RESUMO
Finding a compatible mating partner is an essential step in the life cycle of most sexually reproducing organisms. Fungi have two or more mating types, and only cells of different mating type combine to produce diploid cells. In mushrooms, this is taken to extremes, with the occurrence of many thousands of mating types. But, having gone to such extraordinary lengths to ensure that almost any two mushroom mycelia in the wild can mate, cell fusion is not followed by nuclear fusion and true diploidy. Instead, the fused cells form a characteristic mycelium, known as the dikaryon, in which haploid nuclei are paired but actively prevented from fusing. The mating-type genes, which encode pheromones, pheromone receptors and homeodomain transcription factors, have crucial roles in regulating the complex developmental programme by which the dikaryon is formed.
Assuntos
Basidiomycota/fisiologia , Reprodução , Basidiomycota/citologia , Basidiomycota/genética , Fusão Celular , Regulação Fúngica da Expressão Gênica , Genes Fúngicos/genética , Genes Fúngicos/fisiologia , Genes Fúngicos Tipo Acasalamento , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Feromônios/química , Feromônios/genética , Feromônios/metabolismo , Receptores de Superfície Celular/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Transdução de SinaisRESUMO
A successful mating in the mushroom Coprinus cinereus brings together a compatible complement of pheromones and G-protein-coupled receptors encoded by multiallelic genes at the B mating-type locus. Rare B gene mutations lead to constitutive activation of B-regulated development without the need for mating. Here we characterize a mutation that arose in the B6 locus and show that it generates a mutant receptor with a single amino acid substitution (R96H) at the intracellular end of transmembrane domain III. Using a heterologous yeast assay and synthetic pheromones we show that the mutation does not make the receptor constitutively active but permits it to respond inappropriately to a normally incompatible pheromone encoded within the same B6 locus. Parallel experiments carried out in Coprinus showed that a F67W substitution in this same pheromone enabled it to activate the normally incompatible wild-type receptor. Together, our experiments show that a single amino acid replacement in either pheromone or receptor can deregulate the specificity of ligand-receptor recognition and confer a self-compatible B phenotype. In addition, we use the yeast assay to demonstrate that different receptors and pheromones found at a single B locus belong to discrete subfamilies within which receptor activation cannot normally occur.
Assuntos
Células Quimiorreceptoras/fisiologia , Cromossomos Fúngicos/genética , Coprinus/genética , Proteínas Fúngicas/fisiologia , Sequência de Aminoácidos , Mapeamento Cromossômico , Cruzamentos Genéticos , Proteínas Fúngicas/genética , Teste de Complementação Genética , Mutagênese Sítio-Dirigida , Feromônios/genética , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
The B mating type locus of the basidiomycete Coprinus cinereus encodes a large family of lipopeptide pheromones and their seven transmembrane domain receptors. Here we show that the B42 locus, like the previously described B6 locus, derives its unique specificity from nine multiallelic genes that are organized into three subgroups each comprising a receptor and two pheromone genes. We show that the three genes within each group are kept together as a functional unit by being embedded in an allele-specific DNA sequence. Using a combination of sequence analysis, Southern blotting, and DNA-mediated transformation with cloned genes, we demonstrate that different B loci may share alleles of one or two groups of genes. This is consistent with the prediction that the three subgroups of genes are functionally redundant and that it is the different combinations of their alleles that generate the multiple B mating specificities found in nature. The B42 locus was found to contain an additional gene, mfs1, that encodes a putative multidrug transporter belonging to the major facilitator family. In strains with other B mating specificities, this gene, whose functional significance was not established, lies in a region of shared homology flanking the B locus.
Assuntos
Coprinus/genética , Genes Fúngicos , Genes Fúngicos Tipo Acasalamento , Feromônios/genética , Receptores de Peptídeos/genética , Fatores de Transcrição , Alelos , Sequência de Aminoácidos , Sequência de Bases , DNA Fúngico , Dados de Sequência Molecular , Receptores de Fator de Acasalamento , Análise de Sequência de DNARESUMO
In the mushroom Coprinus cinereus, the multiallelic B mating type genes are predicted to encode a large family of seven-transmembrane domain receptors and CaaX-modified pheromones. We have shown that a single amino acid change Q229P in transmembrane domain VI of one receptor confers a self-compatible mating phenotype. Using a heterologous yeast assay, we have demonstrated that this C.cinereus pheromone receptor is a G-protein-coupled receptor and that the Q229P mutation is constitutively activating. A C.cinereus pheromone precursor was processed to an active species specifically in yeast MATa cells and activated the co-expressed wild-type receptor. Yeast cells expressing the wild-type receptor were used to test the activity of synthetic peptides, enabling us to predict the structure of the mature C.cinereus pheromone and to show that the Q229P mutation does not compromise normal receptor function.
