) digested with %I. The con-6 probe was either plasmid pCON6 (Berlin and Yanofsky, 1985b) digested with PstI, or a con-6 cDNA fragment. The con-8 and cm-10 probes were cDNAs (Roberts et al., 1988; Roberts and Yanofsky, 1989), as were the cox-5, cox-8, rpl-1, and rps-1 probes (Sachs et al., 1986; Kreader and Heckman, 1987). The rpl-1 probe detects a transcript encoding a large ribosomal subunit protein (clone CC2, J. Heckman, Indiana University, Bloomington, personal communication); the rps-1 probe detects a transcript for the o-p-1 gene, which encodes a small ribosomal subunit protein homologous to S. cerevisiae CYHB. The cpc-1 probe was the BgZII-EcoRI fragment of pM031 (Paluh et ab, 1988). The his-3 probe was the BamHI-Hind111 fragment of pNH60 (Legerton and Yanofsky, 1985) or the HindIII-SmaI fragment, obtained from a subclone. The histone H3 probe was the appropriate Sac1 fragment of pNCH3H4 (Woudt et cd., 1983), and the histone H4 probe was the appropriate SucI-XhoI fragment of pNCH3H4; each was obtained from a subclone. The nit-2 probe was the 6-kb EcoRI fragment from cosmid 6:9H (Stewart and Vollmer, 1986; Fu and Marzluf, 1987). The tub2 probe was the Sac1 fragment of pBT3 (Orbach et al., 1986). The trp-1 probe was the EcoRI-Hind111 fragment of pNC2 (Schechtman

and Yanofsky, 1983). The trp-3 probe was pDB1 (Burns and Yanofsky, 1989) digested with BamHI. Probes were labeled with 32Pby the random priming method (Feinberg and Vogelstein, 1983); unincorporated label was removed from probe preparations by spin chromatagraphy through Sephadex G-25 columns (Boehringer-Mannheim, Indianapolis, IN). RNA blots were prepared and hybridized to probes as described (Orbach et aZ.,1990). Blots were washed twice for 5 min and twice for 15-20 min at 65°C with wash buffer containing SSPE (0.1~) and SDS (0.5% or 1%). Measurements of Poly(A) Tail Length Poly(A) levels were analyzed using two techniques (Sachs and Davis, 1989). The lengths of poly(A) tracts at the 3’ ends of poly(A) RNA obtained by oligo(dT)-cellulose chromatography were determined as follows: poly(A) mRNA (5-100 ng) was 3’-end labeled with [“PIpCp and digested with RNase A, which does not cleave within tracts of poly(A). The sizes of the labeled, poly(A) mRNA 3’ ends were determined using autoradiography after electrophoresis of samples through a 12% polyacrylamideF7 M urea gel. Amounts of poly(A) in total RNA were determined by measuring the ability of these preparations to protect [3H]poly(U) from nuclease

120

DEVELOPMENTALBIOLOGY V0~~~~148,1991

digestion in a hybridization-protection assay in comparison to the protection obtained with known amounts of pure poly(A) standard. RNase Protectim

taining the fragments were excised, and the amounts of label they contained were quantitated by scintillation counting. RESULTS

