48. Links between cell biology and fungal development.
Reinhard Fischer, Philipps- Universitat Marburg and Max-Planck- Institut fur terrestrische Mikrobiologie, Karl-von-Frisch-Str., D-35043 Marburg, Germany.
Fungi have been used as model organisms to study cell biology and differentiation of
eukaryotic cells for many years. E.g. molecular anlyses of mitosis, meiosis or organelle movement
revealed generally valuable insights into these evolutionarily conserved processes. Fungal
development has also been studied in several fungi on a molecular level. Besides "true"
developmental genes which are specifically involved in the regulation of morphogenesis, the
number of characterized genes necessary for basic cellular processes and for fungal development
is increasing. The aim of the talk will be to emphasize recent advances in understanding some
links between cell biology and differentiation processes in fungi.
49. Characterization of the car1 gene of Neurospora crassa; A putative peroxisome assembly factor gene.
Kelly A. Howe and Mary Anne Nelson, University of New Mexico.
Peroxisomes are the least characterized organelles of the cell; they received little attention until the discovery of their role in a specific group of human genetic disorders. The most severe of these disorders Zellweger Syndrome which is lethal shortly after birth. The disease is characterized by a lack of peroxisomes in cells and a subsequent loss of vital peroxisomal functions. It is now known that peroxisomes contain enzymes involved in many metabolic pathways of eukaryotic cells, including -oxidation of fatty acids. Peroxisome biogenesis and the role of the organelle in development are still poorly understood, but microorganisms have provided good model systems for the study of peroxisomes. We have cloned a peroxisome assembly factor gene, car1, from the filamentous fungus Neurospora crassa. The car1 gene is a putative peroxisome assembly factor with significant homology to a conserved family of proteins related to the human PAF1 (Peroxisome Assembly Factor 1) protein. The PAF1 protein is known to be involved in peroxisome biogenesis and the PAF1 gene is one of the genes known to be responsible for human Zellweger Syndrome.
The car1 gene has been RIP-disrupted and partially characterized in N. crassa. It's amino acid
sequence encodes two membrane spanning domains and a zinc finger region that are all conserved
among the PAF1-related proteins. A car1 mutant strain has been isolated exhibiting a mild form
of RIP-disruption of the car1 gene and potential partial peroxisome function based on selection
procedures. Here we report the sequence analysis and partial characterization of car1 function in
N. crassa.
50. Involvement of the Aspergillus nidulans anaphase promoting complex/cyclosome in a surveillance pathway blocking entry into mitosis in the absence of NIMA function.
C. Mark Lies, J. Cheng, S. Venkatram, and P. M. Mirabito, School of Biological Sciences, University of Kentucky, Lexington, KY.
In Aspergillus nidulans, initiation of mitosis normally requires the function of the cell cycle regulated NIMA kinase. The requirement for NIMA function can be relieved by mutational inactivation of the bimA or bimE genes, suggesting that BIMA and BIME are components of a G2 checkpoint that prevents entry into mitosis in the absence of NIMA function (the NIMA checkpoint). Genetic and biochemical data indicate that BIMA and BIME are components of the Anaphase Promoting Complex/Cyclosome (APC/C), leading us to propose that the APC/C itself regulates the NIMA checkpoint. If so, then all loss of function APC/C mutants should be defective in this checkpoint. We have tested this hypothesis by using reverse genetics. We have cloned an Aspergillus gene corresponding to the human APC/C gene, HCDC16, which we propose to call bimH. Strains containing an alcA::bimH fusion as their only intact bimH gene die on glucose medium with a complex mitotic phenotype, consistent with BIMH being an essential component of the APC/C. We find that nimA5, bimH- double mutants arrest in mitosis. These results are consistent with a model in which the APC/C is part of a surveillance system that prevents entry into mitosis until NIMA function is complete.
