109. The bad27 mutation in Coprinus cinereus is defective in both Methylation Induced Premeiotically (MIP) and meiotic chromosome synapsis.
Lesley S. Benyon and Patricia J. Pukkila, University of North Carolina at Chapel Hill, Chapel Hill.
In the basidiomycete Coprinus cinereus an epigenetic process known as methylation induced
premeiotically (MIP) results in the preferential methylation of cytosines in the CpG dinucleotides of
repetitive sequences. Here we document the first mutation in this epigenetic system. Strains
homozygous for bad27 (basidiospore development) develop normally but contain almost no de novo
methylation and their sexual spores arrest in pachytene of meiosis I with unsynapsed chromosomes.
To monitor methylation patterns we chose to use the isoschizomers HpaII and MspI. Copies of the
trp1 sequence were inserted by homologous recombination and the levels of MIP evaluated. In two
independent experiments, one involving a tandem triplication of the trp1 sequence and the other a
tandem duplication of trp1, highly significant differences in de novo methylation were detected
between wildtype strains and strains homozygous for the bad27-1 allele. We conclude that the
morphological phenotype of our mutant results from the failure of a homology-based search as does
the failure of the mutant to successfully carry out the de novo methylation process. Thus these two
homology searches have a common genetic basis that includes the gene disabled by our mutation.
110. Molecular analysis of conversion events at the am locus of Neurospora.
Frederick J Bowring and David EA Catcheside, School of Biological Sciences, Flinders University, Adelaide, Australia.
In cross B163, heteroallelic am1 am6 and heterozygous for both conventional genetic flanking markers and closer molecular markers (Bowring and Catcheside 1996), we found that although 24 % of the conversion events that generated prototrophic recombinants were associated with an exchange of flanking genetic markers, the molecular markers were recombined in only 7% of convertants. We concluded that either conversion and crossing over result from different recombination pathways or that resolution of a common intermediate is biased in favour of preservation of the local parental marker association. We report here that natural polymorphisms distinguishing the parents of cross B163 also include sequences within and closely flanking am, supplying markers for determining the structure of conversion tracts in the progeny of cross B163. Conversion of am6, the more distal allele, is more frequent than conversion of am1 and is associated with a peak of conversion frequency ~ 100bp 5' of am. 37 of 61 continuous tracts converting am1 extend less than the 741 bp maximum detectable with the available markers. Distal of am where additional markers are available, all nine crossovers that occurred in convertants were at least 115 kb away. We consider it implausible that exchanges at a distance two orders of magnitude further away than the modal length of conversion tracts are directly associated with the conversion event.
Bowring, FJ and DEA Catcheside. (1996) Gene conversion alone accounts for more than 90% of
recombination events at the am locus of Neurospora crassa. Genetics 142: 129-136
111. Molecular analysis of pan-2 prototrophs in Neurospora crassa.
Mary E. Case, University of Georgia, Athens.
Tetrad analyses of pantothenic acid-requiring mutants pan-2 B3 and pan-2 B5 indicated that 90 %
of aberrant events arose by gene conversions and 10% by reciprocal recombination (Case and Giles,
CSHSQB 23:119-135, 1958). A molecular examination of prototrophs from crosses between pan-2
mutants have confirmed these classical genetic studies. Since the original pan-2 mutants were
isolated in wild type 74 A, five new pan-2 mutants were isolated in the wild type Mauriceville strain.
These new pan-2 mutants were crossed to a marked strain ylo-1 pan-2 B3 trp-2. Prototrophs
obtained from crosses with these mutants were phenotypically categorized as parental, ylo+ trp+ or
ylo trp. or "recombinant" ylo trp+or ylo+ trp with respect to the flanking markers. DNA isolated from
prototrophs from each of the these classes was digested with HindIII. Southern blots were done and
the prototrophs were categorized as having either a 74A or Mauriceville restriction pattern. These
molecular analyses indicate that over 90% of the events giving rise to prototrophs between the pan-2
mutants arose by gene conversion not reciprocal recombination.
112. The use of the REMI technique to tag and mutagenize rad12 and rad12-associated genes in Coprinus cinereus.
Martina Celerin, Teresa M. Niehoff, Miriam E. Zolan. Department of Biology, Indiana University, Bloomington, Indiana 47405
We are exploiting the synchronous meiosis of Coprinus cinereus to better understand the related
processes of DNA repair and meiosis. Our laboratory has identified four genes, rad3, rad9, rad11
and rad12, which are required both for survival of gamma irradiation and for meiosis in C. cinereus.
Microscopic analysis of three rad12 mutants showed that rad12 chromosomes condense but do not
synapse completely during prophase I of meiosis. In addition,rad12 nuclei arrest at late prophase 1.