Assays

Oligonucleotides (5’-GATATCGGATCCATTACACCTCTTATCGCA) and (5’-CAGCTGAATTCAGATACGGCTCATGGAGCA) were used to prime synthesis of a double-stranded DNA product from a plasmid copy of the cpc-1 gene using the polymerase chain reaction (PCR; Saiki et ah, 1985). This product included the cpc-1 intron-exon boundary (nt 1200-1420, Paluh et al., 1988). Oligonucleotides (5’-GATATCGGATCCAAGGTCGTCTCAAGAGAC) and (5’-CAGCTGAATTCTGGTAGCCTCTTGCCACTC) were used to prime synthesis of a double-stranded DNA product from a plasmid copy of the coxd gene. The product included the first cox-5 intronexon boundary (nt 1052-1300, Sachs et al., 1989). PCR reactions (100 ~1) contained 0.5 yg of each oligonucleotide primer, 10 ng of plasmid DNA, and 5 units of Taq polymerase (Perkin-Elmer, Norwalk, CT) and were performed in a reaction mixture containing 10 mM TrisHCl, pH 8.3,50 mM KCl, 1.5 mM MgCl,, and 200 PM of each deoxyribonucleoside triphosphate. Reactions were incubated in a thermocycler (Ericomp, San Diego, CA) using the following conditions: first, reactions were heated to 92°C for 1.5 min; this was followed by 35 cycles of the regimen of 50°C for 2 min, 60°C for 5 min, and 92°C for 1 min; finally, reactions were adjusted to 50°C for 2 min and 60°C for 10 min. PCR-generated DNA fragments were purified by recovery from low-meltingpoint agarose gels, digested with the restriction endonucleases BamHI and EcoRI, and inserted into pSP72 vector DNA digested with BamHI and EcoRI to produce pGO1 (cpc-I insert) and pHC1 (cox-5 insert). The RNase protection assay employed was based on previously described procedures (Zinn et al., 1983). Synthetic, 32P-labeled antisense strand RNA probes were prepared using linearized plasmid templates for in vitro transcription. pGO1 and pHC1 were linearized with HindUI, and T7 and SP6 RNA polymerases were used, respectively, to produce cpc-1 and cox-5 probes. The antisense probe representing the 3’ end of arg-2 mRNA was obtained by synthesizing RNA from the small StzcIEcoRI fragment of pARCG228 (Orbach et aZ., 1990) using an appropriately linearized subclone. Probes were annealed to different samples of total RNA, or, as controls, to different amounts of synthetic (+)-strand RNA. After annealing, samples were digested with RNases A and Tl and electrophoresed on denaturing gels containing 6% polyacrylamide/7 Murea. The radiolabeled RNA fragments which were protected from digestion were visualized by fluorography; the portions of the gels con-

Analyses of RNA Levels during Development

Our initial objective was to determine changes in mRNA levels for conidiation-related genes and amino acid biosynthetic genes as N. crassa cells progressed through the asexual cycle. RNA isolated from N. crassa cultures at different stages of the cycle was examined for transcript levels for the genes listed in Table 1. Northern blot analyses were performed with samples containing equal amounts of total RNA (Fig. 1) or poly(A) mRNA (Fig. 2). The results obtained with total RNA and poly(A) RNA were qualitatively similar, although the fraction of total RNA that was poly(A) RNA was reduced in conidiating cultures and in conidia (see below). con Gene Expression

The relative level of con-Z, con-6, con-g, con-lo, and con-11 transcripts increased in both total RNA and poly(A) preparations from conidiating cultures, and high levels were maintained in both young and old macroconidia (Figs. 1 and 2). Within 0.5 hr of initiating germination, con-8 and con-11 transcript levels dropped precipitously; they remained very low until initiation of conidiation. The level of con-10 transcript initially decreased, then increased transiently following germination (Figs. 1 and 2). The level of con-6 mRNA also increased transiently following initiation of germination. Levels of con-6 and cm-10 transcripts, like those for con8 and con-II, remained low during mycelial growth (6-20 hr after initiating germination). In contrast, the transcript for con-2 was reduced but was readily detectable during mycelial growth. The levels of the l.O-kb con-9 transcript (see Table 1) were highest during the late log and early stationary phases of vegetative growth and did not increase in conidiating cultures (Fig. 1 and Berlin and Yanofsky, 1985b). This transcript was present in substantial amounts in young conidia but was not maintained at these levels in old conidia (Fig. 1). We examined cm-2, con-6, con-& and con-10 RNA levels more thoroughly during conidiation by taking RNA samples at frequent intervals following induction of conidiation (Fig. 3). Under the conditions used, the morphological shift to commitment to conidiation, marked by the formation of major constriction chains (Springer and Yanofsky, 1989), occurred approximately 6-8 hr following induction. Transcripts for con-2 and con-8 were present throughout conidiation, although their levels

Developmer~tnl

SACHS AND YANOFSKY

Germination

(h)

Conidiation

(h)

con 2 con 6

.-

~.

--.

