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Department of Plant Biology, The University of Minnesota, 250 Biological Sciences Center, Saint Paul, MN 55108, USA
Correspondence
Robert Brambl
brambl{at}umn.edu
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ABSTRACT |
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The early years of biochemical genetics |
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) summarized the field, reviewing organic acid metabolism, the chemical nature of the mould mycelium, nutrition, nitrogen metabolism, advances in penicillin production, and new methodologies in mould metabolism. He was determined not merely to catalogue composition and observations, but to provide a comparison of active biochemical systems, or ‘transformations effected by fungi’. His approach was modern in spirit, focusing on experimentation, but even more so when he excitedly described the recent accomplishments in biochemical genetics and the Beadle and Tatum work (Beadle & Tatum, 1941). His interests in the text were concentrated on biochemical activities unique to fungi, but he also recognized the potential usefulness and importance of fungi as model systems for larger questions in biology. In this text, Foster presented a pattern that has been repeated to the present: a dynamic and an occasional conflict between the study of fungi for their inherent, unique activities or for their usefulness and accessibility to study questions that apply to all of biology. Do we study fungi to solve problems in technology, agriculture and human health or to ask and answer larger questions about cell biology, genetics, development and biochemistry? Do we study the physiology of applied science or the molecular biology of model systems?
Despite Foster's enthusiasm and the acclaim eventually awarded to the Beadle and Tatum gene action studies with Neurospora crassa, as Norman Horowitz reminded us, this experimental approach and its reductionist interpretation were not widely accepted at the time (Horowitz, 1991). Geneticists and biologists in general were uncomfortable with simple interpretations of complex phenomena; the idea that a mutant phenotype was anything more than a terminal disturbance of a complex metabolic process encountered severe resistance. Further, an important criticism of the general applicability of the one-gene, one-enzyme hypothesis was the question of whether auxotrophic mutants, reparable by supply of a single growth factor, were representative of mutations in general. Rowland Davis points out that given the absence of knowledge about the structure of proteins, the identity of the genetic material, the organization of chromosomes, and most metabolic pathways, it was not clear how to trace causal relations between mutation and phenotype of complex organisms (Davis, 2000). Nevertheless, it was this determined attempt to trace these causal relations by Beadle and Tatum and their colleagues and students, through genetic, physiological and biochemical studies, that led to the generalized understanding of gene action and interaction as well as to methodological techniques for mutant generation and analysis that were formative and essential to the new field of biochemical genetics. Tatum and C. H. Gray showed that biochemical mutations also could be induced by irradiation in bacteria, thereby providing suitable (and essential) genetic markers that led directly to the work of Tatum's student, Joshua Lederberg, who demonstrated sexuality and recombination in Escherichia coli.
The subsequent studies depended upon isolation and characterization of N. crassa mutant strains by new recruits to the Beadle laboratory, Norman Horowitz, Herschel Mitchell, Mary Houlahan (Mitchell), David Bonner and Francis Ryan, among others. These workers generated a body of evidence supporting the one-gene, one-enzyme hypothesis, and their effort was essential for Beadle's generalization that ‘... a given enzyme will usually have its final specificity set by one and only one gene’ (Horowitz, 1991). Nevertheless, it was not until 1948 that H. Mitchell and J. Lein showed directly that a mutation in the N. crassa tryptophan synthetase locus could lead to an enzyme deficiency. The original mutant strain of N. crassa upon which the first Beadle and Tatum paper was based turned out not to be due to a null mutation as reported; instead, scientists at the Merck Research Laboratories discovered that the expression of the gene depended upon the pH of the growth medium. This unexpected result led to modifications of the continuous mutant screen in the Beadle laboratory to also include simultaneous high-temperature incubation. This led to another very important discovery and the use by Mitchell and Houlahan in 1946 of temperature-sensitive conditional mutant strains, which proved to be even more useful in genetics than the original nonconditional auxotrophic strains.
Other workers in the 1950s asked, if an auxotrophic nutritional requirement could be attributed to loss of activity in a particular enzyme, was the expression of the mutant gene blocked, or was the gene producing an inactive form of the enzyme? Bonner and Charles Yanofsky showed that the mutant form of N. crassa tryptophan synthetase could be immunoprecipitated with antibodies to the wild-type protein and in comparable quantity, clearly supporting the latter alternative. Norman Giles and his colleagues in the 1950s began to study the basis of reversion in biochemical mutants of N. crassa, interallelic complementation, and gene structure and organization. The Giles laboratory mounted a decades-long study of gene organization and regulation in the quinic acid gene cluster of N. crassa (and other fungi), much later showing that the quinic acid cluster represents a group of adjacent coding sequences whose expression is regulated at the level of transcription and is under the combined control of quinic acid and the qa-1 protein. This group found that a single enzyme, a product of one gene, catalysed two distinct reactions in the same pathway (Giles et al., 1991).