We have initiated an approach to isolating the rad12 gene and proteins which may be interacting with
rad12. The strategy involves tagged mutagenesis using restriction-enzyme mediated integration
(REMI) of transforming DNA followed by anchored PCR to retrieve the tagged gene. A self-compatible strain of C. cinereus, defective at both mating loci (Amut Bmut), and a plasmid harboring
the hygromycin marker, linearized with the restriction enzyme KpnI, were used for REMI
transformations. The linearized plasmid and extra KpnI were transformed into competent protoplasts.
Hygromycin-resistant mycelial colonies, resulting from regenerated, transformed cells, were collected
and induced to fruit using an established light and temperature regime. Of 2129 hygromycin-resistant
transformants, 153 fruited as white mushrooms, an indication that these mutants are likely defective
in meiosis and/or spore formation (W.J. Cummings, J. Crodian, L. Brunnick, M.E. Zolan, in
preparation). To determine whether any of the 153 new, likely meiotic mutants are actually tagged
alleles of rad12, we have mated each independently to two of the 15 known alleles of rad12. Of the
130 crosses that have fruited to date, 29 repeatedly produced white mushrooms, thereby
demonstrating lack of complementation between the new mutant and the rad12 mutation. We have
so far identified 13 mutants which contain a single hygromycin insert that co-segregates with the
sporeless, white fruiting body phenotype. Hence, the mutations in these strains are likely tagged.
Currently, semi-random, two-step PCR (ST-PCR; Chun et al., 1996) is being employed to identify
the tagged genes.
113. Involvement of homologous recombination in tandem repeat formation following integrative transformation in Penicillium paxilli.
Yasuo Itoh1, and Barry D. Scott2. 1Faculty of Science, Sinshu University, 390, Matsumoto, Japan. 2Department of Microbiology and Genetics, Massey University, Palmerston North, New Zealand.
We have been attempting to clone genes involved in the biosynthesis of paxilline, a tremorogenic
mycotoxin, by plasmid tagging. One mutant that did not produce the toxin was isolated by ectopic
integration of plasmid pAN7-1. Analysis of the site of integration in this mutant revealed the presence
of an extensive genomic deletion. This phenomenon was also observed in a set of pAN7-1 derived
spore color mutants. We were therefore interested in furthering our understanding of the process of
plasmid integration in this fungus. Transformations were carried out either with or without the
addition of restriction enzyme, and 102 integration events analysed. In the absence of enzyme, 50%
of the integrations were single copy events whereas addition of HindIII increased this ratio to 82 %.
Of the 33 tandem repeat integrants obtained from transformations with and without enzyme, 88%
were organised in a head to tail orientation with the remainder in inverted repeat configurations.
Dephosphorylation of linearized pAN7-1 had no significant effect on either the transformation
frequency or the integration profiles obtained, except that tandem repeats in the inverted repeat
configurations were not obtained using dephosphorylated vector. These results suggest that the
major mechanism for tandem repeat formation in P. paxilli is by homologous recombination of
additional copies of the plasmid using the single integrated copy as substrate. Addition of restriction
enzyme may provide a different pathway for plasmid integration.
114. Class I and Class II synaptic mutants in the basidiomycete Coprinus cinereus.
Janet Knight and Patricia J. Pukkila, University of North Carolina at Chapel Hill.
In the basidiomycete Coprinus cinereus, ultraviolet light can induce mutants that cannot successfully
complete meiosis. These mutants are recognized phenotypically by a white cap which signifies a lack
of spore production. Using electron microscopy and propidium iodide staining as characterization
tools, these mutants can be divided into two distinct classes. Class I mutants are defined as those in
which karyogamy and nucleolar fusion occur normally, and then subsequently arrest with no further
progression through meiosis. These mutants exhibit very little structure in the form of synaptonemal
complexes, and many times do not even initiate axial core assembly. Class 2 mutants, on the other
hand, usually complete axial core formation and occasionally initiate small regions of synaptonemal
complex. Of the ten bad (basidiospore development) mutants examined, four fell into Class I, five into
Class II, and one exhibited wild type synapsis. None of these mutants is sensitive to ionizing radiation.
Comparisons with previously published work indicate that mutants with defects in DNA repair have
a different spectrum of synaptic defects than those observed here.
115. The hotspot paradox and the evolution of meiotic crossing-over.
R. Redfield, R. S. Myers and A. Boulton, Dept. of Zoology, University of British Columbia.
Studies of meiotic recombination have revealed an evolutionary paradox. Molecular and genetic
analysis has shown that crossing over initiates at hotspots, by a recombinational-repair mechanism
that replaces the initiating hotspot with a copy of its homolog. Computer simulations of large
populations show that this mechanism causes active hotspots to be rapidly replaced by inactive alleles
arising by rare mutation. Neither of the known benefits of crossovers (accurate segregation and
genetic recombination) were sufficient to preserve active alleles in the face of this conversion. The
paradox was partly resolved by introducing into the model an additional, non-meiotic function for
hotspots, consistent with their observed association with functional sites in chromatin. Strong
selection for this function could allow active hotspots to persist in spite of frequent conversion to
inactive alleles. However, this explanation is unsatisfactory for two reasons. First, it is unlikely to
apply to obligately-sexual species, because the viability selection needed to preserve many hotspots
per genome (necessitated by observed crossover frequencies) would drive the species to extinction.