Assuntos
Células Quimiorreceptoras/metabolismo , Coprinus/fisiologia , Proteínas de Ligação ao GTP/metabolismo , Coprinus/metabolismo , Expressão Gênica , Genes Fúngicos , Genes Fúngicos Tipo Acasalamento , Dados de Sequência Molecular , Mutação , Peptídeos/química , Peptídeos/farmacologia , Fenótipo , Feromônios/metabolismo , Reprodução , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologiaRESUMO
The A mating type genes of the mushroom Coprinus cinereus encode two families of dissimilar homeodomain proteins (HD1 and HD2). The proteins heterodimerize when mating cells fuse to generate a transcriptional regulator that promotes expression of genes required for early steps in sexual development. In previous work we showed that heterodimerization brings together different functional domains of the HD1 and HD2 proteins; a potential activation domain at the C terminus of the HD1 protein and an essential HD2 DNA-binding motif. Two predicted nuclear localization signals (NLS) are present in the HD1 protein but none are in the HD2 protein. We deleted each NLS separately from an HD1 protein and showed that one (NLS1) is essential for normal heterodimer function. Fusion of the NLS sequences to the C terminus of an HD2 protein compensated for their deletion from the HD1 protein partner and permitted the two modified proteins to form a functional transcriptional regulator. The nuclear targeting properties of the A protein NLS sequences were demonstrated by fusing the region that encodes them to the bacterial uidA (beta-glucuronidase) gene and showing that beta-glucuronidase expression localized to the nuclei of onion epidermal cells. These observations lead to the proposal that heterodimerization regulates entry of the active transcription factor complex to the nucleus.
RESUMO
Pheromone signaling plays an essential role in the mating and sexual development of mushroom fungi. Multiallelic genes encoding the peptide pheromones and their cognate 7-transmembrane helix (7-TM) receptors are sequestered in the B mating type locus. Here we describe the isolation of the B6 mating type locus of Coprinus cinereus. DNA sequencing and transformation analysis identified nine genes encoding three 7-TM receptors and six peptide pheromone precursors embedded within 17 kb of mating type-specific sequence. The arrangement of the nine genes suggests that there may be three functionally independent subfamilies of genes each comprising two pheromone genes and one receptor gene. None of the nine B6 genes showed detectable homology to corresponding B gene sequences in the genomic DNA from a B3 strain, and each of the B6 genes independently alter B mating specificity when introduced into a B3 host strain. However, only genes in two of the B6 groups were able to activate B-regulated development in a B42 host. Southern blot analysis showed that these genes failed to cross-hybridize to corresponding genes in the B42 host, whereas the three genes of the third subfamily, which could not activate development in the B42 host, did cross-hybridize. We conclude that cross-hybridization identifies the same alleles of a particular subfamily of genes in different B loci and that B6 and B42 share alleles of one subfamily. There are an estimated 79 B mating specificities: we suggest that it is the different allele combinations of gene subfamilies that generate these large numbers.