__

Expression

121

in N. crassu

acteristic changes during asexual development (Figs. 1 and 2). In general, RNA levels were low in dormant conidia, and peaked in germinating conidia and young myCelia. The transcript encoding a major regulator of nitrogen metabolism, d-2, had a similar pattern of expression (Fig. 2). The levels of RNA for other genes important for cell growth in minimal medium (~0x4, cox-8, rpl-1, rps-1, histone H3, histone H4, and tub2) also increased following germination. However, transcripts for most of these genes remained at relatively high levGerm (h)

Con (h)

con 6 con 8

arg 2 his 3 FIG. 1. Developmental Northern blots of RNA species in N. crasser total RNA. Samples of total RNA were purified from dormant conidia (Lane C) and from cultures inoculated into minimal medium and grown for the periods indicated under the line labeled “Germination (h).” RNA samples also were obtained from cultures which were harvested after 20 hr growth and induced to conidiate for 3,11, and 28 hr, and from conidia obtained from the 28-hr culture (2%) by harvesting with Soltrol 170 (indicated under the line labeled “Conidiation (h)“). RNA samples (3 pg/lane) were size-fractionated by electrophoresis through formaldehyde-agarose gels and transferred to nylon membranes. Membranes were probed with “P-labeled DNA probes for the N. crassa genes indicated at the left of each row. Only the portions of the autoradiograms corresponding to the mRNA species listed in Table 1 are shown. Additional RNA samples, isolated from a second, independent series of cultures, were analyzed in parallel, with similar results (not shown).

increased modestly after the morphological commitment to conidiation. At commitment, the con-6 transcript began to appear; the con-10 transcript appeared after 10 hr. Thus each of these conidiation-related transcripts had a unique expression pattern during asexual development, but all were present at their highest levels in cultures committed to conidiation. Expression

of Amino Acid Biosynthetic

Genes

Transcript levels for genes involved in amino acid biosynthesis (arg-2, hid’, trp-1, and cpc-,l) underwent char-

trp 1

‘t w-a SW

k.

cpc 1 nit 2

rps 1 tub 2 FIG. 2. Developmental Northern blots of N. crassa poly(A) mRNA. Poly(A) RNA samples (0.4 fig) were analyzed as described in the legend to Fig. 1. Samples were obtained from dormant conidia (Lane C), from cultures grown in minimal medium for the periods indicated under the line labeled “Germ (h)“, and from cultures which were harvested after 24 hr and then induced to conidiate for 4 or 24 hr (indicated under the line labeled “Con (h)“). Only the 1.2-kb histone H4 RNA species is shown.

122

DEVELOPMENTALBIOLOGY Conidiation

(h)

con 6 con8

,-‘: c

con 10 rps 1 FIG. 3. Developmental Northern blots of mRNA species in total RNA from conidiating N. crassa cultures. Total RNA samples were prepared from cultures harvested after 24 hr growth and induced to conidiate for O-24 hr, as indicated under the line labeled “Conidiation (h)“. Equal amounts of RNA (6 fig) were loaded in each lane and analyzed as described in the legend to Fig. 1.

els in older mycelia, at times when transcript levels for amino acid biosynthetic genes were greatly reduced (Figs. 1 and 2; data not shown). In contrast to transcripts detected with con gene probes, transcript levels for genes important for cell growth and amino acid biosynthesis did not increase in RNA samples obtained from cultures undergoing conidiation (Figs. 1 and 2). Although the levels of transcripts for these genes were relatively high in RNA isolated from young macroconidia (Fig. 1, Lane 2SC), they were generally lower in RNA isolated from older macroconidia (Fig. 1, Lane C), implying decay. Expression of Alternative Transcripts