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Nitrogen metabolism |
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Antibiotics and toxins |
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The isolation and chemical characterization of mycotoxins was one of the major achievements of mycologists and chemists in the 1960s. Mycotoxins have caused problems as long as grain crops have been grown and stored, but their chemical nature was not determined until the 1950s and 1960s. In the previous decades in the USSR, a lethal disease in humans and livestock, alimentary toxic aleukia, was shown to be caused by consumption of mould-contaminated grain, and it was subsequently determined that the toxic agent was a member of the trichothecene group, potent inhibitors of eukaryotic protein synthesis. In the early 1960s the loss of a large number of animals in England was associated with consumption of peanut meal incorporated into the animal diets, a dietary ingredient that was shown to be contaminated with Aspergillus flavus or A. parasiticus. The toxic agent was characterized and described as aflatoxin B1, itself a relatively harmless substance until metabolized by the liver cytochrome P450 system, thus creating one of the most potent carcinogens known. The aflatoxins have remained an important public health problem, especially as contaminants of maize and tree nuts, along with other, more recently discovered classes of mycotoxins. G. A. Payne, Nancy P. Keller, D. Bhatnagar and colleagues have studied the biosynthetic pathways and molecular genetics of the polyketide aflatoxins produced by A. flavus and A. parasiticus, and they used a variety of strategies to identify and clone genes for these pathways and to examine their genetic regulation (Brown et al., 1999). (This general area of research experienced an ignominious misapplication of science to politics in the early 1980s when the US government, citing a university-supplied analysis of suspect US military samples, accused the USSR of employing three toxins of the trichothecene family as biological warfare agents in Southeast Asia and Afghanistan. The toxic ‘Yellow Rain’ which supposedly carried the aerial toxin proved to be only beehive waste and pollen.)
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Senescence |
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). The genetic factor responsible for the strain-specific time-of-onset phenotype was inherited through the protoperithecial or maternal parent. These workers and others, such as Donald Cummings, determined in the 1980s that P. anserina senescence is caused by circular, multimeric plasmid-like DNA elements that are amplifications of excised, non-overlapping regions of the mitochondrial genome (Cummings et al., 1979). The stopper mutant strains of N. crassa, which show a stop-start, cell death phenotype, also have deficiencies in mitochondrial cytochromes and develop abnormal mitochondrial DNAs with large deletions or rearrangements. Ragged mutants of Aspergillus amstelodami show an erratic growth resembling the stopper mutants, and rearrangements of mitochondrial DNA are correlated with this phenotype. |
The birth of mitochondrial genetics |
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). These findings presented the first evidence of non-Mendelian inheritance or of cytoplasmic inheritance of a genetic trait. Subsequent work by David Luck and others in the 1960s using other slow-growing strains of N. crassa showed that the trait could be transferred from one strain to another in microinjection experiments, and the cytoplasmic element responsible for the trait was the mitochondrial fraction. Luck also performed labelling experiments to test possible explanations for the origin of mitochondria, and he determined that mitochondria arose from pre-existing mitochondria, not from accretion of other cellular components. Anaerobic yeast cells appeared to have no mitochondria, leading to the view that mitochondria assembled de novo upon aerobic growth. However, Gottfried Schatz and colleagues showed that anaerobic yeast cells do possess rudimentary mitochondria, and these organelles become visible cytologically if certain lipids are provided to the cultures. A number of groups working with S. cerevisiae and N. crassa determined that mitochondria possess quasi-autonomous genetic systems, with circular DNAs, ribosomes and tRNAs that are distinct from the cytoplasmic protein synthesis system, and sensitivity to antibiotics such as chloramphenicol that have no effect upon cytoplasmic protein synthesis. The slow-growth phenotypes of N. crassa and S. cerevisiae mutant strains and the loss of mitochondrial respiratory system components like cytochromes proved to be caused by loss or rearrangement of mitochondrial DNA or defective gene expression and protein synthesis in mitochondria. In N. crassa, for example, R. Brambl found that several mitochondrial tRNAs were not synthesized in mi-1, apparently a consequence of a mutant transcriptional promoter for a region of the mitochondrial genome that also encoded the small rRNA, as shown by Alan Lambowitz, explaining also the low concentration of mitochondrial ribosomes in this mutant strain.
Alexander Tzagoloff, L. A. Grivell, Nancy Martin and G. Bernardi, and many others, studied the organization and the expression of mitochondrial genes in S. cerevisiae and the processing and splicing of transcripts (Tzagoloff & Myers, 1986; Grivell, 1995). A. Lambowitz, E. Agsteribbe and U. L. RajBhandary performed early studies of the N. crassa mitochondrial genome, and C. Scazzocchio, G. Turner and H. Küntzel studied that of Aspergillus nidulans.
Several laboratories in the 1970s working with S. cerevisiae, such as those of Schatz and Tzagoloff, or with N. crassa, such as those of Walter Neupert, Sigurd Werner and Walter Sebald, showed that the proteins encoded by mitochondrial genes provided one to several subunit peptides to each of the respiratory membrane complexes and to the ATP synthase, whereas the remainder of the subunit peptides were encoded by nuclear genes, synthesized on cytosolic ribosomes and imported into mitochondria for assembly with their mitochondrially synthesized counterparts. In the next two decades the Schatz and Neupert laboratories in particular were preoccupied with the process of protein import into the organelles and with determining the structure and function of the surprisingly complicated translocase complexes of the outer and inner mitochondrial membranes (Hoppins et al., 2004; Neupert, 1997). These comprehensive studies with fungal systems provided the foundation for studies of mammalian mitochondria and of the role of mitochondria in human disease.