Assuntos
Coprinus/genética , Genes Fúngicos , Genes Fúngicos Tipo Acasalamento , Peptídeos/genética , Feromônios/genética , Receptores de Peptídeos , Fatores de Transcrição , Sequência de Aminoácidos , Mapeamento Cromossômico , Fator de Acasalamento , Dados de Sequência Molecular , Precursores de Proteínas/genética , Receptores de Fator de Acasalamento , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Transformação GenéticaRESUMO
The recognition of compatible mating partners in the basidiomycete fungi requires the coordinated activities of two gene complexes defined as the mating-type genes. One complex encodes members of the homeobox family of transcription factors, which heterodimerize on mating to generate an active transcription regulator. The other complex encodes peptide pheromones and 7-transmembrane receptors that permit intercellular signalling. Remarkably, a single species may have many thousands of cross-compatible mating types because the mating-type genes are multiallelic. Different alleles of both sets of genes are necessary for mating compatibility, and they trigger the initial stages of sexual development--the formation of a specialized filamentous mycelium termed the dikaryon, in which the haploid nuclei remain closely associated in each cell but do not fuse. Three species have been taken as models to describe the molecular structure and organization of the mating-type loci and the genes sequestered within them: the pathogenic smut fungus Ustilago maydis and the mushrooms Coprinus cinereus and Schizophyllum commune. Topics addressed in this review are the roles of the mating-type gene products in regulating sexual development, the molecular basis for multiple mating types, and the molecular interactions that permit different allelic products of the mating type genes to be discriminated. Attention is drawn to the remarkable conservation in the mechanisms that regulate sexual development in basidiomycetes and unicellular ascomycete yeasts, Saccharomyces cerevisiae and Schizosaccharomyces pombe, a theme which is developed in the general conclusion to include the filamentous ascomycetes Neurospora crassa and Podospora anserina.
Assuntos
Basidiomycota/fisiologia , Genes Fúngicos/fisiologia , Genes Fúngicos Tipo Acasalamento , Sequência de Aminoácidos , Genes Homeobox/fisiologia , Dados de Sequência Molecular , Feromônios/fisiologia , Reprodução , Transdução de Sinais/fisiologiaRESUMO
A protein with characteristic properties of a fungal hydrophobin (CoH1) was isolated from the monokaryotic stage of the basidiomycete Coprinus cinereus. A cosmid clone containing the corresponding gene (coH1) was identified using a cDNA probe derived by RT-PCR. Hybridization and sequence analysis identified a second gene, coH2, just 4.1 kb downstream of coH1 encoding a hydrophobin (CoH2) with 64% sequence identity. Both coH1 and coH2 are subject to developmental regulation. They are expressed in vegetative monokaryotic cells but not in the asexual oidia produced on the surface of monokaryons. Transcripts of the genes were barely detected in dikaryotic mycelium and were absent from fruit bodies. Loss of aerial growth due to a mutation known as oid-1 was correlated with lack of both hydrophobins.
Assuntos
Coprinus/genética , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Família Multigênica , Mutação , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Coprinus/metabolismo , Proteínas Fúngicas/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Ligação Genética , Dados de Sequência Molecular , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Propriedades de SuperfícieRESUMO
The nucleotide sequence of the structural gene for isocitrate lyase (acu-7) is presented and features of its coding sequence and predicted protein are described. Several motifs were identified within the promoter region which are potentially involved in transcriptional regulation. Surprisingly, some of these occur within the coding sequence of an adjacent gene of unrelated function that terminates within 371 bp upstream from acu-7. The sequence of this second gene identified an N-acetylglucosamine-1-phosphate transferase.
Assuntos
Coprinus/enzimologia , Isocitrato Liase/genética , Sequência de Aminoácidos , Sequência de Bases , Coprinus/genética , DNA Fúngico , Indução Enzimática , Genes Fúngicos , Dados de Sequência Molecular , Família Multigênica , Regiões Promotoras GenéticasRESUMO
Fungal cells need to recognize that they are genetically different in order to mate. Cells signal to each other by secreting small peptide pheromones which are detected by cell surface receptors. New transcriptional regulators are formed that alter the pattern of gene transcription. Basidiomycete fungi are unusual in having several thousands of different mating types. In these fungi, mating partner recognition demands a remarkable degree of specificity on the part of the proteins and peptides involved.
Assuntos
Fungos/genética , Proteínas de Membrana/fisiologia , Feromônios/genética , Fatores de Transcrição/genética , Fungos/fisiologia , Proteínas de Ligação ao GTP/fisiologia , Expressão Gênica , Meiose , Feromônios/fisiologia , Reprodução , Fatores de Transcrição/fisiologiaRESUMO
The A mating type locus of Coprinus cinereus determines mating compatibility by regulating essential steps in sexual development. Each A locus contains several genes separated into two functionally independent complexes termed A alpha and A beta, and the multiple alleles of these genes generate an estimated 160 A mating specificities. The genes encode two classes of homeodomain-containing proteins designated HD1 and HD2. In this report we describe two newly cloned loci, A2 and A5, and compare them with A42, A43 and A6 that we have described previously. An A beta-null locus, retaining just a single active HD1 gene from the alpha-complex, was generated by mutation. Using this as a transformation host, gene combinations that promote A-regulated development were identified. We demonstrate that each A locus contains members of three paralogous pairs of HD1 and HD2 genes. Different allelic versions of gene pairs are compatible but paralogous genes are incompatible. The genes present in four uncloned A loci were deduced using Southern analyses and transformations with available cloned genes. The combined analysis of nine A factors identifies sufficient A gene alleles to generate at least 72 A mating specificities.
