Fungal Genetics and Biology 41 (2004) 285–292

www.elsevier.com/locate/yfgbi

Fungal Genetics and Biology 41 (2004) 285–292Fungal Genetics and Biology 41 (2004) 285–292

Commentary

Comparative sequence analysis of Sordaria macrosporaand Neurospora crassa as a means to improve genome annotationq

Minou Nowrousian, Christian W€urtz, Stefanie P€oggeler, and Ulrich K€uck*

Lehrstuhl fur Allgemeine und Molekulare Botanik, Ruhr-Universitat Bochum, 44780, Bochum, Germany

Received 15 August 2003; accepted 22 October 2003

Abstract

One of the most challenging parts of large scale sequencing projects is the identification of functional elements encoded in a

genome. Recently, studies of genomes of up to six different Saccharomyces species have demonstrated that a comparative analysis of

genome sequences from closely related species is a powerful approach to identify open reading frames and other functional regions

within genomes [Science 301 (2003) 71, Nature 423 (2003) 241]. Here, we present a comparison of selected sequences from Sordaria

macrospora to their corresponding Neurospora crassa orthologous regions. Our analysis indicates that due to the high degree of

sequence similarity and conservation of overall genomic organization, S. macrospora sequence information can be used to simplify

the annotation of the N. crassa genome.

� 2003 Elsevier Inc. All rights reserved.

Keywords: Sordaria macrospora; Neurospora crassa; Filamentous fungi; Genome annotation; Exon–intron boundaries; Synteny; Comparative

genomics

1. Introduction

Over the last few years, several fungal genomes

have been fully sequenced, and even more will be se-

quenced in the near future (Galagan et al., 2003; Goffeau

et al., 1996, http://www-genome.wi.mit.edu/seq/fgi/

candidates.html, http://www.ncbi.nlm.nih.gov/cgi-bin/

Entrez/map00?taxid¼ 5085). However, the most chal-lenging part in genome analysis today is usually not

generating the sequence but annotating it. Features like

open reading frames, exon–intron boundaries and reg-

ulatory elements within a genome are often difficult to

predict correctly from genomic sequence information

alone. Identification of transcribed regions can be im-

proved if EST1 sequences are available, but EST data-

bases tend not to contain rarely transcribed genes, sooften no cDNA sequence information is available.

qSupplementary data associated with this article can be found, in

the online version, at doi: 10.1016/j.fgb.2003.10.005.* Corrresponding author. Fax +49-234-321-4184.

E-mail address: ulrich.kueck@ruhr-uni-bochum.de (U. K€uck).1 Abbreviations used: EST, expressed sequence tag; ORF, open

reading frame; indel, site corresponding to an insertion or deletion.

1087-1845/$ - see front matter � 2003 Elsevier Inc. All rights reserved.

doi:10.1016/j.fgb.2003.10.005

Comparison with public databases is an additional

means of identifying open reading frames, but for many

genes, no putative homologs are available in the data-

bases; e.g., many fungal EST sequencing projects have

found that only less than or�50% of the ESTs generated

have already characterized homologs among other or-

ganisms (e.g., Nelson et al., 1997; Prade et al., 2001; Zhu

et al., 2001). Microarray data or other large scale tran-scriptome data can help to discover genes that are reg-

ulated similarly at the transcriptional level and therefore

might contain common promoter elements (e.g., Hughes

et al., 2000; Ren et al., 2000; Roth et al., 1998), but this

approach to identify regulatory elements is labor-inten-

sive and requires an EST library or annotation of open

reading frames as a prerequisite to generate microarrays.

Information from within the genome as well ascomparisons to databases and experimental data have

been used to annotate the genome of Saccharomyces

cerevisiae, the first eukaryote to be sequenced (Goffeau

et al., 1996) and the one for which annotation has

progressed furthest (http://www.yeastgenome.org/).