An important level of regulation of gene expression in mitochondria appears to be post-transcriptional, a gene expression entirely dependent upon nuclear-encoded enzymes and regulatory factors (Brambl, 2004). Thomas Fox and colleagues have found that in S. cerevisiae, translation of at least five of the eight major mitochondrial mRNAs for subunits of cytochrome b and cytochrome c oxidase requires the action of one or more nuclear-encoded, gene-specific translational activator proteins (Costanzo & Fox, 1990). The proteins seem to function by interacting with the mitochondrial ribosome, with specific regions of the 5'-leaders, and with the inner membrane. The Brambl laboratory found evidence of translational control of mitochondrial gene expression in N. crassa. The three genes for the mitochondrial cytochrome c oxidase subunits are transcribed at sharply divergent times during conidial germination in N. crassa. Nevertheless, all three subunit peptides are first synthesized at the same point late in germination, at the time when processed transcripts for the three genes are simultaneously recruited into the mitochondrial polyribosomes (Bittner-Eddy et al., 1994). The 5'-leaders of these mitochondrial mRNAs have conserved stem–loop structures that bind proteins that may regulate their recruitment into the translational apparatus (Kleidon et al., 2003). The nuclear gene cya-5 of N. crassa encodes such a candidate mRNA-binding protein that is required for the mitochondrial translation of stable, mature transcripts for subunit 1 of cytochrome c oxidase, a finding that is parallel to the finding in yeast.
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Mating systems |
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mating-type genes from inactive, storage loci to an active locus, MAT (Rine & Herskowitz, 1987). The lack of expression in these storage loci is caused by trans-acting regulators encoded by SIR genes. The gene products that are encoded by both MATa and MAT are proteins that regulate the switch to the alternate mating-type. The replacement of genetic material being expressed at the MAT locus by sequences in a distant storage locus represents one of the first molecular explanations of gene conversion, a process first described in N. crassa in 1955 by H. Mitchell.
The A and a mating-type loci in N. crassa also regulate heterokaryon formation and heterogenic incompatibility, a process that causes death of the A and a hyphae at the point of fusion. The A and a mating-type genes of N. crassa were the first to be isolated from a filamentous ascomycete in the late 1980s, by several cooperating groups, by S. J. Volmer, Chuck Staben and C. Yanofsky and by Louise Glass and Robert Metzenberg. These A and a mating-type genes consist of dissimilar DNA sequences at the same chromosomal locus, and haploid strains contain a single copy of either A or a sequence, unlike S. cerevisiae. These genes encode transcriptional activators that regulate genes in a cell-specific signalling pathway and that regulate expression of pheromones and pheromone receptors (Glass & Nelson, 1994).
Several groups in the 1980s and 1990s, including Robert Ullrich and Charles Novotny (using Schizophyllum commune), Lorna Casselton (Coprinus cinereus), and Regine Kahmann and Flora Banuett (Ustilago maydis), studied the complicated, multiallelic mating systems of the basidiomycetes (Casselton & Kües, 1994; Kämper et al., 1994). They found that mating interaction in this group of fungi requires coordinated activities of two gene complexes, one of which encodes homeodomain transcription factors that heterodimerize upon mating to generate an active transcriptional regulator that drives other mating responses, and the other of which encodes peptide pheromones and pheromone receptors. The homeodomain proteins 1 and 2 of the basidiomycete fungi resemble the a1 and 2 homeodomain mating-type proteins of S. cerevisiae, reflecting a conservation of the mechanisms that regulate mating and sexual development in basidiomycetes and unicellular ascomycete yeasts. The ascomycete proteins act as transcriptional repressors, but they also require additional proteins to complete this function; it is not known how the counterparts in the basidiomycetes regulate transcription in the mated cells.
Studies of mating-type switching in S. cerevisiae and of mycelial heterothallic mating-type loci of the ascomycetes N. crassa and P. anserina, along with those of the basidiomycetes, gave rise to an amazement that complex morphology and complex behaviour could be the result of such simple, single genetic specifications. The mating processes of these fungi have been especially useful for understanding cell signalling pathways in higher organisms, including mammals, and for understanding the roles of protein–protein interactions in regulation of gene expression.
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Chitin synthesis: the chitosome |
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). They are the major reservoir of zymogenic chitin synthetase in fungi, and they have a unique size, a low buoyant density and a shell that is shed upon chitosome activation by substrate. The initial discovery that visible chitin microfibrils could be made from UDP-GlcNAC validated the then-uncertain sugar nucleotide pathway hypothesized for chitin synthesis, and it showed that the crystalline fibrous component of the fungal cell wall could be assembled in vitro by these microvesicles. The smallest component of the chitosome capable of synthesizing chitin in vitro is the reversibly dissociated 16S subunit (
500 kDa), but the catalytic subunit of chitin polymerization has not yet been identified. This discovery of exocytotic chitosomes and a particulate chitin synthetase activity contrasted with Enrico Cabib's alternative description of a plasma-membrane-bound chitin synthetase in yeast (Cabib et al., 1996). Eventually, it was shown that yeast cells do contain chitin synthetase in conventional chitosomes with a low buoyant density, and also in another population of large vesicles, derived from the plasma membrane. The rapid growth of the fungal hyphal tip is dependent upon a polarized secretory apparatus, with an accumulation of at least two populations of vesicles at the tip: the microvesicles containing chitin synthesis zymogen, and the macrovesicles containing enzymes and polymers that contribute the amorphous phase of the fungal cell wall. |
Spores and spore germination |
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). A seemingly inordinate amount of effort was devoted, especially by the physiologists and mycologists, to defining dormancy, dormancy subtypes and ontogenetic diversity of spores, and even to descriptions of spore germination itself and its ultrastructural correlates (Turian & Hohl, 1981). Since the early discovery of the requirement for heat activation of ascospores of N. crassa and Ascobolus by B. O. Dodge, several workers, such as Sussman and David Cotter, have attempted to understand the physical basis of this activation, a process that remains unexplained.