Assuntos
Mapeamento Cromossômico , Coprinus/genética , Genes Fúngicos , Genes Homeobox , Genes Fúngicos Tipo Acasalamento , Alelos , Southern Blotting , Transformação GenéticaRESUMO
The A mating type-genes of the mushroom, Coprinus cinereus, encode two classes of homeodomain-containing proteins distinguished as HD1 and HD2 on the basis of conserved, but distinctly different motifs. Compatible mating partners bring together versions of the proteins that can heterodimerize, thereby generating an active transcription factor complex that commits mated cells to sexual development. We have previously described a rare mutation in which an HD2::HD1 gene fusion generates a 'fused dimer' lacking much of HD1 including the homeodomain yet capable of constitutively promoting development [Kües et al., EMBO J. 13 (1994b) 4054-4059]. Here, we exploit this mutation to help identify contributions made by each protein class to normal heterodimer function. We show that the HD2 homeodomain is essential; deletion within the HD1 homeodomain can be tolerated in a normal heterodimer, as well as in the mutant fusion protein, but not substitution of a critical amino acid. We define, by deletion analysis, an essential C-terminal region of the HD1 and demonstrate its potential activation function by the ability to activate transcription in yeast when fused to the GAL4 DNA-binding domain. We also identify a potential role in transcriptional repression for the predicted C-terminal helix of HD1 proteins.
Assuntos
Coprinus/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Fúngicas , Proteínas de Homeodomínio/metabolismo , Sequência de Aminoácidos , Biopolímeros , Proteínas de Ligação a DNA/genética , Proteínas de Homeodomínio/genética , Dados de Sequência Molecular , Saccharomyces cerevisiae/genética , Transcrição GênicaRESUMO
The A mating-type locus of the mushroom Coprinus cinereus contains three or more paralogous pairs of genes encoding two families of homeodomain proteins (HD1 and HD2). A successful mating brings together different allelic forms of at least one gene, and this is sufficient to trigger initial steps in sexual development. Previous studies have suggested that development is regulated by heterodimerization between HD1 and HD2 proteins. In this report, we describe 5[prime] gene deletions and 5[prime] end exchanges showing that the N-terminal regions of the proteins are essential for choosing a compatible partner but not for regulating gene transcription. Using an in vitro glutathione S-transferase association assay, we demonstrated heterodimerization between HD1 and HD2 proteins and found that heterodimerization only occurs between compatible protein combinations. The N-terminal regions of the proteins were sufficient to mediate dimerization, and N-terminal swaps resulted in a predicted change in dimerization specificity. By analyzing the N-terminal amino acid sequences of HD1 proteins, we identified two potential coiled-coil motifs whose relative positions vary in paralogous proteins but are both required for in vivo function.
RESUMO
The A mating type locus of the mushroom Coprinus cinereus regulates essential steps in sexual development. The locus is complex and contains several functionally redundant, multiallelic genes that encode putative transcription factors. Here, we compare four genes from an A locus designated A42. Overall, the DNA sequences are very different (approximately 50% homology), but two classes of genes can be distinguished on the basis of a conserved homeodomain motif in their predicted proteins (HD1 and HD2). Development is postulated to be triggered by an HD1 and an HD2 gene from different A loci. Thus, proteins encoded by genes of the same locus must be distinguished from those encoded by another locus. Individual proteins of both classes recognize each other using the region N-terminal to the homeodomain. These N-terminal specificity regions (COP1 and COP2) are predicted to be helical and are potential dimerization interfaces. The amino acid composition of the C-terminal regions of HD1 proteins suggests a role in activation, and gene truncations indicate that this region is essential for function in vivo. A corresponding C-terminal region in HD2 proteins can be dispensed with in vivo. We will discuss these predicted structural features of the C. cinereus A proteins, their proposed interactions following a compatible cell fusion, and their similarities to the a1 and alpha 2 mating type proteins of the yeast Saccharomyces cerevisiae.
