A large cluster of highly expressed genes is dispensable for growth and development in Aspergillus nidulans.

A Large Cluster of Highly Expressed Genes Is Dispensable for Growth and

Development in

Aspergillus nidulans

Rodolfo Aramayo, Thomas H. Adams and William

E.

Timberlake

Departments of Genetics and Plant Pathology, University of Georgia, Athens, Georgia 30602

Manuscript received December 19, 1988 Accepted for publication February 7 , 1989

during A. nidulans development.

A

whose biological properties make it valuable for studying mechanisms controlling eukaryotic cell me- tabolism, growth and differentiation (PONTECORVO et al. 1953; RAPER, FENNELL and AUSTWICK 1965; CLUTTERBUCK 1974; MACDONALD and HOLT 1976; ARST and BAILEY 1977; COCKER and GREENSHIELDS 1977; KINGHORN and PATEMAN 1977; MCCULLOUGH, PAYTON and ROBERTS 1977; MOSS 1977; PATEMAN and KINGHORN 1977; SMITH et al. 1977; THOMAS

1977; ZONNEVELD 1977; HAWKINS, GILES and KINGH- ORN 1982; ARST and SCAZZOCCHIO 1985; GREEN and SCAZZOCCHIO 1985; HYNES et al. 1985; MAY et al. 1985; OAKLEY, 1985; PATEMAN et al. 1985; BEEVER and LARACY 1986; CADDICK, BROWNLEE and ARST

1986; TIMBERLAKE and HAMER 1986; TIMBERLAKE 1987; TIMBERLAKE and MARSHALL 1988). T h e ge- nome of A. nidulans is compact (2.6 X l o 4 kb), about 5.5 times the size of the Escherichia coli genome (BAIN- BRIDGE 197 1 ; TIMBERLAKE 1978; KOHARA, AKIYAMA and ISONO 1987). It contains only 3% repetitive DNA, most of which codes for rRNA and tRNA precursors (TIMBERLAKE 1978). Laboratory strains appear to lack mobile genetic elements. Structural genes are very closely spaced and either lack or have only a few short introns (ORR and TIMBERLAKE 1982; GWYNNE et al. 1984; HAWKINS, DA SILVA and ROBERTS 1984; MUL-

1987; ADAMS, BOYLAN and TIMBERLAKE 1988). All of these features suggest that despite its metabolic and morphological diversity the A. nidulans genome has been streamlined through evolutionary pressures.

During asexual spore (conidium) production, 6% of the A. nidulans genome, corresponding to 1200 di- verse mRNAs (TIMBERLAKE 1980), is selectively tran- LANEY et UI!. 1985; UPSHALL et al. 1986; BOYLAN et

d .

Genetics 122: 65-7 1 (May, 1989)

ABSTRACT

We investigated the functions of the highly expressed, sporulation-specific SpoCl genes of Asper- gillus nidulans by deleting the entire 38-kb SpoC 1 gene cluster. T h e resultant mutant strain did not differ from the wild type in ( 1 ) growth rate, (2) morphology of specialized reproductive structures

formed during completion of the asexual or sexual life cycles, (3) sporulation efficiency, (4) spore viability or ( 5 ) spore resistance to environmental stress. Thus, deletion of the SpoCl gene cluster,

representing 0.15% of the A. nidulans genome, had no readily detectable phenotypic effects. Impli- cations of this result are discussed in the context of major alterations in gene expression that occur

scribed. Mature conidia contain approximately 200 spore-specific mRNAs that collectively comprise >11% of the mass of mRNA stored in the spore. T h e functions of these mRNAs are unknown. However, based on their abundance in spores, the evolutionary conservation of at least some of the genes encoding them (MULLANEY and KLICH 1987) and the compact- ness of the A. nidulans genome, we have speculated that they code for proteins having important functions in conidium differentiation, maintenance or germi- nation (MILLER et al. 1987).

One group of genes that encodes spore-specific transcripts has been subjected to extensive analysis (TIMBERLAKE and BARNARD 1981; GWYNNE et al.

66 R. Aramayo, T. H. Adams and W. E. Timberlake

MATERIALS A N D METHODS

A. nidulans strains and genetic techniques: T h e geno- types of strains used in this study are given in Table 1 .

Standard A. nidulans genetic techniques (PONTECORVO et al. 1953; CLUTTERRUCK 1974; KAFER 1977) and transforma- tion procedures (YELTON, HAMER and TIMRERLAKE 1984) were employed.