VOLUME148, 1991

tone transcripts, which arise from single copy genes (Woudt et al., 1983), have not been characterized. Changes in Poly(A) RNA Levels Several observations indicated that either the fraction of total RNA consisting of polyadenylated transcripts or the lengths of poly(A) tails decreased during conidiation. With many probes, relatively low hybridization signals were observed with total RNA samples from conidia or conidiating cultures compared to the signals obtained with poly(A) samples (Figs. 1 and 2). In addition, the fraction of total RNA that was recovered as poly(A) RNA after purification by oligo(dT)-cellulose chromatography was reduced in samples from dormant conidia and from conidiating cultures (data not shown). To determine the explanation for these observations, we measured levels of poly(A) by hybridization-protection (Table 2). Relative levels of poly(A) RNA increased following germination, with total RNA from ‘7-hr mycelium containing twice as much poly(A) RNA as dormant conidia. Assuming that the average poly(A) mRNA is 1700 nt long, and that the average poly(A) tail contains 50 A residues, then 2% of the total RNA isolated from 7-hr mycelium would be poly(A) mRNA. The differences in poly(A) levels shown in Table 2 were due to changes in the fraction of RNA containing poly(A) tails rather than to changes in poly(A) tail lengths. Poly(A) tail lengths were measured directly in poly(A) RNA preparations obtained from these different developmental stages (Fig. 4). The distribution of poly(A) tail lengths remained essentially unchanged through the life cycle; major poly(A) species in N. crassa have tails approximately 20-70 nucleotides long. Our results agree with previous measurements of poly(A) tail length in vegetative mycelium (Lucas et ah, 1977). TABLE 2 CHANGESIN THE PoLY(A) CONTENTOFN. crassa RNA DURINGASEXUAL GROWTH’

Relative level of poly(A) Growth (hr) Some genes were represented by multiple mRNA species, including con-8 (Figs. 1 and 2), trp-I (Fig. 2), cox-5 C (1) (Figs. 1 and 2), histones H3 and H.4 (Figs. 1 and 2), and 1.5 0.5 2 rps-1 (Figs. 1 and 2). Several of these transcripts were 1.5 2.2 3 differentially expressed during the asexual cycle. For 1.9 7 example, a larger ~0x6 transcript was evident in dor1.7 11 mant conidia but was absent in mycelia. Preliminary 1.2 24 analyses of coxd cDNA clones indicate that some tran0.5 4 (con) 0.3 24 (con) scripts in dormant conidia contain intron sequences; thus the larger species may represent unspliced or difDRNA samples are described in the legend to Fig. 2. The amounts of ferentially spliced cox-5 transcript. A large histone H3 poly(A) in samples of total RNA were measured by protection of transcript was more abundant in mRNA from vegeta- [‘H]polyU from nuclease digestion (Methods). Levels of poly(A) are tively growing mycelia than in mRNA from conidiating expressed relative to the level in dormant conidia (12.6 ng poly(A)/ cultures. The structures of the multiple Neurospora his- A, unit total RNA).

SACHS AND YAN~FSKY

Effects of Amino Acid Starvation Mycelia

wild type

on RNA Levels in

The response of wild-type, arg-W, and Acpc-1 strains to amino acid starvation was determined during expoGerm (h)

123

Developmental Expression in N. wassn

Con (h)

+

/

Acpcl G

*,

argl 2s 9-%

e,G

rpl 1 his 3 trp 3 cpc 1 Acpc 1 arg 2 cox 5

26

FIG. 4. Direct visualization of N. crassa poly(A) tails in RNA isolated throughout the asexual life cycle. Poly(A) mRNA (5-100 ng) was X,-end labeled with [32P]pCp and digested with RNase A. The sizes of the labeled, poly(A) mRNA S’ends were determined after electrophoresis through a 12% polgacrylamide/7 M urea gel and autoradiography (Sachs and Davis, 1989). The sizes of HhaI-digested pBR322 DNA fragments (Lane M) are indicated. Lanes contain poly(A) mRNA samples from: dormant conidia (C); conidia germinated for 0.5,1.5,3,7,11, and 24 hr; from cultures induced to conidiate for 4 and 24 hr; and from purified, 24-hr conidia (24C).