Assuntos
Coprinus/genética , Proteínas Fúngicas/genética , Genes Fúngicos/genética , Genes Fúngicos Tipo Acasalamento , Proteínas de Homeodomínio/genética , Sequência de Aminoácidos , Sequência de Bases , DNA Complementar/genética , Genoma Fúngico , Proteínas de Homeodomínio/classificação , Fator de Acasalamento , Dados de Sequência Molecular , Peptídeos/genética , Estrutura Secundária de Proteína , Reprodução/genética , Análise de Sequência de DNA , Fatores de Transcrição/genética , Transformação GenéticaRESUMO
We have identified the seven genes that constitute the A43 mating-type factor of Coprinus cinereus and compare the organisation of A43 with the previously characterised A42 factor. In both, the genes that trigger clamp cell development, the so-called specificity genes, are separated into alpha and beta loci by 7 kb of noncoding sequence and are flanked by homologous genes alpha-fg and beta-fg. The specificity genes are known to encode two classes of dissimilar homeodomain (HD1 and HD2) proteins and have different allelic forms which show little or no cross-hybridisation. By partial sequencing we identified a divergently transcribed HD1 (a1-2) and HD2 (a2-2) gene in the A43 alpha locus. a2-2 failed to elicit clamp cell development in three different hosts, suggesting that it is non-functional. a1-2 elicited clamp cells in an A42 host that has only an HD2 gene (a2-1) in its alpha locus, thus demonstrating that the compatible A alpha mating interaction is between an HD1 and an HD2 protein. The A43 beta locus contains three specificity genes, the divergently transcribed HD1 and HD2 genes b1-2 and b2-2 and a third HD1 gene (d1-1) that was shown by hybridisation and transformation analyses to be functionally equivalent to d1-1 in A42. An untranscribed footprint of a third A42 HD1 gene, c1-1, was detected between the A43 b2-2 and d1-1 genes by Southern hybridisation.
Assuntos
Coprinus/genética , Proteínas Fúngicas/genética , Genes Fúngicos , Genes Fúngicos Tipo Acasalamento , Proteínas de Homeodomínio/genética , Sequência de Aminoácidos , Dados de Sequência Molecular , RNA Fúngico/genética , Transformação GenéticaRESUMO
The A mating type genes of the mushroom Coprinus cinereus encode two classes of putative transcription factor with distinctive homeodomain motifs (HD1 and HD2). A successful mating brings together different allelic forms of these genes and this triggers part of a developmental sequence required for sexual reproduction. In this report we provide evidence that this developmental programme is promoted by a physical interaction between the two classes of homeodomain protein. Rare dominant mutations conferring self-compatibility map to the A locus and result in constitutive operation of the A-regulated developmental pathway. Our molecular analysis of one of these mutations shows that it has generated a chimeric gene by inframe fusion of an HD2 and an HD1 gene. Fusion has overcome the normal incompatibility between two proteins coded by genes of the same A locus and generated a protein that is sufficient to promote development in the absence of any other active A mating type genes. The fusion protein retains most of the HD2 sequence, but only the C-terminal part of the HD1 protein. It has only the HD2 homeodomain motif as a potential DNA binding domain fused to an essential C-terminal region of the HD1 protein, which in a normal HD1-HD2 protein complex may be the major activation domain.
Assuntos
Coprinus/genética , Proteínas Fúngicas/genética , Genes Fúngicos/genética , Genes Homeobox/genética , Genes Fúngicos Tipo Acasalamento , Proteínas de Homeodomínio/biossíntese , Sexo , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Coprinus/citologia , Proteínas Fúngicas/biossíntese , Rearranjo Gênico , Proteínas de Homeodomínio/genética , Dados de Sequência Molecular , Mutação , Proteínas Recombinantes de Fusão/biossíntese , Reprodução/genética , Deleção de Sequência , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genéticaRESUMO
The A mating factor of Coprinus cinereus determines compatibility in mating by regulating part of a developmental sequence that leads to dikaryon formation. The A genes that trigger development encode two different classes of homeodomain proteins, and for a successful mating, a protein of one class, HD1, must interact with a protein of the other class, HD2. In this report we show that C. cinereus A genes that encode HD2 proteins, a2-1 and b2-1, can elicit A-regulated development in the heterologous host C. bilanatus. Transformation rates were very low, suggesting that the genes were poorly transcribed. The fact that the HD2 genes are functionally expressed implies successful heteromultimeric association of putative DNA-binding proteins coded by the two Coprinus species. This interaction was sufficient to satisfy the need for different A factors in the formation of a fertile C. bilanatus dikaryon, but fertile dikaryons were more readily produced in matings with the a2-1 gene transformants. The C. cinereus A genes, b1-1 and d1-1, which encode HD1 proteins, were either not expressed or their proteins were non-functional in C. bilanatus. These experiments raise some interesting questions regarding HD1-HD2 protein interactions.
