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Biological Research

Print version ISSN 0716-9760

Biol. Res. vol.34 n.2 Santiago  2001

http://dx.doi.org/10.4067/S0716-97602001000200012 

Genetic and cytological characterization of a
developmental mutant of Aspergillus nidulans induced
by 5-azacytidine

COSTA, MARCO ANTONIO1, SILVA, NELMARA CRISTINA GONÇALVES2 AND CASTRO-PRADO, MARIALBA AVEZUM ALVES2

1 Universidade Paranaense, Instituto de Ciências Farmacêuticas e Bioquímica, Praça Mascarenhas de Morais S/N, Umuarama, Paraná, Brasil, 87500-000

2 Universidade Estadual de Maringá, Departamento de Biologia Celular e Genética, Av. Colombo 5790, Maringá, Paraná, Brasil, 87015-900.

Corresponding Author: Marialba A. A. de Castro-Prado.Universidade Estadual de Maringá, Departamento de Biologia Celular e Genética. Av. Colombo 5790, Maringá, PR, Brasil. CEP: 87015-900. FAX: (55) 44 263655. e-mail: maacprado@uem.br

Received: January 16, 2001. In revised form: June 29, 2001. Accepted: July 9, 2001.

ABSTRACT

An analysis of a new medusa mutant of Aspergillus nidulans obtained by 5-azacytidine-treatment and named B116 is provided. The B116 mutant was phenotypically characterized by the production of conidiophores with reduced pigmentation and vesicles bearing multiple tiers of sterigmata. A single nuclear gene located on chromosome I is responsible for phenotypical changes in the mutant. The 5-azacytidine-altered locus, designated medA102, is recessive in heterozygous diploid and the medusa mutant is a Dp(II,I) duplication bearer that renders the strain mitotically unstable.

Key terms: conidiation, developmental program, medusa.

INTRODUCTION

The orderly differentiation of a multicellular reproductive structure named conidiophore characterizes asexual reproduction in the filamentous fungi Aspergillus nidulans. Conidiophore begins to develop five hours after induction of a competent mycelium. It consists of an aerial hypha, a multinucleated vesicle and two tiers of uninucleate sterigmata, the metulae and phialides (11, 33).

The acquisition of developmental competence and the regulation of conidiation are genetically controlled (1, 6). The conversion of vegetative growth into conidiation requires several genes, including six fluffy genes designated fluG, flbA, flbB, flbC, flbD and flbE. FluG is apparently needed for the production of a small diffusible molecule that signals initiation of conidiophore development, whereas flbA is required to regulate a response to the fluG signal (24, 25, 38). All these genes are necessary for normal brlA mRNA accumulation (3, 36).

brlA (bristle) is a complex locus consisting of two overlapping transcripts called brlAa and brlAß, which encode redundant proteins (BrlAp). This protein contains tandem cys2-his2 zinc-fingers typical of DNA binding proteins. It is a primary transcriptional regulator of development, required for conidiation and necessary for spore formation (2, 13, 27). Null brlA mutants make only conidiophore stalks that elongate themselves indeterminately. The absence of BrlAp in these mutants prevents further formation of the conidiophore vesicles and gives the colony a 'bristle' appearance (1, 3,14).

Sequential activation of brlA, abaA (abacus) and wet (wet-white conidia) establishes the central regulatory pathway required in the transition from the vegetative cycle to conidiation (34, 36).

The abacus protein (AbaAp) encodes a DNA-binding protein that activates the brlAa transcription, reinforces its own transcription and activates wetA. It is required for phialide differentiation and developmental determination. The wet-white conidia protein (WetAp) is required for conidia maturation through the activation of numerous spore-specific genes (5, 34).

Two other important genes, stuA (stunted) and medA (medusa), are necessary to correct spatial organization of the conidiophores. The Stunted protein (StuAp) is a member of a family of fungal transcription factors that is necessary for development and cell cycle progression (37). The medusa protein (MedAp) regulates the expression of the two brlA transcripts and is required as a coactivator of abaA expression. medA mutants have delayed differentiation of phialides and conidia, resulting in production of multiple layers of sterigmata (4, 11,18). They are also self-sterile (11, 13).

5-azacytidine (5AC) is a cytosine analogue with a nitrogen atom in position 5 of the pyrimidine ring. Incorporation of 5AC into DNA leads to DNA hypomethylation and chromosome decondensation in eukaryotic cells (21, 23, 30). It was developed as a chemotherapeutic drug for the treatment of cancer, mainly leukaemia (20).