FIG. 5. The effects of His starvation and Arg supplementation on transcript levels in wild-type, &PC-1 and arg-1T strains. Cultures of wild-type, Acpc-1 and urg-I? mycelia were grown for 14 hr in minimal medium (-), or they were grown for 12 hr in minimal medium followed by an additional 2 hr of growth after addition of AT to the medium to a final concentration of 6 m&f, or they were grown for 14 hr in minimal medium supplemented with 0.4 mg/ml arginine (R). Equal amounts of RNA (4 gg) were loaded in each lane and analyzed as described in the legend to Fig. 1 using a2P-labeled DNAs representing the genes indicated in each row. The truncated cpc-1 transcript produced in the kpc-1 strain is designated Acpcl.

nential vegetative growth, and following germination. RNA was prepared from cultures grown in minimal medium and from parallel cultures starved for His by growth in the presence of 3-amino-1,2,4-triazole (AT) prior to harvesting. In 14-hr cultures of wild-type mycelia which had been starved of His for 2 hr, his-3 and trp-3 mRNA levels were higher than in unstarved mycelia; the cpc-1 mRNA level also generally increased slightly (Fig. 5). The level of arg-2 mRNA was not appreciably affected, while the levels of co&j and rpl-1 mRNA decreased slightly. The levels of arg-2, cpc-1, and cox-5 transcripts in 12-hr wildtype mycelia starved for His for periods of 0.25,1, and 2 hr were quantified using an RNase protection assay (Table 3) as described under Methods. The relative levels of arg-2 and cpc-1 mRNA increased within 0.25 hr of starvation, with peak increases observed after 1 hr. Thus a transient increase in these transcript levels is observed following starvation. In contrast, the relative level of cox-5 mRNA decreased to one-third of its original value within 1 hr of starvation. These data agree with the results of Northern analyses with these RNA samples (data not shown). Northern analyses of his-3 and trio-3 transcript levels indicated that they also increased dur-

124

DEVELOPMENTALBIOLOGY

TABLE 3 CHANGESIN RELATIVELEVEL OFcpc-I, arg-2, AND COX-5TRANSCRIPTS IN WILD-TYPE MYCELIA IN RESPONSETO His STARVATION cpm in protected RNA probe Hours of His starvation 0

cpc- 1

1

850 (1) 2560 (3) 4020 (4.7)

2

1900 (2.2)

0.25

arg-2 (1) 870 (1.6) 550

1230 (2.2) 750 (1.4)

cox-5

1310 (1) 1430 (1.1) 360 (0.3) 340 (0.3)

Note.Parallel cultures of wild-type mycelia were grown for 12 hr in minima1 medium. AT was added to the medium (final concentration, 6 mM) and the cultures incubated an additional 0.25, 1, or 2 hr. RNA was prepared from 12 hr mycelium immediately prior to AT addition (0), and His-starved mycelia harvested after growth with AT as indicated. Relative levels of cpcl, arg-2, and coz-5 mRNA were determined using the RNase protection assay (Methods). The relative level of each transcript, compared to its level in unstarved hyphae, is given in parentheses. ing starvation, and were highest 2 hr after addition of AT (data not shown; prolonged starvation was not examined). Thus wild-type cultures respond to His starvation by increasing their relative levels of RNAs important for amino acid biosynthesis, while reducing their relative levels of other RNA species. Starvation of cpc-1 Strains In minimal medium, transcript levels for amino acid biosynthetic genes were similar in Acpc-1 and wild-type mycelia (Fig. 5). However, the Acpc-1 strain differed from wild-type in its response to His starvation (Fig. 5). Neither the his-3 nor trp-3 mRNA level increased in starved Acpc-1 mycelia. Levels of rpl-1 mRNA increased, in contrast to wild-type, where they decreased. The Acpc-1 strain produces a 1.5-kb truncated cpc-1 mRNA lacking the CPCl protein coding region. The Acpc-1 transcript level increased upon AT addition; increases in the cpc-1 transcript level were also observed in starved mycelia of strains bearing the mutant alleles cpc-1 (CD15) or cpc-1(CD86) (Paluh et al., 1988; data not shown). Therefore, although a functional cpc-1 gene is necessary for the cross-pathway control response of biosynthetic genes, as noted previously (Paluh et ab, 1988), cpc-1 expression is regulated in response to amino acid starvation by factors other than CPCl. Starvation of the arg-12” Strain The argl? strain is a bradytroph which is chronically starved for Arg when grown in minimal medium; crosspathway control is activated in this strain under these conditions (Davis, 1986). We observed that arg-19 mycelium grown in minimal medium contained higher levels