Nucleic acid manipulations: DNA and R N A were iso- lated as described (TIMRERLAKE 1986). Blot hybridizations were carried out as recommended by the membrane man- ufacturer (Hybond-N, Amersham).

Plasmid constructions: The following plasmids were con- structed by using standard procedures (MANIATIS, FRITSCH and SAMBROOK 1982; AUSREL et al. 1987).

1. pDC 1 : A 1.8-kb Sphl-BamHI fragment containing t h e A. nidulans argB+ gene (BERSE et al. 1983; UPSHALL et al.

1986) inserted into the SphI and BamHI sites of pIC20R (MARSH, ERFLE and WYKFS 1984).

2. pDC2: A 0.8-kb SalI-EcoRI fragment from XAn- SpoC1-R2 (GWYNNE et al. 1984) inserted into the Sal1 and GcoRI sites of pIC2OH (MARSH, ERFLE and WYKES 1984).

3. pDC3C: A 2.6-kb BamHI fragment from XAnSpoC1- D5 (GWYNNE et al. 1984) inserted into the BamHI site of pIC2OH.

4. pDC4: A 0.8-kb Xhol-SphI fragment from pDC2 in- serted into the XhoI and SphI sites of pDCl.

5. pDC5R: A 2.6-kb BamHI fragment from pDC3C in- serted into the BamHI site of pDC4.

6. pDC6: A 5.2-kb SmaI-XhoI fragment from pDC5R inserted into the SmaI and XhoI sites of pIC20H.

Spore survival experiments: Conidia were harvested and subjected to various treatments. They were then serially diluted, spread onto agar-solidified minimal medium con- taining appropriate nutritional supplements, incubated in the dark for 4 days at 37" and colonies were counted. Ultraviolet light treatment was done according to BARRON and MACNEILL (1 962). For high temperature' treatment, spores were suspended in H2O at a concentration of 1.5 X

1 O7 ml and incubated at 60" for various times. For extended viability tests, spores were suspended in H20 or maintained in the dry state and incubated at room temperature (22- 25") or at 37" for various times. For exposure to potentially toxic substances, spores were suspended in H z 0 at a concen- tration of 1.5 X lo7 ml and incubated for 2 min at room temperature in the presence of 0.1% (w/v) Nonidet P-40, 0.1 % Tween 80, 0.1 % SDS, 0.1 % Na+ deoxycholate, 0.1 % Triton X-100, 0.15 M NaCI, 5 M NaCI, CHCls-, CsH50HaC2- or CsHeO-saturated H20. Only treatments with the last three compounds significantly decreased spore viability.

Sporulation efficiency and spore germination: Coni- dium production was quantified as described by YACER, KURTZ and CHAMPE (1982), except that 1 X lo5 spores/ Petri dish were incubated at 37" and the number of spores/ cm2 was determined at 30, 32, 34, 36 and 38 h after inoculation. The fraction of spores germinating, the timing of germination and the morphology of germlings were determined by microscopic examination.

RESULTS

We wished to determine if removal of any SpoCl genes caused a phenotypic alteration. We therefore deleted the entire SpoC 1 region by using a transplace- ment strategy (ROTHSTEIN 1983; Figure 1 A). Plasmid pDC6 containing A. nidulans DNA fragments from

FIGURE 1 .-(A) Diagram of the SpoCl region. A map of the A.

nidulans SpoCl region is given and the sequences used to delete

the cluster are shown below it. The sequences corresponding to the probes are: Probe 1 , 8-kb BamHl fragment from XAnSpoCI-S3; probe 2, XAnSpoC1-L8; probe 3. XAnSpoC1 and probe 4, 7 kb

Sal1 fragment from XAnSpoCl-R2. (GWYNNE el al. 1984). Restric-

tions sites are: B (BamHI), S (Sall). E (EcoRI), Sm (Smal) and X

( X h o l ) . The only X and Sm restriction sites shown are those that

were used to isolate the fragment to delete the cluster. (B) Southern blot ;~nalysis. DNA from the wild-type (WT) and a ASpoC1 (M)