51. Swollen cell (swo) mutants of Aspergillus nidulans.
Michelle Momany, Gretel Abramowsky, Jered Brown, and Patrick Westfall, University of Georgia, Athens, GA.
Fifty-two mutants showing limited growth and a swollen cell phenotype at restrictive
temperature (swo mutants) have been identified from a collection of temperature-sensitive A.
nidulans mutants based on stereoscope examination. Phenotypes of putative swo mutants at
restrictive temperature range from conidia which swell but do not send out germ tubes to hyphae
with irregular swellings. Such phenotypes may be associated with defects in cell wall synthesis.
Analys's of progeny from swo x wild-type crosses showed that 17 of the putative swo mutants
resulted from single gene mutations. The swo mutants are currently being analyzed for response
to osmoticum and known inhibitors of cell wall synthesis.
52. Dynamics of actin and chitin ring formation during cytokinesis in Aspergillus nidulans.
Michelle Momanyl and John E. Hamer2, 1University of Georgia, Athens, GA and 2 Purdue University, West Lafayette, IN
Previous work has shown that septation in the filamentous fungus Aspergillus nidulans
superficially resembles cytokinesis in animal cells in that both are dependent on mitosis and
require actin. We show that a ring of actin is the precursor to the chitin ring seen in mature septa
and that actin and chitin co-localize during an intermediate phase of septum formation. We
present evidence supporting a contractile actin ring in septation and suggest a structural role for
the chitin ring. By employing the microtubule depolymerizing drug benomyl we show that
microtubules are required for both actin ring formation and the progression from the early actin
ring to the chitin ring. In contrast to studies in yeast cells, our results suggest that an intact
mitotic spindle is required for actin ring formation in A. nidulans and that a mitotic checkpoint
may prevent further progression of cytokinesis in the absence of an intact mitotic spindle.
53. Overlapping binding sites for the CreA repressor and AmdX and AmdA activator proteins in the 5' region of the amds gene of Aspergillus nidulans.
Rachael L. Murphy, Alex Andrianopoulos, Janynke L. Brons, Meryl A. Davis and Michael J. Hynes, Department of Genetics, University of Melbourne, Parkville 3052, Victoria, AUSTRALIA.
The acetamidase gene (amdS) of Aspergillus nidulans is subject to complex transcriptional control. One important regulator is the creA gene product which mediates carbon catabolite repression of amdS. The amdA and amdX positively acting genes were identified as regulators of amdS following the isolation of gain-of-function alleles of these genes.
The CreA, AmdA and AmdX proteins each contain two C2H2 zinc finger DNA binding motifs. Alignment of these domains reveals extensive similarity particularly in residues known to be involved in contacting DNA. Consequently, the three proteins are predicted to bind to very similar target sites which are compatible with the published CreA binding site consensus sequence.
We present results showing that in vitro DNA binding of the CreA, AmdA and AmdX zinc
fingers to the amdS 5' region can be localised to two sites containing predicted consensus binding
sequences. The in vitro data is consistent with results of in vivo experiments suggesting that
CreA, AmdA and AmdX act through these sites. Competition for binding site occupancy by
multiple regulators may therefore play a role in the regulation of amdS and possibly other CreA-regulated genes.
54. Grisea, a nuclear gene of Podospora anserina involved in the control of differentiation and senescence, encodes a putative transcription activator.