VOLUME148,199l

of arg-2, his-$ and trp-3 mRNA than wild-type mycelium (Fig. 5), consistent with cross-pathway induction. However, the level of cpc-1 mRNA did not increase appreciably. arg-1X’ mycelium showed additional responses when starved for His: his-3, trp-3, and cpc-1 mRNA levels increased, and levels of arg-2 mRNA decreased. Eflects of Arg Addition on Expression of Amino Acid Biosynthetic Genes RNA levels for the genes surveyed in Fig. 5 were also determined for mycelia grown in the presence of excess Arg (Fig. 5, Lanes marked ‘R’). Arg-grown wild-type mycelia had higher levels of his-3 and cpc-1 mRNA than minimal-grown mycelia, mimicking amino acid starvation. Levels of his-3 mRNA were unchanged in Arggrown Acpc-1 mycelia, although levels of Acpc-1 mRNA increased. Apparently at least some fraction of the response of amino acid genes to excess Arg was cpc-1 dependent. In Arg-grown argl? mycelia, levels of arg-2 mRNA were greatly reduced, as they were in wild-type and Acpc-1 mycelia (Fig. 5 and Orbach et aL, 1990). The relative levels of his-J, trp-3, and cpc-1 mRNA were increased in Arg-grown arglX mycelia, exceeding the levels in wild-type. Thus, Arg supplementation appears to both induce a cpc-1 dependent starvation response and reduce the level of arg-2 transcript. Effects of Amino Acid Starvation in Germinating Conidia The response to His starvation was also examined in germinating conidia, a developmental stage when basal levels of transcripts for amino acid biosynthetic genes were exceptionally high in wild-type cells (Figs. 1 and 2). In wild-type cells germinating under conditions of His starvation, levels of his-3, trp-3, cpc-1, and arg-2 mRNA were appreciably higher than in unstarved cells (Fig. 6). The results of Northern analyses were confirmed and extended by quantifying arg-2, coxd, and cpc-1 mRNA levels in both dormant and germinating conidia using the RNase protection assay (Table 4). Compared to levels in dormant conidia, levels of arg-2 mRNA increased 17-fold during germination in minimal medium, and 24fold during germination under conditions of His starvation. cpc-1 RNA levels increased 24-fold (minimal medium) and 95-fold (His starvation) during germination. The level of coz-5 transcript increased modestly (4.6fold) during germination in minimal medium but was reduced when germinating conidia were starved for His. Levels of rpl-1, his-3, arg-2, and cox-5 transcripts were similar in Acpc-1 and wild-type strains germinating in minimal medium (Fig. 6). However, in the Acpc-1 strain,

SACHSANDYANOFSKY

wild type ~ 14

125

Developmental Expression in N. crassa

during asexual growth. In addition, we examine the effects of amino acid starvation on transcript levels in wild-type cells and in cells bearing mutations affecting amino acid metabolism.

-Acpcl 14

rpl 1

Developmental Patterns of RNA Expression

his 3 argl 2s

trp 3 cpc 1 Acpc 1

arg 2

cox 5 FIG. 6. The effects of His starvation on RNA levels in germinating wild-type, kpc-I and arg-12” conidia. Wild-type, kpc-1 and arg-19 conidia were germinated for 2 hr in minimal medium (p), or for 0.5 hr in minimal medium and were then incubated for an additional 1.5 hr after adding AT to a final concentration of 6 mM RNA samples (4 pg/lane) were probed with =P-labeled DNAs representing the N. crassu. genes indicated in each row. Acpcl is the truncated cpc-1transcript detected in the Acpc-1 strain.