Several tumor-suppressor genes have been reported to be silenced by DNA methylation in cancer cells. 5AC-treatment of cancer cells in vitro may result in re-expression of genes previously silenced by DNA methylation process (10, 16).

5AC is also a mutagen that primarily induces C-to-G transversions, although C-to-T transitions and C-to-A transversions have also been induced by the analog (22, 29). It has been shown that in Escherichia coli the DNA-cytosine methyltransferase has little effect on 5AC-stimulated C-to-G transversions (17).

The present study was undertaken in order to characterize the B116 developmental mutant of A. nidulans obtained by 5AC. Genetic analysis of this mutant revealed that a single nuclear gene is responsible for its morphological changes The mutant allele showed itself to be recessive in the heterozygous diploid and received the provisional name of medA102. The cytological analyses of the B116 mutant led us to the characterization of a new medusa mutant.

MATERIAL AND METHODS

Aspergillus nidulans strains and media

A. nidulans strains used are described in Table I. The morphological mutant B116 was obtained in our laboratory after 5-azacytidine treatment of the 115 duplication strain (9, 12). Complete medium (CM) and minimal medium (MM) were described by Van de Vate and Jansen (1978) (35) and Pontecorvo et al. (1953) (26). Selective medium (SM) consisted of MM supplemented, according to the requirements of each strain. Solid medium contained 1.5 % agar. Incubation was at 37°C.

5-azacytidine treatment

Conidia from the 115 strain were inoculated into the center of six Petri plates containing 100 mM 5AC. The plates were incubated at 37°C for five days. The treated strain yielded a visible mitotic sector with different morphology from the original haploid 115 strain.

Cytological characteristics B116 mutant

For microscopic observation of the mutant strain, spores were inoculated over a dialysis membrane supported by solidified complete medium and the plates were incubated at 37°C for 30, 36 and 42 hs. After this period, dialysis membranes containing one or more colonies were stained with cotton-blue-lactophenol and examined under the light microscope.

Genetic Techniques

General methodology followed Pontecorvo et al (1953) (26).The diploids were prepared by Roper's method (28). The mapping of the mutant allele from B116 mutant was determined by the haploidization of the B116 // A288 diploid strain in Complete Medium (19). Heterokaryons were prepared in liquid MM plus CM (2,0%). Cleistothecia were obtained from the heterokaryons after 21 days of incubation in sealed Petri dishes containing solid MM supplemented, according to the requirements of the crossed strains.

RESULTS

1- Cytological characterization of the B116 mutant

The effects of medA102 mutation on development were analyzed from cytological preparations examined under the light microscope. Abnormal conidiophore vesicles bearing multi-layered metulae were recorded from colonies of the B116 mutant, growing over a dialysis membrane. The mutant also produced secondary conidiophores and vesicle bearing multiple and undifferentiated branches (Fig. 1).

Figure 1. Conidiophore morphology of A757 (A) and B116 strains (B-D). A, normal conidiophore, B, vesicle of conidiophore with multiple and undifferentiated branches, C, vesicle of conidiophore with multiple layers of sterigmata, D, vesicle of conidiophore showing multiple branches and secondary conidiophores. The conidiophore vesicle diameter corresponds to 10.0 µm.

2- Mapping of the medA102 mutant allele by sexual and parasexual cycles

The B116//A288 diploid strain presented normal conidiation and was haploidized spontaneously in CM. Mitotic segregants were analyzed phenotypically for the mapping of the mutant allele for conidiogenesis (Table II). Analysis of the B116 x A610 cross was undertaken to analyze chromosome IV meiotic segregation (Table III).

Cleistothecia were obtained from B116 x A507, B116 x G188 and B116 x G1101 crosses. Analysis of progeny of these crosses showed linkage between the medA102 and the fpaB37, galD5 and acuM301 mutant alleles from chromosome I (Table IV). Results mapped the medA102 mutation in chromosome I (Tables II, III and IV).

The B116 x G0255 cross was employed to analyze the sexual cycle of the B116 mutant which failed to produce cleistothecia in this cross. This had already been expected since medusa mutants are self-sterile and indicates allelism of medA15 and medA102 mutations. On the other hand, the development of sexual cycle-specific Hülle cell remained unaffected in the B116 mutant strain.

3-Mitotic and meiotic instability of the B116 mutant strain

The B116 medusa mutant showed mitotic instability, spontaneously producing w mitotic variants, mutant for conidiation (Fig. 2). The altered morphology of a w mitotic variant, named V1, showed linkage with the galD5 marker (Table V).

w meiotic segregants were also recovered among the progeny of the B116 x G817 cross, homozygous for w+ (Fig. 3). Results suggest the presence of Dp(II,I) duplication in the genome of the B116 mutant expressing the w recessive marker. The Dp(II,I) duplication is a long segment from chromosome II duplicated and transposed to chromosome I, including the Acr, w and meth+ markers (12).