Nevertheless, the gene count between different methods

of analysis has varied considerably (e.g., Malpertuy

et al., 2000). Recently, it was shown for several

286 M. Nowrousian et al. / Fungal Genetics and Biology 41 (2004) 285–292

Saccharomyces species that comparison of closely relatedwhole genome sequences can be of immense value for the

annotation, because it greatly increases the signal-to-

noise ratio for the discovery of open reading frames,

introns, and regulatory sequences (Cliften et al., 2003;

Kellis et al., 2003). Sequences that are of functional

significance are usually more conserved between species,

and therefore lack of sequence conservation within pre-

dicted features might indicate that the prediction doesnot necessarily reflect reality. On the other hand, short

open reading frames can be identified more readily be-

cause they are marked by conserved ‘‘islands’’ within less

conserved sequence surroundings. Short ORFs are

sometimes overlooked during the annotation of a single

genome due to the fact that thresholds for defining

the length of open reading frames often exclude them.

The comparison of the S. cerevisiae genome to that ofthree other ascomycetous yeasts indicated that the total

number of genes might be �5500. The authors identified

43 previously undetected small open reading frames,

whereas �500 predicted open reading frames most likely

do not represent functional genes (Kellis et al., 2003).

The comparison of yeast species exemplifies the use-

fulness of sequencing closely related species for im-

proving annotation. The second fungal genome to becompletely sequenced and for which annotation has al-

ready progressed significantly is that of Neurospora

crassa (Galagan et al., 2003). No species closely related

to N. crassa has been fully sequenced and annotated yet.

Here, we demonstrate that, similarly to the recent

analyses of closely related yeast genomes, genomic se-

quence information from the closely related filamentous

ascomycete Sordaria macrospora can be used to improveannotation of the N. crassa genome.

2. Sequence identitiy between S. macrospora and N.crassa ORFs

The ascomycete S. macrospora is a close relative of N.

crassa, both of which belong to the family of Sordaria-ceae (order Sphaeriales). Both are characterized by

black perithecia of similar size which contain eight-

spored asci; but in contrast to N. crassa, S. macrospora

does not produce any conidiospores and is homothallic

(self-fertile) which means that no mating partner is

needed to develop mature fruiting bodies. Therefore,

mutants with defects in the sexual cycle can easily be

isolated and serve as recipient strains for transforma-tions to restore the wild type phenotype. Consequently,

S. macrospora has been used as a model system to study

fruiting body development, and several genes essential

for perithecia formation as well as components of the

mating type, pheromone, and pheromone receptor

pathways have been isolated (Masloff et al., 1999;

Nowrousian et al., 1999; P€oggeler, 2000; P€oggeler and

K€uck, 2000, 2001; P€oggeler et al., 1997). S. macrospora

has also been used to analyze chromosome pairing and

recombination during meiosis. This has involved ex-

tensive cytological studies as well as work on the mo-

lecular basis of meiotic events (Le Chevanton and

Zickler, 1991; Thompson-Coffe et al., 1999; van Heemst

et al., 1999; Zickler et al., 1985, 1992).

Sequence analyses of S. macrospora genes isolated so

far have already indicated that homology between S.

macrospora and N. crassa is not restricted to exons but

extends to intronic and other non-coding sequences.

Here, we present a comparison of 85 genes comprising

186 full or partial exon sequences and 98 intron se-

quences from S. macrospora with their N. crassa ho-

mologs (for a complete list of genes used in for this

comparison, see Table 1 supplementary material).

N. crassa sequence information was taken from theNeurospora genome at http://www-genome.wi.mit.edu/

annotation/fungi/neurospora/ (Galagan et al., 2003).

S. macrospora sequences used for this comparison are

from previously published genes, or were isolated in our

laboratory either by complementation of mutants or by

PCR from S. macrospora genomic DNA using oligo-

nucleotide primers derived from N. crassa sequences.

Sequence comparisons were performed using LALIGN(Huang and Miller, 1991) at http://www.ch.embnet.org/

software/LALIGN_form.html.

As expected, sequence identities were highest within

exons with an average of 89.5% nucleic acid identity; but

even within introns, an average sequence identity of

70.4% was found. As mentioned above, several of the

genes used in this analysis were isolated from S. mac-

rospora based on their sequence homology to N. crassa

or other filamentous fungi. To exclude the possibility

that this has caused a bias for genes with a higher than

average sequence similarity to N. crassa, we performed a

separate comparison with four genes that were isolated

from S. macrospora by mutant complementation. These

genes comprise �11 kb of exon and 0.5 kb of intron se-

quences (acl1, pro1, pro11, and spo76, see Table 1 in the

supplementary material). The results were similar tothose derived from the complete dataset; the average

exon sequence identity was 89.5%, and nucleic acid

identity for introns was 70.2%.