Relative RNA levels for genes isolated on the basis of their preferential expression in conidiating cultures (Berlin and Yanofsky, 1985b) were exceptionally high in both newly formed and older conidia (Fig. 1). Although several of these genes have been characterized (Roberts et ah, 1988; Roberts and Yanofsky, 1989), the functions of their products remain unknown. Some (con-6, con-g, cm10,and con-11) were highly expressed only after cultures became morphologically committed to conidiation (Fig. 3); others (cm-2 and con-8) also were expressed in vegetatively growing mycelium, or early in conidiation. The different con genes examined had different patterns of expression during germination. Levels of con-8 and con11 transcripts decreased sharply within the first 0.5 hr of initiating germination, while levels of con-6 and con10 transcripts increased transiently during germination, then decreased. The rapid depletion of some cm2 transcripts early in germination is probably due to degradation. Jervis and De Busk (1975) reported that a minor, conidium-specific N. crassa tRNAph” was rapidly lost during germination, indicating that mechanisms exist for the developmental regulation of degradation of TABLE

the responses to His starvation during germination (Fig. 6) were fully (trp-3, arg-2) or partially (his-3, cpc-1) eliminated. Interestingly, the relative level of arg-2 transcript was considerably reduced when Acpc-1 conidia were starved for His during germination (Fig. 6 and Table 4). In germinating Acpc-1 conidia, the level of cox5 mRNA was reduced during His starvation (Fig. 6 and Table 4). The level of rpl-1 mRNA increased in the Acpc1 strain but not the wild-type strain during His starvation (Fig. 6), as was observed when His starvation was imposed during mycelial growth (Fig. 5). The levels of cpc-1 and arg-2 mRNA were higher in arg-19 than in wild-type conidia germinating in minimal medium, while the level of cox-5 transcript was similar in both strains (Fig. 6 and Table 4). Levels of cpc-1 and arg-2 mRNA decreased in His starved germinating arg-lP conidia (Fig. 6 and Table 4); this finding contrasts with the results obtained with 14-hr arg1P mycelia, in which His starvation led to increased levels of cpc-1 RNA (Fig. 5). DISCUSSION

In this report we examine changes in transcript levels for N. crassa genes involved with a variety of processes

4

CHANGESINRELATIVELEVELSOF~~~-~,C~~-~,ANDC~-~TRANSCRIPTS INGERMINATINGSPORESINRESPONSETOHISSTARVATION cpm in protected probe Strain Wild-type

4CPC-

1

arg-1B

RNA

Cell type Conidium Germinating His-starved germinating Conidium Germinating His-starved germinating Conidium Germinating His-starved germinating

120 2850 11410 ND ND ND 120 7110 1560

210 3590 4940 400 4510 1330 380 8990 4710

150 700 450 150 970 830 80 730 540

Note. RNA was prepared from (i) dormant conidia, (ii) conidia germinated for a period of 2 hr in minimal medium, and (iii) conidia germinated for a period of 2 hr: 0.5 hr in minimal medium and then exposed to AT for 1.5 hr at a final concentration of 6 mM(His-starved germinants). Transcript levels for arg-2, cpc-1, and coz-5 were measured in wild-type, Acpc-1 and arg-lb cells by RNase protection assays. Since the segment of the cpc-1 gene corresponding to the antisense probe is deleted from the 4cpc1 strain, 4cpc-1 mRNA was measured but not found with the cpc-1 probe. ND, no cpm above background detected.