Heinz D. Osiewacz, Corina Borghouts, Erik Kimpel, Johann Wolfgang Goethe-Universitat, Botanisches Institut, Marie-Curie-Str. 9, D-60439 Frankfurt, Germany
In Podospora anserina all wild-strains are characterized by a limited lifespan which is
controlled by both, mitochondrial genetic traits as well as by a number of nuclear genes. Recently,
we reported the cloning of the nuclear gene grisea. Mutation of this gene leads to a significant
increase in lifespan and affects morphogenesis (spore color, sexual reproduction). A molecular
characterization of grisea suggested that it codes for a copper-activated transcription factor. This
factor appears to be important for a tight regulation of the cellular copper level and is thus linked
to metabolic pathways leading to the generation of reactive oxygen species. Four point mutations
were identified in the sequence of the cloned grisea region of the mutant. Three of these changes
are located in the sequence upstream of the start codon and one mutation at the 5'-boundary
between the first exon and the single intron of this gene. The latter mutation was shown to result
in an RNA splicing defect. The characteristics of long-lived mutant grisea and those of the
putative GRISEA protein suggest a number of candidate target genes, the expression of which
may be controlled by GRISEA. Data of approaches to identify and to characterize individual
genes of this type and to unravel unknown parts of the molecular network involved in the genetic
control of differentiation and senescence in P. anserina will be presented.
55. Mutations in the Aspergillus nidulans bncA gene uncouple the nuclear division and cell division cycles.
Renata Castiglioni Pascon*, Aline A. Pizzirani-Kleiner*, Bruce L. Miller**. *Universidade de Sao Paulo, S.P., Brazil;
** University of Idaho, Moscow, ID.
The Aspergillus nidulans bncAl (binucleate conidia) mutant was first described as a single
mutation located on chromosome IV that caused the formation of approximately 25% binucleated
and 1% trinucleated conidia. Further analysis has shown that this mutation also affects
conidiophore morphology. Metulae and phialides are elongated and have incorrect number of
nuclei. Philalides also show internal septation. Diploid construction and cytological analysis of
the conidia indicate that bncAl is a recessive mutation. Heterokaryon analysis using a binucleated
strain and a uninucleated strain with contrasting auxotrophic markers produced prototrophic
conidia at a high frequency compared to control heterokaryons, suggesting that more than one
nucleus of each genotype migrated from the phialide to the conidium. The vegetative growth rate
of the bncAl mutant was the same the wild-type. DAPI/Calcofluor double staining showed the
nuclei of bncAl to be fairly normal in number, distribution and shape in the hyphal tip, but in the
older elements nuclei appear to undergo continued, asynchronous divisions or, are mostly
misshapen and fragmented. In general the mutant strain has a greater number of nuclei per hyphal
element. The cell division cycle was followed during conidial Germination. We observed a high
frequency of young trinucleated germlings and older germlings with odd numbers of nuclei.
Results suggest that unlike the wild type, early nuclear divisions are not synchronous in the same
germline. The information available suggests that the gene product of bncAl is required during
the early staizes of germination and throughout development, affecting both vegetative tissue and
the asexual reproductive apparatus. Moreover, bncAl may have a role in controlling nuclear
division and septation. These questions will be addressed.
56. The Inquiry Track approach to learning principles of genetics.
Patricia J. Pukkila and Marshall Hall Edgell, University of North Carolina at Chapel Hill.
Advanced genetics courses for both undergraduate and graduate students usually provide
active educational experiences that capture a sense of how professionals pursue the discipline.
The challenge is to bring these useful approaches into large "lecture" courses at the beginning
level. Our goals are for these students to feel entitled to seek information, to analyze the methods
used to derive the "facts", to assess the validity of conflicting viewpoints, and to communicate
their conclusions effectively. Accordingly, we have asked students to use reading guides to
synthesize information from the textbook prior to class, to compare their answers to these
assignments with those of neighboring students in the class, to participate in class discussions that
develop a consensus, to revise their assignments to abandon erroneous views and/or incorporate
new information, to explore cause and effect using computer simulations, to prepare written
analyses of the conceptual difficulty of past exam questions, to research and then discuss
conflicting points of view concerning issues in the field that are currently unsolved, and to take
the intellectual risks that are necessary to learn to think effectively in this discipline. Our methods
have dramatically increased student-student and student-faculty interactions in classes of 150
students, and we conclude that the Inquiry Track approach could be effective in classes of any
size.