In the 1960s several groups began investigating the process of fungal spore germination, in the context of developmental biology. These fungal workers, like those using echinoderm and amphibian eggs, plant seeds or bacterial spores, searched for controls of the resumption of transcriptional and translational activities characteristic of the activation of dormant cells. These studies asked if the regulation of dormancy and activation was controlled at the level of transcription or translation. J. Van Etten and his colleagues – and others, such as Richard Staples and T. Yanagita – compared components of protein synthesis systems extracted from dormant and germinated spores, since it was known that protein synthesis is essential for spore germination, finding that all components were present in the dormant spores, but with generally lower levels of enzyme activities. One of the more attractive questions remained, however: do the dormant spores contain mRNA? Is new transcription required for germination, or if present, does the dormant cell's mRNA encode sufficient information for germination? James Lovett, P. Allen, Larry Dunkle and Richard Staples, along with Van Etten and Brambl, used diverse approaches to answer this question (Brambl et al., 1978). (The highly influential generalization in developmental biology, that cell differentiation is associated with differential gene expression, was expected to apply to spore germination, but it was not clear where this gene expression occurred, during either spore formation or spore germination.) The question seemed to be clearly answered by these workers when mRNA was detected in polyribosomes in dormant spores. The Brambl and Van Etten laboratories separately showed that this mRNA encoded proteins qualitatively different from the same mRNA fraction of germinated spores. However, it was only after this finding that the Brambl group along with Peter Russell showed that an additional RNA was synthesized early in N. crassa conidial germination that was essential for germination (Brambl et al., 1987).
Gottlieb, Sussman and their colleagues measured respiration during germination, particularly in terms of carbohydrates used as substrates for germination, self-inhibitors of germination, and enzymic components of pathways such as glycolysis and the TCA cycle. Using well-characterized inhibitors and techniques to measure oxygen consumption and respiratory enzyme activity, it was easy to establish that aerobic respiration was essential to spore germination. Many years later, Brambl examined the mitochondrial cytochrome electron-transport chain in the dormant pycnidiospores of Botryodiplidia theobromae and found that, based upon enzyme activity and upon low-temperature spectroscopy, cytochromes bc1 and a were absent but were assembled upon germination, concomitantly with the development of aerobic respiration. Subsequently, it was established that the cytochrome c oxidase was assembled early in germination from several enzyme subunit precursors synthesized and stored in the cytoplasm during dormancy and imported into mitochondria upon initiation of germination for assembly with mitochondrially synthesized subunits (Brambl, 1981, 1985). In the dormant ascospores of N. crassa, evidence exists for a similar pattern of assembly of the ATP synthase from precursors of subunits accumulated in cytoplasm. The mitochondrial ATP synthase was also absent in the dormant spores of B. theobromae, but its assembly appears to depend upon de novo translation of mRNA for the nucleus-encoded subunits of this enzyme. The dormant, nonhydrated conidia of N. crassa contain a complete mitochondrial respiratory system and electron-transport system that is active immediately upon spore activation, illustrating the diversity of mechanisms among spore types.
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Respiration |
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Transport systems |
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). Thus, the fungal plasma membrane potential is maintained by a proton flux, generated by the plasma-membrane ATPase, rather than sodium and potassium fluxes as in animal cells. Barry and Emma Jean Bowman, initially working with Carolyn Slayman, began a long-term study of the vacuolar ATPase of N. crassa. The Slayman laboratories, with A. Lambowitz, also characterized the differences in concentrations of respiratory cytochrome components of wild-type and poky strains of N. crassa, and described the cyanide-resistant respiration of this organism, a study that led eventually to the identification of the SHAM-sensitive, CN-insensitive alternate oxidase. Four amino acid transport systems were identified (by Gabriel Lester, Martin Pall, Gib DeBusk and others) and shown to have broad substrate specificities, like those of animal systems, rather than the highly specific permeases of bacteria. Amino acid biosynthesis is controlled by feedback inhibition of biosynthetic enzymes and by compartmentalization in organelles. Matthew Sachs and others have explored cross-pathway controls of amino acid biosynthesis, in which starvation for a single amino acid causes expression of genes for biosynthetic enzymes of other amino acids in addition to those of the missing amino acid. Absolute levels of free amino acids do not cause this control, which instead is the result of lowering the ratio of charged tRNA to uncharged tRNA, the latter of which limits polypeptide synthesis.