Second, it fails to explain how natural selection could maintain such a genetically costly mechanism
of recombination. Resolution of the paradox may require significant changes to the commonly-accepted models of meiotic recombination.
116. A meiosis-specific initiation site for recombination in the promoter region of the niiA-niaD gene cluster of A. nidulans.
Henk W.J. van den Broek, Hans Thijs and Theo Goosen. Dept. of Genetics, Agricultural University Wageningen, Dreyenlaan 2, 6703 HA Wageningen, The Netherlands.
Genetic markers typically show Mendelian segregation (2:2) in meiosis, but with a low frequency deviations are observed which are generally viewed as the consequence of gene conversion, in which one DNA duplex (the acceptor) is altered using a non-sister duplex (the donor) as a template. This interaction (which probably reflects the chromosomal search for homology during meiotic prophase 1) requires the formation of a heteroduplex tract between strands of non-sister chromatids that may include mismatches and the physical linkage of the homologues by Holliday junctions. Repair of these mismatches could result in aberrant segregation patterns.
Usually, polarity of gene conversion is observed (markers on one side of a locus are converted at higher frequencies than markers on the other side), which is interpreted as the result of fixed initiation sites for heteroduplex formation and distance dependent resolution. In the ARG4 and HIS4 genes of yeast and the niiA-niaD gene cluster of A. nidulans these initiation sites are located in the promoter regions of these genes.
We have introduced molecular markers in the niiA-niaD gene cluster and used the resulting strains
in two-point crosses. The results confirm the location of the initiation site in the promoter region and
demonstrate that this site is meiosis specific. Furthermore they show that conversion tracts are small
and that mutations included in a tract are always co-converted. Initiation of mitotic recombination
events does not take place at a specific site.
117. Molecular analysis of recombination events associated with the cog recombinator of Neurospora.
P Jane Yeadon and David EA Catcheside, School of Biological Sciences, Flinders University, Adelaide, Australia.
Multiple polymorphisms distinguish Emerson and Lindegren strains of Neurospora crassa within the histidine-3 gene and in its distal flank. Restriction site and sequence length polymorphisms in an overlapping set of PCR products covering this region have been used to identify the parental origin of DNA in histidine-prototrophic recombinant progeny of crosses between the strains. 29% of conversion tracts are interrupted. Where the absence of rec-2+ permits activity of the recombination hotspot cog, conversion appears to originate at cog and conversion tracts are up to 5.6 kb long. The chromosome bearing cogL the dominant allele which confers a high frequency of recombination, is almost invariably the recipient of information. In progeny from rec-2+crosses, conversion tracts are much shorter, most are not initiated at cog and either chromosome seems equally likely to be converted. Conversion is only infrequently associated with crossing over, suggesting that a conversion intermediate containing paired Holliday junctions may usually be resolved by a topoisomerase, or by scission of one junction and migration of the second to the resulting nicks. The low level of association between conversion and crossing over could be due to occasional scission of both junctions, resulting in a crossover in half of these events.
118. A Novel MCBL plates:co-induce nuclear membrane fusion and chromosome nondisjunctional recombination.
Yun-Can Ai, Fan-Mei Meng, Jin-Xian Luo,(Zhongshan University,Guangzhou,PR China), Christian P Kubicek, Robert Mach, Tanja Krupica,(TU Wien,Wlen,Austria)
MCBI, plates were constructed with Czapek's Minimal medium containing 0.1% (w/v) d-Camphor and 0.5-1.0 ppm Benomyl based on an assumption that d-Camphor could induce nuclear membrane fusion while Benomyl could induce chromosome nondisjunctional recombination.After the PEG-mediated fusion of UV-inactived prototrophic protoplasts between Trichoderma reesei and Aspergillus niger, the temporary heterokaryon could be induced on these plates at the same time. Thus serveral very stable fusants with dominance could be obtained from this fusion cross, for the first time. Three typical recombinants --after they had been undergone segregation over five years-- were confirmed under the approches of PFEE for chromosomes, RAPD-PCR for gene finger-prints especially Southern Blotting with cbhl, cbh2, egl, eg2, bgll probes for cellulose genes analysis.These results demonstrat a novel way for overcoming the several problems, such as incompatibility and no recombination, arose from the ordinary procedures It therefore would provide a novel approach for fungal genetics and biotechnology.