57. Genetics of two incompatibility genes at het-6 in Neurospora crassa.
Myron L. Smith, Nadereh Mir-Rashed, Cristina O. Micali, Sheryl P. Godkin, David J. Jacobson* and N. Louise Glass#. Carleton University, Ottawa, Canada. *Stanford University, Stanford, CA. #U. of British Columbia, Vancouver, Canada
The heterokaryon incompatibility system of Neurospora crassa provides an excellent model
for the molecular, genetic and biochemical study of non-self recognition. N. crassa has at least 11
heterokaryon incompatibility (het) loci (including mt). Cell fusion between two individuals that
differ at any one of these loci triggers an incompatibility response that results in little, or no
growth of heterokaryons. This response reduces cytoplasmic contact and may, therefore, prevent
the spread of disease elements among individuals. We have cloned and sequenced two genes,
het-6V and het-6J, at the het-6 locus on LGII that appear to be involved in heterokaryon
incompatibility function. A difference in alleles at het-6V results in no appreciable growth of
heterokaryons. Heterokaryons with different alleles at het-6J can grow for a few days before a
rapid and complete growth cessation occurs. From DNA sequence data and transcript analysis,
het-6V contains no introns and putatively encodes a 400 amino acid polypeptide. A 300+ open
reading frame has been identified at het-6J . Neither het-6V nor het-6J ORFs have striking
sequence identity to data-bank entries. Subclones that confer the het-6J incompatibility function
(based on transformation assays) also complement strains carrying the temperature sensitive
mutation, un-24. Sequence analysis of the mutant un-24 allele is nearly complete. Southern blot
analyses of N. crassa populations reveal multiple RFLPs in the regions of het-6V and het-6J. Both
genes have homologous counterparts in other Neurospora species. We will present a preliminary
analysis of the distribution of alleles at both genes in a world-wide population sample of N.
crassa.
58. Tyrl5 phosphorylation of p34cdc2 regulates septation and development in Aspergillus.
Xiang S. Ye, Sarah Lee McGuire1, Tom Wolkow2, John E. Hamer2 and Stephen A. Osmani, Geisinger Clinic. 1Millsaps College; 2 Purdue University.
Conidia of A. nidulans undergo polarized growth, producing multinucleate filamentous
hypha which are then compartmentalized by septation. Septation is normally dependent on
mitosis as germinating spores unable to complete mitosis fail to septate. We demonstrate that the
dependency of septation on mitosis is established by Tyrl5 phosphorylation of p34cdc2. p34cdc2 is
inhibited after Tyrl5 phosphorylation by ANKAwee1 and activated by Tyrl5 dephosphorylation by
NIMTcdc25. At 42 C nimT23 cells arrest at G2 with Tyrl5 phosphorylated p34cdc2 and no septation
occurs. At 37.5 C, nimT23 cells undergo mitosis and grow at near wt rate but are unable to form
septa. Addition of 6 mM HU, which increases Tyrl5 phosphorylated p34cdc2 levels but does not
arrest mitosis, also suppresses septation. Conversely, deletion of ANKAwee1 or a Tyrl5 mutant
p34cdc2 fully complements the nimT23 septation defect and, furthermore, cells unable to tyrosine
phosphorylate p34cdc2 undergo septation without mitosis. Such strains form multiple septa in the
conidiophore stalk and vesicle, which normally never septate. Tyrl5 mutant p34cdc2 strains also
form highly abnormal conidiophores and conidiate poorly. Thus, septation and cellular
differentiation are regulated through Tyr 1 5 phosphorylation of p34cdc2 in A. nidulans.
59. Analysis of loci on linkage group VIL of Neurospora crassa.
Thomas J. Schmidhauser and Dan Chen. The University of Southwestern Louisiana.
Genetic Mapping places the lys-5 and un-4 loci on LGVIL of Neurospora crassa with un-4 mapping 2 mu to the right of lys-5. We have cloned both genes and present cloning and RFLP analysis data. DNA sequence analysis of cDNA isolates is also presented.