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Stress responses and heat-shock proteins |
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). The synthesis of these proteins is not confined to the stress response, since they or relatives are essential proteins that are required for metabolism during normal cell growth and development. They have come to be seen as molecular chaperones, required for folding and assembly of proteins and protein complexes, with certain chaperones, such as Hsp70 and Hsp60, having specific functions that depend upon cellular localization. Distinct forms of Hsp70 are present in the cytoplasm, the endoplasmic reticulum and mitochondria, where they participate in synthesis and trans-membrane movement and folding of substrate proteins, whereas the Hsp60 is confined to the mitochondrion. Nora Plesofsky and her colleagues have studied the expression of the N. crassa gene for Hsp30, a protein related to the vertebrate lens -crystallin, and its function in apparently stabilizing activities of hexokinase and of protein complexes of the outer mitochondrial membrane, such as the MOM complex for import of proteins into mitochondria. |
Lignin degradation |
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). The white- rot basidiomycetes, such as Phanerochaete chrysosporium, are unique in their ability to efficiently mineralize lignin, which itself is unsuitable as a sole carbon source, while simultaneously degrading cellulose and hemicellulose. The enzymes that accomplish lignin degradation have an extremely high oxidation potential, and they are extracellular and non-specific. The lignin peroxidase, manganese peroxidase and hydrogen peroxide (a product of glyoxal oxidase) generate highly reactive free radicals that cause a series of spontaneous cleavage reactions. (There is no role for laccases in lignin degradation in P. chrysosporium, but they evidently function in related organisms.) In P. chrysosporium, previously not known to be accessible experimentally, the recent development of transformation systems, site-specific mutagenesis and heterologous expression systems have led to enormous progress in understanding of lignin degradation. The multiple gene sequences encoding these individual enzymes have been analysed with respect to their structure, evolution and regulation of expression. |
Polarized growth and the role of microtubules |
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). They found that the cytoskeletal systems that orient and move nuclei in germinating conidia are highly conserved from fungi to animal cells. In early experiments they found that nuclear migration in these conidia was blocked by the microtubule inhibitor benomyl. That the inhibition was due to its effect on microtubules was shown with mutants having genetic lesions in β-tubulin which produce either resistance to benomyl or high sensitivity. Subsequently, dynactin, dynein (a minus-end microtubule-stimulated ATPase-motor), and other components on the plus ends of the astral microtubules interact with the cell cortex to generate forces that act on the minus ends of microtubules embedded in a 
-tubulin (discovered in A. nidulans)-containing structure in the nucleus, the nuclear microtubule organizing centre or the spindle pole body (Oakley, 1985).
Hyphal morphogenesis and the mechanism of polarized growth of fungi at the hyphal tip has deeply interested mycologists and cytologists for decades, and until recently the components of this system were unknown. In the 1960s Tatum and his colleagues generated and characterized a number of morphological mutants which, decades later, with techniques of molecular genetics, were shown to be mutant in signalling pathways, such as protein kinases, or components of the cytoskeleton or dynein motor complexes. With abundant vesicles contributing to a highly structured tip growth apparatus, the Spitzenkörper, it has been assumed that this apparatus functioned to incorporate cell wall components into vesicles and to deliver these vesicles to the plasma membrane at the hyphal tip for exocytosis and assembly of the wall components. Salomón Bartnicki-García, Berl Oakley, Steven Harris, Michelle Momany and others have contributed both mathematical models and experimental evidence showing that microtubules and actin microfilaments have important roles in this vesicle traffic (Harris et al., 1999). Recently, generation of fluorescent fusions of proteins involved in tip growth has made it possible to study their distribution by time-lapse microscopy. The components of a stable, highly structured but dynamic tip growth apparatus are held together by actin cables. The microtubules are important for long-distance delivery of vesicles to the Spitzenkörper; actin microfilaments organize vesicles within the Spitzenkörper (which may be a vesicle supply centre), control their delivery to the apical plasma membrane of the hypha, and position the tip growth apparatus. Endocytosis of secretory components occurs at a ring of the plasma membrane just below the apex.
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Dimorphism: yeast versus hyphal growth |
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). Originally, dimorphism seemed to be a useful model for morphogenesis and regulation of apical growth, but more recently it has also become important in terms of growth of clinically and agriculturally important fungi. The conversion of cells from a yeast-like to a mycelial habit of growth was studied initially in terms of the inducing cultural or environmental conditions. The yeast form of M. rouxii results from growth under anaerobiosis in an atmosphere containing at least 30 % CO2; in the absence of CO2 and regardless of O2, growth is mycelial. A causal relationship seemed to exist between respiratory or oxidative metabolism and mycelial growth, but only later did it become evident that the concentration and nature of the carbon source is the primary determinant of yeast-like growth in M. rouxii. Disruption of mitochondrial functions also stimulates yeast-like growth. In M. rouxii, cAMP levels decrease during the yeast-to-hyphal transition induced by exposure to O2 and this transition was blocked by addition of dibutyryl-cAMP. Certain amino acids, such as cysteine or proline, induce yeast-like growth in fungi such as Candida albicans and Histoplasma capsulatum, fungal pathogens in which dimorphism is important as a clinical phenomenon.
In 1992 G. R. Fink's group reported that in an atypical, non-laboratory strain of S. cerevisiae, nitrogen starvation (or poor carbon sources and some physical stresses) caused formation of filamentous chains of cells called pseudohyphae, with a resulting filamentous growth that differs morphologically and behaviourally according to diploidy or haploidy (Roberts & Fink, 1994). This pseudohyphal or invasive growth requires two signalling pathways, a MAP kinase cascade and a cAMP-dependent pathway, involving as a central element the GTP-binding protein Ras2. Pathogenic C. albicans may grow reversibly as a budding yeast or as pseudohyphae, and it also develops true hyphae (without constrictions that mark septa) and germ tubes, in which growth depends upon apical extension. In C. albicans, hyphal development is induced by stresses such as nitrogen starvation, high temperature and high pH.