4- Mitotic and meiotic instability of the 41-N segregant

The 41-N segregant, obtained in the B116 x A507 cross, is normal to conidiation and forms colonies with crinkled morphology. This segregant occasionally produces improved mitotic sectors with normal morphology and growth rate. w+ and w segregants were also obtained in the 41-N x G188 cross, homozygous for w+ (Fig. 4). Results suggest that the 41-N segregant and B116 mutant are Dp (II,I) duplication bearers.

Figure 2. Mitotic instability of the B116 mutant Colony of B116 medusa mutant producing white mitotic variant mutant for conidiation

Figure 3. Meiotic instability of the B116 medusa mutant Production of w (white) segregants among the progeny of the B116 x G817 cross.

Figure 4. Meiotic instability of the 41-N segregant. Production of w (white) segregants among the progeny of the 41-N x G188 cross.

DISCUSSION

Temporal exposition of 115 strain of A. nidulans at low doses of 5-azacytidine resulted in the isolation of mutant cells clone for conidiogenesis. The mutant had the same characteristics of other A. nidulans mutants, described in literature as medusa (11,34), or rather, formation of multiple layers of sterigmata, conidiogenesis delay, dependence on a recessive change and impairment of sexual cycle.

The nuclear location of the allele involved in the medusa condition of the B116 mutant was performed by analyzing of the diploid strain formed with the mutant and the A288 mapping strain. Mitotic segregants derived from the B116 // A288 diploid strain showed independent segregation between the morphologic alterations of the B116 mutant and the markers from chromosomes II to VIII. On the other hand, all segregants with normal conidiogenesis were also bi+ (A288 paternal class) and most of the medusa segregants showed bi phenotype (B116 paternal class). med+bi segregants were not reported (Table II). As a role, this result and those in Tables III and IV mapped the gene responsible for the mutant morphology of the B116 strain on chromosome I. The mutant allele received the provisional name of medA102 because it showed allelism with the medA15 mutation (13).

Previous papers showed that low concentrations of 5AC might alter the developmental program of A. nidulans, converting a high percentage of the cell population into fluffy phenotypic variants. They were mitotically and meiotically stable and mapped on a single nuclear gene (fluF1) (32). The authors suggested that 5AC did not act through random mutagenic action but, rather, that fluF constituted a specific target for this drug during the initial stages of asexual differentiation (31, 32).

The medusa condition of the B116 mutant may be the outcome of heritable organizational or mutational alterations induced by 5AC in the medA gene. These alterations might impair the production of the medA transcript during conidiation.

A nidulans strains bearing duplicate chromosomal segments are mitotically unstable, giving rise to deteriorated or improved sectors during vegetative growth. These sectors exhibit addition (deteriorated sectors) or deletion (improved sectors) of genetic material (7, 8). In current work, the production of w mitotic variants by the B116 mutant and improved sectors by the 41-N segregant suggest that they are DP (II,I) duplication bearers (Fig. 2). The mitotic variants probably result from the total or partial loss of one of the duplicated segments. On the other hand, the w meiotic segregants obtained in the B116 x G817 and 41-N x G188 crosses may be the result of the pairing of the homologous segments from chromosomes I and II in a quadrivalent configuration: the normal segment of chromosome II and the duplicated one, present on chromosome I. After pairing, the occurrence of a double crossing-over followed by normal disjunction and distribution in the meiotic products results in the segregation of the recessive marker w involved in the Dp(II,I) duplication (Fig. 5).

Present results demonstrate that 5AC is effective in the induction heritable alterations in the A. nidulans genome which may alter the developmental program of this filamentous fungi. Molecular cloning of the B116 affected gene will probably help clarify the mechanism of 5AC action in the A. nidulans genome.

Figure 5. Schematic representation of the 41-N x G188 cross in quadrivalent configuration during meiosis. The segregation of the parental (1) and the recombinant (2) chromatids towards the same meiotic pole results in wmeth+ segregants. ( ), duplicated segment.

ACKNOWLEDGMENTS

We are indebted to Dr. A. J. Clutterbuck for the Glasgow strains used in this work, and to Mrs. Luzia A. S. Regasse and Mrs. Sonia A. de Carvalho for their technical assistance. Our work was financially supported by CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico). We are especially thankful to God.

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