Similarly to the yeasts (Kellis et al., 2003), exon

boundaries for the genes used in this comparison were

usually marked by an increase in sequence identity and

the absence of indels. Intron lengths in both fungi are

similar, ranging from 47 to 545 bp in S. macrospora and45–542 bp in N. crassa for the introns compared in this

study. Average lengths were 106 nt for S. macrospora

and 101 nt for N. crassa introns, which is a bit lower

than the average intron size of 134 nt that was calculated

for the N. crassa genome (Galagan et al., 2003). As was

reported previously (P€oggeler, 1997), consensus se-

quences for intron donor, acceptor, and branch site are

Fig. 1. Comparison of intron 3 and adjacent regions of the S. macrospora (S.m.) and N. crassa (N.c.) pro11 genes. Intron sequences are indicated in

small case and are underlined. A region that is annotated as part of intron 3 in N. crassa, but most likely is part of exon 4 is shaded in gray. For

further information see text.

30.0

40.0

50.0

60.0

70.0

80.0

90.0

75.0 80.0 85.0 90.0 95.0 100.0% identity exons

% id

entit

y in

tron

s

Fig. 2. Conservation of introns is not linked to conservation of exonic

sequences. For 57 genes, sequence identity of exons was calculated

separately from that of the introns for each gene. A table with the

genes used in this comparison can be found in the supplementary

material (Table 2). The graph depicts the nucleic acid identity of in-

tronic sequences (y-axis) versus exonic sequences (x-axis) for the 57

M. Nowrousian et al. / Fungal Genetics and Biology 41 (2004) 285–292 287

almost identical in S. macrospora and N. crassa (data

not shown). Intron positions within open reading frames

in S. macrospora were highly conserved when compared

to N. crassa in all cases investigated. In some cases, therewere slight variations of the exact intron start or end,

but these might indicate annotation errors rather than

true biological differences. An example for this can be

seen at the 30 end of the third intron of the pro11 gene

(Fig. 1, P€oggeler and K€uck, 2003). The flanking exons

are highly similar whereas within the intron, the

homology is significantly lower and several indels

are present (Fig. 1). In the N. crassa homolog(NCU08741.1), the intron 30 end was annotated another

24 nt downstream of the predicted 30 end within S.

macrospora, but these 24 nt are 100% identical to the

corresponding S. macrospora sequence. This indicates

that they might constitute exonic sequence rather than

intron sequence. Sequencing of the S. macrospora pro11

cDNA confirmed the exon/intron boundary at the po-

sition indicated in Fig. 1.

genes. Black triangles indicate genes for which only partial S. mac-

rospora sequence information was available for comparison. Open

squares indicate complete genes used for this comparison.

3. Conservation of introns is not linked to conservation of

exon sequences

As described before, introns and exons of a given

gene are colinear and highly similar in S. macrospora

and N. crassa. An interesting question that can be askedin a case like this is whether intron and exon conserva-

tion are correlated. In other words, would a high degree

of exon similarity between two orthologous genes also

mean a high degree of intron similarity? To answer this

question, average exon and intron similarities were cal-

culated separately for 57 genes that have introns (Table

2 in the supplementary material). Fig. 2 shows a graph

of exon identities and corresponding intron identities. If

there were a simple correlation between exon and intron

identity, one would expect a linear distribution. How-

ever, as indicated in Fig. 2, this is not the case. Nor

could we identify any other statistically significant cor-

relation between intron and exon identities (data not

shown). Thus, within the genes included in our study, it

seems that there is no apparent correlation between in-

tron and exon sequence identity within a given gene.Usually, exons are under strong selective pressure to

preserve their coding capacities, whereas introns simply

have to retain their splicing signal sequences; thus, this

finding might not be unexpected. However, comparing

288 M. Nowrousian et al. / Fungal Genetics and Biology 41 (2004) 285–292

intron sequence identities might be a means of identi-fying regions within a genome that accumulate muta-

tions more readily than others. This might help to

identify regions which are more susceptible to mutagenic

influences or are less efficiently repaired by DNA repair

mechanisms. Analyses like this might even be more re-

vealing in genomes with higher intron content, e.g., the

human genome.