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RNA pol III transcripts, as well as the presumed pol II con-6, cm-8, con-10, and con-11 transcripts we examined. The levels of many mRNA species important for N. crassa growth were lower in old conidia than in young conidia (Fig. 1). Presumably, different RNAs are programmed for preferential stabilization or destabilization as conidia mature and dormancy is established. Several observations are consistent with these possibilities. At least one gene (COX-5)is represented by a larger transcript unique to dormant conidia (Figs. 1 and 2). Also, it has been observed that the relative ratio of nonpolyadenylated to polyadenylated transcript for the proteolipid subunit of the F,F, ATPase is greater in dormant conidia than in germinating conidia (Brambl et al., 1987). The relative contributions of synthesis and degradation in establishing transcript levels at different developmental stages are presently unknown. We observed that the levels of transcripts encoding products important for growth (e.g., enzymes responsible for amino acid biosynthesis, P-tubulin, ribosomal subunits, and cytochrome oxidase subunits) increase significantly during germination. New RNA synthesis appears to be essential for germination (Schmit and Brody, 1976;Brambl et ab, 1987). Thus, although mRNAs representing all of the genes we have analyzed are detectable in N. crassa conidia, the levels in dormant spores may not be sufficient to complete the germination process. Levels of N. crassa tub.2 transcript increase rapidly during germination and remain high during mycelial growth (Fig. 2; also see Hoang-Van et al, 1989). Levels of COZ-5and co%-8 mRNA are highest in the mid-log phase of mycelial growth; the cellular level of cytochrome oxidase activity also peaks at this time (Alberghina and Guarnieri, 1975). The level of one con gene transcript, the 1-kb con-9 mRNA, reaches peak levels during lateexponential growth, although it is also high in young conidia. Presumably these are two periods during development when the product of this transcript is required. The levels of pyruvate kinase and its mRNA have been shown to be highest during the period of late exponential growth as well (Devchand and Kapoor, 1987). Measurements of sequence complexity in A. nidulans mRNA pools during development showed there were significant differences in mRNA pools between conidiating cultures, conidia, and vegetatively growing hyphae in this organism as well (Timberlake, 1980). Eflects of Amino Acid Starvation on mRNA Levels Germinating conidia contain very high levels of mRNAs for amino acid biosynthetic enzymes, suggesting that the genes for these enzymes are transcriptionally hyperactive during this period. This observation is

VOLUME 148,199l

consistent with the finding that pools of free amino acids are lower in 1 to 3-hr germinating conidia than in dormant conidia or mycelia (Schmit and Brody, 1975). A functional cpc-1 gene, which is necessary for the response to starvation for single amino acids, appears to be largely dispensable for the increased transcription of these genes observed during germination (Fig. 6). Therefore, other regulatory mechanisms must be responsible for the large increases in transcript levels for amino acid biosynthetic genes observed during germination. A functional cpc-1 gene was not necessary to obtain elevated levels of arg-2 and his-3 transcript during germination in minimal medium, but was necessary to obtain full expression of trip3. When His starvation was imposed during germination, functional cpc-1 was required for the observed increases in transcript levels for all of the amino acid biosynthetic genes we examined. In actively growing mycelium in minimal medium, basal transcript levels for amino acid biosynthetic genes appeared unaffected by the cpc-1 deletion (Fig. 5), although a functional cpc-1 gene was required for the increased expression of these genes in response to His starvation. These data suggest that cpc-1 expression may be necessary for establishing basal transcript levels for some but not all amino acid biosynthetic genes. Expression of cpc-1 may be principally used to provide the increases in transcript levels of genes important for amino acid biosynthesis during amino acid starvation. Thus the regulation of transcript levels for amino acid biosynthetic genes in N. crassa by cpc-1 resembles the comparable situation in S. cerevisiae, in which GCN4 expression plays an essential role in the response to amino acid starvation. GCN4 is also necessary for establishing basal transcript levels for some, but not all, amino acid biosynthetic genes (Arndt et al., 1987). Finally, our observation that amino acid starvation results in an increase in the transcript level for a mutant allele of cpc-1 which lacks the CPCl polypeptide coding region, and the recent identification of another N. crassa gene necessary for cross pathway control, cpc-2 (Kruger et al., 1990), indicate that the response to amino acid starvation in this organism also is complex. We thank David Perkins for advice and encouragement. We thank Alan Sachs for help with the poly(A) measurements and Joyce Heckman for providing clones and data prior to publication. We thank Dan Ebbole, Oded Yarden, Matthew Springer, Richard Weiss, and Carl Yamishiro for helpful discussions and comments on the manuscript. These studies were supported by a grant from the National Institutes of Health (GM-41296). M.S.S. was supported by a fellowship from the American Cancer Society. C.Y. is a Career Investigator of the American Heart Association. REFERENCES ALBERGHINA, F. A. M., and GUARNIERI, D. (1975). Change of the cytochrome oxidase level during exponential growth in Neurospora crussu. Ezperientia 31,914-915.

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