TABLE 1

Aspergillus nidulans strains

~ ~~

Designation Genotype Origin

P" 1 (bioA I '; argB2; mefG I b; velA I ') P. WEGLENSKI, Department

o f Genetics, Warsaw Uni- versity. Pol;md

A.JC 473.14 ( ~ e l A 2 ~ ; a r g B 2 pxnA4'; velA I . JOHN CLUTTERRUCK, Depart-

n i a D l 7 ) ment of Genetics, Glasgow

University, Scotland

FGSC26 (bioA I ; velA I ) Fungal Genetics Stock Center

FGSC237 (yelA2, pabaAl; trpC801, Fungal Genetics Stock Center

FGSC I94 (yelA2, bioA I ; velA I ) Fungal Genetics Stock Center

velA I )

FGSC3.57 (bioA I : wA 3; velA I ) Fungal Genetics Stock Center

TRAOO7 (bioAI; argB2, ASpoC1::argB'; This study; PW 1 transformed

metGI; velAI) w i t h the 5.2-kb Smal-Xhol

fragment from pDC6

RKAOO2 (bioA I ; argB2, ASpoC1 ::argB': Progeny from TRAOO7 X

RRAOO7 (yelA2; metG I ; velA 1 ) Progeny from KRAOO2 X

KKAOOS (yelA2; ASpoC1::argB'; metGI: Same as KRA007

RRAOOS (metG I ; velA I ) Same a s RRA007

RRAOIO (ASpoC 1 ::argB+; mrtG 1 ; Same as KKA007

metG I ; velA I ) AJC 473.14

FGSC237

velA I )

velA I )

' bioAI = biAI; b metGI = methGI; ' velAI = v e A l ; d yelA2 = yA2; ' pxnA4 = pyroA4.

FIGURE 2.-RNA blot :lnal\sis. (;I) Total R N A ( I O pg, lanes 1

; ~ n d 3: 20 pg. lanes 2 and 4) from a ASpoC1 (lanes I and 2) or the wild-type strain (lanes S and 4) w a s fractionated by denaturing agarose gel electrophoresis and blots were hybridized with radiola- bclecl DNA fragments corresponding to a portion of the SpoCl region (XAnSpoC1 and XAnSpoC1-SS). (B) The membrane was \c;lshed and rehybridized w i t h ;In argB probe to confirm loading of the lanes.

chromosomal regions adjacent to the SpoCl cluster fused to a fragment containing the argB+ gene, was constructed. T h e composite SmaI-XhoI A . nidulans D N A fragment was isolated and used to transform an

argB- A . nidulans strain (PW 1, Table 1) to arginine-

independence. T h e chromosomal integration event illustrated in Figure 1A was expected to evict the

entire SpoCl region, including the 1.1-kb direct re- peats (RPT3).

DNA was isolated from 64 transformants and gel blot hybridization analysis was used to determine if both RPT3 sequences had been deleted. A single transformant (TRA007) lacked both repeats and was characterized further. Figure 1 B demonstrates that the entire SpoCl region was deleted from TRA007. T h e hybridization pattern observed for wild-type DNA was as expected (Figure lB, lanes 1, 3, 5 and 7; GWYNNE et al. 1984), while no hybridization to the SpoCl probes (Figure 1A) was observed for TRA007 DNA (Figure lB, lanes 2, 4, 6 and 8). Equal loading of DNA was confirmed by hybridizing the same mem- brane, without removing the previous probes, to ra- diolabeled DNA that corresponds to 6rlA (Figure 1 B,

lanes 3 , 4 , 7 and 8; ADAMS, BOYLAN and TIMRERLAKE 1988). Evidence that the deletion was directed by

integration of the SmaI-XhoI fragment into the SpoCl region was obtained by demonstrating genetic linkage of the argB+ gene to the deletion event (data not shown).

Conidial RNA gel blots were hybridized with two SpoCl internal clones containing most of the SpoCl messages (XAnSpoC1 and XAnSpoCl-S3; GWYNNE et al. 1984) and washed at moderate (0.3 M Na+, 65") or high (0.03 M Na+, 65") stringencies to detect SpoC 1 -related RNAs. As shown in Figure 2, no SpoC 1 complementary transcripts were detected.