4. S. macrospora and N. crassa share a high degree of

synteny that simplifies the identification of open reading

frames

Comparison of larger regions of S. macrospora and

N. crassa DNA revealed that sequence identity between

the two species is present even outside of open readingframes. The regions show nearly complete synteny and

can be readily aligned at the nucleotide level (Figs. 3A

4A). Within the 15 kb region shown in Fig. 3A, five

genes (pho88, rad14, pro11, trnN, and etp) can be

identified in the same orientation in both organims. A

sixth predicted N. crassa gene, NCU08740.1, has no

Fig. 3. Synteny between S. macrospora (S.m.) and N. crassa (N.c.) in a 15 k

tergenic regions as gray boxes. (A) Nucleic acid identity between the two geno

the S. macrosporaDNA. (B) The N. crassa open reading frame NCU08740.1

macrospora genome. Sequence identity between the adjoining genes pro11 a

macrospora and N. crassa DNA. For more information see text.

homolog in S. macrospora, in fact, the correspondingORF and adjoining sequences are absent from this

region of the S. macrospora genome (Fig. 3B). There

are several possibilities for this apparent absence of the

gene from the region. It might, for example, be located

elsewhere in the S. macrospora genome, or it is not

present in S. macrospora at all. The latter possibility

would indicate that it is a gene that is not necessary for

S. macrospora, but might be useful for N. crassa. Al-ternatively, NCU08740.1 might not be a true open

reading frame. It is a rather short ORF of 200 nt, 125

nt of which comprise a predicted intron sequence, and

the derived polypeptide sequence is 24 amino acids

long. Such short open reading frames are often difficult

to predict from DNA sequence information alone.

Further information about whether this is a true gene

might be gained by comparing sequences from otherclosely related species.

Another predicted Neurospora ORF which cannot be

verified in S. macrospora is NCU06784.1 (Fig. 4).

NCU06784.1 is part of a larger region of �10 kb which

is strongly homologous in both organisms. Upstream

and downstream from NCU06784.1 are the acl2 and

b genomic region. Exons are given as black, introns as white, and in-

me regions. A region which is shown in detail in (B) is indicated above

and flanking regions are not present at their corresponding site in the S.

nd trnN as well as intergenic sequences are indicated between the S.

Fig. 4. Comparison of the acl-gene containing regions from S. macrospora (S.m.) and N. crassa (N.c.). Exons are given as black, introns as white, and

intergenic regions as light gray boxes. Exons of the open reading frame NCU06784.1 for which no S. macrospora homologue can be identified are

given in dark gray. (A) A syntenic region of about 10 kb contains the acl1 and acl2 genes (Nowrousian et al., 2000). Nucleic acid identity is indicated

between the two sequences. A part which is shown in detail in (B) is indicated above the S. macrospora DNA. (B) Intergenic region between acl1 and

acl2. The upper part of (B) shows sequence identities determined separately for putative exons, the intron, and upstream and downstream regions of

N. crassa ORF NCU6784.1 to their S. macrospora counterparts. The lower part of (B) gives sequence identity between S. macrospora and N. crassa in

various parts of the intergenic regions as determined by local alignment using LALIGN (Huang and Miller, 1991). (C) Sequence alignment of N.

crassa ORF NCU06784.1 and 100 nt of upstream and downstream regions to its corresponding S. macrospora counterpart. Putative translation start

and stop codons are given in bold, intronic sequences in small case. Indels within the putative ORF that do not contain a multiple of three nu-

cleotides, and therefore would result in frame-shift mutations, are shaded in gray.

M. Nowrousian et al. / Fungal Genetics and Biology 41 (2004) 285–292 289

acl1 genes, respectively. In this case, a region with high

homology to NCU06784.1 is present in S. macrospora,

but no bona fide open reading frame can be identified.At the position of the ATG in Neurospora is a GTG in

Sordaria. GTG as a start codon has been reported

within filamentous fungi (e.g., Guti�errez et al., 1991),

but several indels that are not multiples of three inter-

rupt the S. macrospora open reading frame (Fig. 4C)

which makes it unlikely that this is a real gene in S.