In the basidiomyete Ustilago maydis, the cause of maize smut, the haploid, yeast-like cells undergo mating and develop a dikaryotic filamentous cell; a haploid yeast phase can undergo direct conversion to a haploid hyphal phase. Flora Banuett and her colleagues have studied the U. maydis yeast-like non-pathogenic form and filamentous pathogenic form, finding that the fuz1 gene encodes a Zn-finger domain protein that through interactions with other proteins regulates specific transcriptional activity and morphogenesis (Banuett & Herskowitz, 1994). Disruption of genes encoding adenylate cyclase or the catalytic subunit of cAMP-dependent protein kinase led to constitutive filamentous growth, which could be suppressed by the lack of MAPKK (Fuz7), leading to the suggestion that morphogenesis in this organism is regulated by both cAMP and MAP kinase signal transduction pathways, reminiscent of the much earlier observations with the phycomycete M. rouxii.
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Signalling |
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dimer functions in many roles in the signalling process, including membrane localization of the G protein subunit. The Gβ subunit in yeast functions as a regulator of the pheromone response in haploid cells by activation of the MAP kinase cascade. Deletion of this subunit gene in Cryptococcus neoformans causes sterility, and deletion in other species leads to disruption of conidiation, hyphal growth, pigmentation, defects in the cAMP glucose-sensing pathway and aberrant reproductive structures. Katherine Borkovich and colleagues have shown that the G protein β dimer is essential for normal asexual sporulation and female fertility in N. crassa (Kays & Borkovich, 2004). An understanding of regulation of non-senescent, necrotic and apoptotic death in mycelial fungi has been slow to develop, although recently, Nora Plesofsky and her colleagues have shown that regulated cell death resulting from physical and nutritional stress in N. crassa may be signalled by stress-specific sphingolipids through the OS-2 MAP kinase pathway (Plesofsky et al., 2008). |
Circadian rhythms |
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Chromosomes and telomeres |
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). Twenty years later, analysis (by J. A. Huberman and colleagues) of replicating chromosomal DNA of S. cerevisiae by fibre autoradiography showed that replication initiates at multiple initiation sites, giving rise to replication forks, along each DNA molecule, and these replication forks move bidirectionally away from the initiation sites. Subsequent studies of yeast helped define the essential attributes of the eukaryotic chromosome, including the autonomously replicating sequences, centromeres and telomeres; the identification of proteins needed for chromosomal replication; the structure–functional analysis of centromeric sequences; and telomere dynamics and replication. Carol Newlon and her colleagues have contributed much to our knowledge of yeast chromosome replication, with the development of tools to study these processes and identification of proteins that function at the replication fork. McClintock anticipated the need for specialized structures at the ends of chromosomes to maintain their stable transmission and to prevent end-to-end fusions. Many decades later, telomeres were identified as the structures at the ends of chromosomes whose function is to stabilize these chromosomes and prevent stepwise loss of genetic material as a result of the incomplete replication of DNA strands with each round of replication. The discovery of the ribonucleoprotein telomerase, with its enzyme-associated RNA template, which recognizes and extends the G-rich strand of the terminal repeat by adding T-G polydeoxyribonucleotide repeats, came from studies of the ciliate Tetrahymena, but this insight was soon expanded by studies of telomeres and the proteins involved in telomere function in S. cerevisiae by laboratories of V. A. Zakian, B. Tye, Judith Berman, J. E. Haber, E. H. Blackburn, D. Shore, and many others (Enomoto et al., 2002). |
Gene silencing |
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AT transitions (Galagan & Selker, 2004). This mutagenic process correlated positively with closeness of linkage of the duplications and is more severe and more likely with duplications of 1 kbp or more. This process is thought to be an inactivating defence against transposable elements and a mechanism to force long homologous sequences to diverge increasingly with repeated rounds of sexual reproduction until they are no longer sufficiently homologous to attract this mutagenesis.
Quelling is a process discovered by Giuseppe Macino and colleagues (Catalanotto et al., 2004), in which the expression of certain genes is reduced when multiple, complete or incomplete copies of these genes are introduced into the genome of N. crassa, much like transgene silencing in green plants. This reversible gene-silencing results from the RNA interference pathway that causes both transgene-induced as well as double-stranded RNA-induced gene silencing. A transgene-specific transcript is converted to double-stranded RNA, which is cleaved to 25-nucleotide inhibitory RNAs that block gene expression through an RNA-induced silencing complex. Slightly shorter inhibitory RNAs, originating from the rRNA locus, tRNA genes, intergenic regions of the genome or open reading frames, have been shown by Yi Liu and colleagues to be induced by the DNA-damage response. These inhibitory RNAs could help regulate DNA damage checkpoints by inhibition of protein synthesis until DNA repair.
In N. crassa, repeat induced point mutation and quelling destroy or silence non-single-copy DNA sequences in haploid mycelial cells. R. Metzenberg and colleagues identified a novel phenomenon, meiotic silencing, as a third mechanism for silencing expression of unpaired sequences, occurring during mating in the transient diploid zygote cell, following karyogamy (Shiu et al., 2001). DNA that is unpaired early in meiosis, in prophase I, induces silencing of all other DNA that is homologous to it, paired or unpaired.