6 8 R. Aramayo, T. H. Adams and W. E. Timberlake

Co-isogenic strains

Mixture <;rc.ell-sprcd Yellowspored

I RRAOO9 ( m e t G I " : v e l A I b ) RRA007 (yelA2': metCI: uel.41) 2 UUAO09 ( m e t G I : v e l A I ) RRAOOR CwlAP; ASpoC1::argB +:

3 RRA010 (ASpoCI::argB+: RKA007 (yeIA.2: m e t G I ; v e M I ) metC I ; velA I )

metGI: velA I )

n metC I = methC: b vel,\ I = ueA I : ' yelA2 = ~qA2.

I IIWlb\YYY\

FIGURE 4.-Sensitivity of conidia to UV light. A mixture of green, yellow and white conidia was exposed to UV light as de- scribed under MATERIAU A N D METHODS. Samples taken at the times indicated were serialy diluted, plated and the fraction of green- spored survivors was calculated relative to either yellow- or white- spored srlrvivors. After 90 sec 3.70% of the green, 1.25% of the

yellow and 0.39% of the white conidia remained viable. One stand- ard deviation is indicated (T).

phores, conidia or germlings of strain TRA007 and the strain from which it was derived were detected by light microscopy (Figure 3). Both strains were identi- cal with respect to colonial morphology, radial growth and spore production rates. Conidia from the ASpoCl

FIGURE J.-"orphology of coni- cliophores, spores ; ~ n d gernllings. Kcpresent;ltive s1ructures from the wild-type (panels A, <: ant1 E) and ;I

ASpoCI strain (panels B, D ;Ind F) were photogr;tphecl a t 4 0 0 X by using differential interfercnce contrast op- tics. Conidiophores. panels A and 13:

ungerminated conidia, panels C and D: conidia gernlinatetl for (i 11, p;lnrls E m d F.

FIGURE 5.-Sensitivity of SpoC1+ and ASpoCI conidia to U V light. Mixtures green and yellow of conidia from SpoC1+ and

ASpoC1 strains (Table 2) were exposed to U V light and analyzed a s described in the legend of Figure 4.

strain swelled during early germination and adhered to one another and to plastic Petri dishes, as did the wild type.

Dispensable Genes in

classes changes. If there is a difference in sensitivity due to the tested genotypic variable (e.g., deletion of SpoCl), the difference should be consistent in recip- rocal spore color mixtures.

T h e feasibility of this approach was tested by mixing co-isogenic green (FGSC26), yellow (FGSC 194) and white (FGSC357) (Table 1) spores and exposing the mixtures to UV. Figure 4 shows that with increasing irradiation the fraction of green spores increased in comparison to either yellow or white spores, demon- strating that yellow and white spores are more sensi- tive to UV light than are wild-type green spores. Thus, spore color mixtures provide a convenient, internally controlled method for detecting a differential sensitiv- ity of conidia to environmental factors.

Three spore mixtures were prepared as shown in Table 2 and subjected to potentially detrimental con- ditions (exposure to UV light, high temperature, de- tergents, high salt concentration and organic chemi- cals) or stored for extended periods (2 weeks) either dry or suspended in H 2 0 (see under MATERIALS AND METHODS). Following treatment, the fraction of sur-

viving spores and the proportion of spores in each color class was determined. Figure 5 shows that the fraction of green spores in each of the spore mixtures increases as a function of UV light treatment. There was a similar change in the fraction of green spores in all populations as a function of irradiation, indicating that deletion of the SpoCl cluster had no effect on sensitivity to UV light. Negative results were obtained with all of the other treatments. Thus, these experi- ments failed to detect any phenotypic change associ- ated with deletion of the A. nidulans SpoCl gene cluster.

DISCUSSION

T h e results presented here demonstrate that the A . nidulans SpoC 1 gene cluster is dispensible for growth and development under laboratory conditions. This region represents 0.15% of the genome and codes for numerous poly(A)+ RNAs, that collectively comprise 2 2 % of the mRNA mass produced and stored in conidia. T h e high level of SpoCl gene transcription and the probable translation of SpoCl mRNAs con- stitutes a substantial energy expenditure during coni- dium formation. Because conidiation likely exists as a dispersal mechanism to expand the organism’s popu- lation when conditions are optimal for growth, it seems probable that evolutionary pressures would fa- vor efficient reproductive mechanisms that minimize the energy expended in spore differentiation. T h e strong regulation and high level of SpoCl gene expression would be expected to have been eliminated if the genes provided no biological advantage. In addition, the DNA sequence of at least the central portion of the SpoCl gene cluster is highly conserved

among diverse Aspergillus species (MULLANEY and KLICH 1987), supporting the notion that SpoCl genes have important functions in the Aspergilli.

