Growth, Differentiation and Sexuality

418 D. Zickler
clearly defined in C. cinereus, in which replication
also occurs before karyogamy (Kanda et al. 1990;
Pukkila 1994). Strikingly, C. cinereus goes through
meiosis even if replication is defective (Kanda et al.
1990; Pukkila et al. 1995; Merino et al. 2000).
B. Premeiotic “Checking and Cleaning”
Mechanisms
The period between fertilization and meiosis is also
one during which several mycelial fungi “check
and clean their genomes”. To do so, they have
developed different premeiotic mechanisms that
scanthegenomeforDNAsequencespresentin
more than a single copy and larger than 450 bp.
These mechanisms, which occur before premeiotic
S-phase (likely during or just before the dikaryotic
stage; Fig. 20.1B), are triggered by the duplication
itself and mutate or silence extra DNA sequences.
They include:
1. The irreversible repeat-induced-point mutation
(RIP) mechanism, which is associated
with de novo methylation of cytosine residues
and converts C/G base pairs to A/T pairs in
the duplicated sequences of N. crassa, P. anserina,
M. grisea and Leptosphaeria maculans
(Cambareri et al. 1989; Hamann et al. 2000;
Graia et al. 2001; Ikeda et al. 2002; Selker 2002;
Idnurm and Howlett 2003; Bouhouche et al.
2004). RIP operates on linked and unlinked
DNA sequences, and is more or less frequent
in the different organisms studied to date.
A cytosine methyltransferase-homologue gene
(rid) is essential for RIP in N. crassa (Freitag
et al. 2002). In N. crassa and P. anserina,
the frequency of progeny affected by RIP is
highly increased in late-expelled ascospores
vs. early-expelled ascospores (Singer et al.
1995; Bouhouche et al. 2004). Similar increases
are also observed in P. anserina when the
steps between fertilization and karyogamy are
delayed in the absence of ami1/apsA, agene
required for correct nuclear movements and
positioning (Bouhouche et al. 2004).
2. The methylation induced premeiotically mechanism
(MIP), found in A. immersus (Rhounim
et al. 1992) and C. cinereus (Freedman and
Pukkila 1993). MIP methylates de novo all
gene-sized duplications at their cytosine
residues, and maintains this methylation without
further requirement for the methylated
sequence to remain duplicated. The inactive
state segregates in a Mendelian way (Rhounim
et al. 1992). As no mutation is associated with
MIP,thegeneinactivationprocessisreversible,
and inhibitors of DNA methylation accelerate
the gene reactivation (reviewed in Colot and
Rossignol 1999; Faugeron 2000). This de
novo methylation is triggered by the putative
C5-DNA-methyltransferase masc1 (Malagnac
et al. 1997).
3. Mechanisms that lead to gene/sequence losses,
rather than silencing. Premeiotic recombination
between cis-duplicated sequences leads
to deletion of the interstitial sequence in N.
crassa and P. anserina (e.g., Selker et al. 1987;
Coppin-Raynal et al. 1989). Chromosome de
novo deletions were found in Nectria haematococca
(Miao et al. 1991), Cochliobolus heterostrophus
(Tzeng et al. 1992) and C. carbonum
(Pitkin et al. 2000). Changes in the size of the N.
crassa nucleolar organizer region (Butler and
Metzenberg 1989) as well as meiosis-associated
deletion in heteroallelic repeats (MDHR) of M.
grisea are likely due to intrachromosomal recombination
events (Farman 2002).
In order to trigger RIP and MIP, the two elements
oftheduplicatedsequencemustbeinthesame
haploid nucleus, and tetrad analyses show that
the “checking for duplications” mechanism occurs
before karyogamy and prior to the premeiotic
S-phase (Fig. 20.1B; review in Rossignol and
Faugeron 1995; Selker 2002). However, the precise
step at which inactivation occurs remains
unknown. Interestingly, MIP strongly reduces the
frequency of allelic crossing-over in the hotspot b2
gene of A. immersus when b2 is methylated in one
or both parents (Maloisel and Rossignol 1998).
In these crosses, methylation transfer between
homologous chromosomes occurs during meiotic
prophase, and the methylated derivative of a gene
can be transferred to the unmethylated active b2
parental allele at frequencies comparable to those
of the gene conversion frequencies seen within b2.
Interestingly also, methylation transfer and gene
conversion are mechanistically related: (1) they
share the same 5 ′ to 3 ′ polarity along the b2 gene
(decrease of frequency from one end of the gene
to the other), and (2) they are similarly reduced
by ectopic positioning and nucleotidic divergence
(Colot et al. 1996). This suggests that, aside from
inactivating possible transposons, methylation
and MIP likely contribute to the stabilization of the
genome by preventing homologous recombination