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Concluding perspective: the emergence of fungal molecular biology |
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Another author invited to prepare this review certainly would have included topics and names of individuals that are not mentioned here or given sufficient attention. I have discussed topics that others might have minimized and I have omitted topics that others may consider to be far more important, this being a disappointment or a relief, depending upon one's point of view. The individuals cited here, for the most part, are those who have had some direct or indirect influence on my own experiences and career, many of whom I began to meet as a student. This account is by no means a history of this era; but a history begins as stories inside the personal narratives of those who tell it. This is a recollection of topics I've enjoyed learning about and teaching and in some cases have had the remarkable good fortune to investigate with my own friends and laboratory colleagues.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Beadle, G. W. & Tatum, E. L. (1941). Genetic control of biochemical reactions in Neurospora. Proc Natl Acad Sci U S A 27, 499–506.
Bittner-Eddy, P., Monroy, A. & Brambl, R. (1994). Expression of mitochondrial genes in the germinating conidia of Neurospora crassa. J Mol Biol 235, 881–897.[CrossRef][Medline]
Bracker, C. E., Ruiz-Herrera, J. & Bartnicki-Garcia, S. (1976). Structure and transformation of chitin synthetase particles (chitosomes) during microfibril synthesis in vitro. Proc Natl Acad Sci U S A 73, 4570–4574.
Brambl, R. (1981). Respiration and mitochondrial biogenesis during fungal spore germination. In The Fungal Spore: Morphogenetic Controls, pp. 585–604. Edited by G. Turian & H. R. Hohl. London: Academic Press.
Brambl, R. (1985). Fungal spore germination and mitochondrial biogenesis. In The Molecular Genetics of Filamentous Fungi, UCLA Symposia on Molecular and Cellular Biology, vol. 34 (New Series), pp. 207–223. Edited by W. Timberlake. New York: Alan R. Liss.
Brambl, R. (2004). Regulation of expression of mitochondrial genes. In The Mycota III: Biochemistry and Molecular Biology, 2nd edn, pp. 129–140. Edited by R. Brambl & G. A. Marzluf. Berlin: Springer.
Brambl, R., Dunkle, L. D. & Van Etten, J. L. (1978). Nucleic acid and protein synthesis during fungal spore germination. In The Filamentous Fungi, vol. III, Genetics, Physiology, and Biochemistry of Development, pp. 94–118. Edited by J. E. Smith & D. R. Berry. New York: Wiley.
Brambl, R., Plesofsky-Vig, N., Hammet, J. R. & Russell, P. J. (1987). Preserved polyadenylated ribonucleic acid in dormant conidia of Neurospora crassa and new RNA synthesis during spore germination. Exp Mycol 11, 317–330.[CrossRef]
Brown, M. P., Brown-Jenco, C. S. & Payne, G. A. (1999). Genetic and molecular analysis of aflatoxin biosynthesis. Fungal Genet Biol 26, 81–98.[CrossRef][Medline]
Cabib, E., Shaw, J. A., Mol, P. C., Bowers, B. & Choi, W.-J. (1996). Chitin biosynthesis and morphogenic processes. In The Mycota III: Biochemistry and Molecular Biology, pp. 243–267. Edited by R. Brambl & G. A. Marzluf. Berlin: Springer.
Casselton, L. A. & Kües, U. (1994). Mating-type genes in homobasidiomycetes. In The Mycota I: Growth, Differentiation, and Sexuality, pp. 307–321. Edited by J. G. H. Wessels & F. Meinhardt. Berlin: Springer.
Catalanotto, C., Pallota, M., ReFalo, P., Sachs, M., Vayssie, L., Macino, G. & Cogoni, C. (2004). Redundancy of the two dicer genes in transgene-induced posttranscriptional gene silencing in Neurospora crassa. Mol Cell Biol 24, 2536–2545.
Costanzo, M. C. & Fox, T. D. (1990). Control of mitochondrial gene expression in Saccharomyces cerevisiae. Annu Rev Genet 24, 91–108.[CrossRef][Medline]
Cullen, D. & Kersten, P. J. (2004). Enzymology and molecular biology of lignin degradation. In The Mycota III: Biochemistry and Molecular Biology, 2nd edn, pp. 249–273. Edited by R. Brambl & G. A. Marzluf. Berlin: Springer.
Cummings, D. J., Belcour, L. & Grandchamp, C. (1979). Mitochondrial DNA from Podospora anserina. Mol Gen Genet 171, 239–250.[CrossRef][Medline]
Davis, R. H. (2000). Neurospora. Contributions of a Model Organism. New York: Oxford University Press.
Dunlap, J. C., Loros, J. J., Denault, D., Lee, K., Froehlich, A., Colot, H., Shi, M. & Pregueiro, A. (2004). Genetics and molecular biology of circadian rhythms. In The Mycota III: Biochemistry and Molecular Biology, 2nd edn, pp. 209–229. Edited by R. Brambl & G. A. Marzluf. Berlin: Springer.
Enomoto, S., Glowczewski, L. & Berman, J. (2002). MEC3, MEC1, and DDC2 are essential components of a telomere checkpoint pathway required for cell cycle arrest during senescence in Saccharomyces cerevisiae. Mol Biol Cell 13, 2626–2638.
Foster, J. W. (1949). Chemical Activities of Fungi. New York: Academic Press.
Galagan, J. E. & Selker, E. U. (2004). RIP: the evolutionary cost of genome defense. Trends Genet 20, 417–423.[CrossRef][Medline]
Giles, N. H., Geever, R. F., Asch, D. K., Avalos, J. & Case, M. E. (1991). Organization and regulation of the Qa (quinic acid) genes in Neurospora crassa and other fungi. J Hered 82, l-7
Glass, N. L. & Nelson, M. A. (1994). Mating-type genes in mycelial ascomycetes. In The Mycota I: Growth, Differentiation, and Sexuality, pp. 295–306. Edited by J. G. H. Wessels & F. Meinhardt. Berlin: Springer.