In view of the preceding considerations, the lack of evidence for SpoCl gene functions during growth and development of A. nidulans might be considered surprising. However, neither the size of this deletion nor the elimination of tightly regulated genes are without precedent. For example, a large (>130 kb) segment of the A. nidulans genome distal to areA is dispensible for growth (CADDICK et al. 1986). Many genes that are specifically expressed by Saccharomyces cerevisiae during ascosporogenesis have been inacti- vated without affecting growth or sporulation (YA- MASHITA and FUKUI 1985; GARBER and SECALL 1986; GOTTLIN-NINFA and KABACK 1986; PERCIVAL-SMITH and SEGALL 1986; PETKO and LINDQUIST 1986). Re- sults from random mutagenesis (GOEBL and PETES 1986) showed that 70% of insertions in the S. cerevisiae genome caused no detectable phenotypic change. Di- rected mutational studies of snRNA genes (PARKER et al. 1988) showed that simultaneous inactivation of 6 snRNA genes had little effect on growth. Thus, a significant number of genes are apparently dispensible in fungi.

Many fungal genes with redundant functions have been described (e.g., MORRIS, LAI and OAKLEY 1979; LINDQUIST 1986). All homologous copies must be mutated before there is a phenotypic effect. We were unable to detect other DNA sequences or transcripts complementary to SpoC 1 in the ASpoC 1 strain by gel blot hybridization. However, some duplicated genes, such as A. nidulans @-tubulin genes, are highly diver- gent (MAY et al. 1985, 1987), indicating that func- tional homology may be difficult or impossible to detect by cross-hybridization.

SpoCl genes may have subtle functions that were not detected in our experiments. For example, A . nidulans grows on substrates as diverse as decaying leaf litter, citrus fruits, and rhinocerous bronchia

(THOMAS 1977; KUTTIN et al. 1984). Conidia may therefore have enzymes in their walls that hydrolyze complex nutrient sources present in their natural en- vironment. Conidia from the phytopathogenic fungus Nectria haematococca, for instance, contain cutinase, an enzyme that digests the waxy cuticle of plant stems and leaves (KOLLER, ALLAN and KOLATTUKUDY 1982). Our experiments were not designed to detect such exotic catalytic activities.

Finally, a large number of fungal genes code for enzymes that catalyze the formation of secondary metabolites such as P-lactam antibiotics and aflatoxins

70 R. Aramayo, T. H. Adams and W. E. Timberlake

Moreover, closely related fungal isolates may have or lack the ability to produce particular secondary me- tabolites without any significant corresponding effect on growth or development (MACDONALD and HOLT

1976; MAKINS, HOLT and MACDONALD 1983). In ad- dition, secondary metabolites may be synthesized by the action of metabolic “networks” rather than by linear metabolic pathways (ZAHNER, ANKE and ANKE

1983). The extensive redundancy of such networks makes them essentially immutable. Some of the 1200 sporulation-specific genes found in A. nidulans, in- cluding SpoCl genes, may be responsible for second- ary metabolite synthesis. Even though such genes may have subtle or redundant functions, and may be of little consequence to the organism itself, their activi- ties in synthesis of beneficial (e.g., antibiotics) and detrimental (e.g., carcinogens) compounds are of great importance to humans.

This work was supported by U.S. Public Health Service grant

GM37886 and U.S. Department of Agriculture grant 88-37262- 3566 to W.E.T. R.A. received support from Empresa Brasikira d e Pesquisa Agropecuiria (Centro Nacional d e Recursos Geneticos e Biotecnologia) and Conselho Nacional d e Desenvolvimento Cienti- fico e Tecnolbgico-Brazil.

We wish to thank S T E V E KARL for his excellent help in statistical analysis of the data, members of the laboratory for their helpful suggestions and CHARLES MIMS and WYATT ANDERSON for their critical reviews of the manuscript.

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