Assuntos
Coprinus/genética , Genes Fúngicos , Genes Fúngicos Tipo Acasalamento , Clonagem Molecular , Coprinus/crescimento & desenvolvimento , Fenótipo , Reprodução , Transformação GenéticaRESUMO
The A mating type factor of Coprinus cinereus regulates part of a developmental sequence that leads to the conversion of the asexual monokaryon into the fertile dikaryon. The A42 factor is a complex of seven genes, at least four of which are involved in determining the specificity of mating interactions. In this report we show that the A42 genes are constitutively expressed in both monokaryons and dikaryons. This has important implications with respect to intracellular recognition of a compatible mating, which requires an interaction between proteins already present within the cells of the mating partners, and for the subsequent maintenance of dikaryotic growth.
Assuntos
Coprinus/genética , Genes Fúngicos , Genes Fúngicos Tipo Acasalamento , Peptídeos/genética , Transcrição Gênica , Northern Blotting , Clonagem Molecular , Cruzamentos Genéticos , DNA Fúngico/genética , DNA Fúngico/isolamento & purificação , Escherichia coli/genética , Fator de Acasalamento , Feromônios/genética , Plasmídeos , RNA Fúngico/genética , RNA Fúngico/isolamento & purificação , RNA Mensageiro/genética , RNA Mensageiro/isolamento & purificação , Mapeamento por RestriçãoRESUMO
We have isolated a gene from Coprinus cinereus which cross-hybridises to the facA and acu-5 genes of Aspergillus nidulans and Neurospora crassa, respectively. These genes encode acetyl-CoA synthetase, an enzyme which is inducible by acetate and required for growth on acetate as sole carbon source. We have designated the C. cinereus gene acs-1 and have used transformation to demonstrate its functional homology to the ascomycete genes by complementation of an N. crassa acu-5 mutation. The acs-1 gene has never been identified by mutation; mutations leading to loss of acetyl-CoA synthetase function map to another gene, acu-1. Using Northern analyses we have shown that acu-1 has a regulatory function that is required for acetate-induced transcription of acs-1 and of another acetate utilisation gene, acu-7, the isocitrate lyase structural gene.
Assuntos
Acetatos/farmacologia , Acetil-CoA Carboxilase/genética , Coprinus/enzimologia , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Genes Reguladores/genética , Ácido Acético , Northern Blotting , Clonagem Molecular , Coprinus/genética , Escherichia coli/genética , Biblioteca Genômica , Isocitrato Liase/genética , Plasmídeos/genética , Mapeamento por Restrição , Homologia de Sequência do Ácido NucleicoRESUMO
The pAN7.1 plasmid containing the E. coli hygromycin B phosphotransferase gene was used to transform protoplasts of the ectomycorrhizal fungus Hebeloma cylindrosporum. Hygromycin-resistant transformants were selected at a frequency of one to five per micrograms of transforming DNA. Southern blot analyses revealed multiple copy integration of the transforming plasmid into the genome. The selection system was used to introduce other genes of interest by co-transformation. Two plasmids, one containing tryptophan biosynthesis genes and the other the NADP-glutamate dehydrogenase gene from the saprophytic basidiomycete Coprinus cinereus, were successfully introduced into the H. cylindrosporum genome with up to 70% efficiency of co-transformation. The hygromycin resistance phenotype was stably maintained during growth of transformants on hygromycin-free medium. All transformants retained their ability to form mycorrhizae with the habitual host plant Pinus pinaster, making them suitable for future physiological studies.