Grivell, L. A. (1995). Nucleo-mitochondrial interactions in mitochondrial gene expression. Crit Rev Biochem Mol Biol 30, 121–164.[Medline]
Harris, S. D., Hofmann, A. F., Tedford, H. W. & Lee, M. P. (1999). Identification and characterization of genes required for hyphal morphogenesis in the filamentous fungus Aspergillus nidulans. Genetics 151, 1015–1025.[Medline]
Hoppins, S. C., Taylor, R. D. & Nargang, F. E. (2004). Import of proteins into mitochondria. In The Mycota III: Biochemistry and Molecular Biology, 2nd edn, pp. 33–51. Edited by R. Brambl & G. A. Marzluf. Berlin: Springer.
Horowitz, N. H. (1991). Fifty years ago: the Neurospora revolution. Genetics 127, 631–635.[Medline]
Kämper, H., Bölker, M. & Kahmann, R. (1994). Mating-type genes in heterobasidiomyces. In The Mycota I: Growth, Differentiation, and Sexuality, pp. 323–332. Edited by J. G. H. Wessels & F. Meinhardt. Berlin: Springer.
Kays, A. M. & Borkovich, K. A. (2004). Signal transduction pathways mediated by heterotrimeric G proteins. In The Mycota III: Biochemistry and Molecular Biology, 2nd edn, pp. 175–207. Edited by R. Brambl & G. A. Marzluf. Berlin: Springer.
Kleidon, J., Plesofsky, N. & Brambl, R. (2003). Transcripts and transcript-binding proteins in mitochondria of Neurospora crassa. Mitochondrion 2, 345–360.[CrossRef][Medline]
Kück, U., Stahl, U. & Esser, K. (1981). Plasmid-like DNA is part of mitochondrial DNA in Podospora anserina. Curr Genet 3, 151–156.[CrossRef]
Lambowitz, A. M., Slayman, C. W., Slayman, C. L. & Bonner, W. D., Jr (1972). The electron transport components of wild type and poky strains of Neurospora crassa. J Biol Chem 247, 1536–1545.
Marzluf, G. A. (2004). Regulation of nitrogen metabolism in mycelial fungi. In The Mycota III: Biochemistry and Molecular Biology, 2nd edn, pp. 357–368. Edited by R. Brambl & G. A. Marzluf. Berlin: Springer.
May, G. S., Weatherbee, J. A., Gambino, J., Tsang, M. L.-S. & Morris, N. R. (1985). Identification and function of beta tubulin in Aspergillus nidulans. In The Molecular Genetics of Filamentous Fungi, UCLA Symposia on Molecular and Cellular Biology, vol. 34 (New Series), pp. 239–251. Edited by W. Timberlake. New York: Alan R. Liss.
Neupert, W. (1997). Protein import into mitochondria. Annu Rev Biochem 66, 863–917.[CrossRef][Medline]
Oakley, B. R. (1985). The molecular biology of microtubules in Aspergillus. In The Molecular Genetics of Filamentous Fungi, UCLA Symposia on Molecular and Cellular Biology, vol. 34 (New Series), pp. 225–238. Edited by W. Timberlake. New York: Alan R. Liss.
Perkins, D. D. (1992). Neurospora: the organism behind the molecular revolution. Genetics 130, 687–701.[Medline]
Plesofsky, N. (2004). Heat shock proteins and the stress response. In The Mycota III: Biochemistry and Molecular Biology, 2nd edn, pp. 143–174. Edited by R. Brambl & G. A. Marzluf. Berlin: Springer.
Plesofsky, N. S., Levery, S. B., Castle, S. A. & Brambl, R. (2008). Stress-induced cell death is mediated by ceramide synthesis in Neurospora crassa. Eukaryot Cell 7, 2147–2159.
Rine, J. & Herskowitz, I. (1987). Four genes responsible for a position effect on expression from HML and HMR in Saccharomyces cerevisiae. Genetics 116, 9–22.[Medline]
Roberts, R. L. & Fink, G. R. (1994). Elements of a single MAP kinase cascade in Saccharomyces cerevisiae mediate two developmental programs in the same cell type: mating and invasive growth. Genes Dev 8, 2974–2985.
Shiu, P. K. T., Raju, N. B., Zickler, D. & Metzenberg, R. L. (2001). Meiotic silencing by unpaired DNA. Cell 107, 905–916.[CrossRef][Medline]
Slayman, C. L. & Slayman, C. W. (1962). Measurement of membrane potentials in Neurospora. Science 136, 876–877.
Stewart, P. R. & Rogers, P. J. (1978). Fungal dimorphism: a particular expression of cell wall morphogenesis. In The Filamentous Fungi, vol. III, Genetics, Physiology, and Biochemistry of Development, pp. 164–196. Edited by J. E. Smith & D. R. Berry. New York: Wiley.
Turian, G. & Hohl, H. R. (1981). The Fungal Spore: Morphogenetic Controls. London: Academic Press.
Tzagoloff, A. & Myers, A. M. (1986). Genetics of mitochondrial biogenesis. Annu Rev Biochem 55, 249–285.[CrossRef][Medline]
Weber, D. J. & Hess, W. M. (1976). The Fungal Spore: Form & Function. New York: Wiley.
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The early years of...