macrospora. Additional hints that NCU06784.1 is not a

true ORF come from the fact that the presumptivecoding and non-coding regions in this case do not sig-

nificantly differ in their degree of homology, as is the

case for other genes compared. In fact, within the 3 kb

intergenic region between acl1 and acl2, there are several

regions of equally high or higher nucleic acid identity

than the predicted ORF NCU06784.1 (Fig. 4B). As acl1

and acl2 are divergently transcribed, the 3 kb intergenic

region most likely contains promoter sequences whichregulate the expression of both genes. The high degree of

overall conservation of this region might indicate regu-

latory mechanisms common to S. macrospora and

N. crassa instead of marking an open reading frame.

One way to shed light on questions like this would beto include sequence information from further close

relatives of S. macrospora and N. crassa into the

analysis. Especially information about the absence or

presence of indels as well as the degree of conservation

of exons within a less conserved sequence environ-

ment might help to identify the most likely open

reading frames from genomic DNA sequence informa-

tion alone.

5. Which additional fungal genomes might be sequenced

for a comparative genomics approach?

Sequencing and annotation of the N. crassa genome

has already greatly advanced our knowledge of fungal

genome organization (Galagan et al., 2003). Annotationof genome sequences from closely related species will be

much easier with the N. crassa genome present, but also

290 M. Nowrousian et al. / Fungal Genetics and Biology 41 (2004) 285–292

the N. crassa annotation itself will become much morereliable with the possibility of comparing two or more

genomes. A prerequisite for this is that the compared

sequences are similar enough to show a sufficient degree

of synteny. The comparisons presented here indicate

that the S. macrospora genome is eminently suitable for

this purpose, because it is similar enough to be readily

aligned at the nucleotide level even outside of coding

regions, but has aquired a sufficient degree of dissimi-larity especially in non-coding regions to provide an

adequate signal-to-noise enrichment for distinguishing

functional from non-functional sites. Another point of

interest might be the fact that in S. macrospora, no in-

dication of RIP (repeat-induced point mutation) has

been found yet (Le Chevanton et al., 1989). RIP has

originally been discovered in N. crassa where it inacti-

vates duplicated sequences during the sexual phase ofthe life cycle (Selker et al., 1987). It was also shown to

exist in a milder form in Podospora anserina (Graia

et al., 2001; Hamann et al., 2000) and Magnaporthe

grisea (Ikeda et al., 2002). RIP is thought to be re-

sponsible for the surprisingly low number of multigene

families and duplicated sequences observed in N. crassa

(Galagan et al., 2003). Therefore, a comparison of the

N. crassa genome with that of S. macrospora will bemost interesting with respect to the divergent evolution

of two closely related genomes one of which displays a

very active form of RIP while the other does not.

However, as has been demonstrated for several Sac-

charomyces species, comparative genomics gains power

with the number of species investigated (Cliften et al.,

2003; Kellis et al., 2003). Which additional filamentous

fungi might be suitable candidates for such an approach?The genomes of the pyrenomycetes P. anserina and M.

grisea are at present being sequenced and annotated. Both

species aremuchmore distant relatives toN. crassa than is

S. macrospora, and previous analyses have shown that

synteny between N. crassa and P. anserina or M. grisea,

respectively, is limited (Hamer et al., 2001; Silar et al.,

2003). In both P. anserina and M. grisea, intergenic re-

gions are not conserved even within syntenic regions.Thus, the genomes of P. anserina and M. grisea will cer-

tainly advance our knowledge of filamentous fungi�s bi-ology, but they are less suited for a comparative genomics

approach with N. crassa. In a white paper describing the

aims of the fungal genome initiative, one of the organisms

included in a list of fungi for initial sequencing is Neu-

rospora discreta (http://www-genome.wi.mit.edu/seq/fgi/

FGI_whitepaper_Feb8.pdf). Comparisons of matingtype genes as well as the gpd gene, the ITS/5.8S rRNA

region and four anonymous nuclear loci from several

Neurospora and Sordaria species indicate thatN. discreta

belongs to a group of closely related Neurospora species

including N. crassa, but is more distantly related to

N. crassa than are, for example, Neurospora tetrasperma

and Neurospora sitophila (Dettman et al., 2001, 2003;

P€oggeler, 1999; Randall and Metzenberg, 1995).Sequence similarity between the mating type and gpd

genes from N. crassa and N. discreta are between 94 and

98%, whereas between S. macrospora and the two Neu-

rospora species they lie between 78 and 90% (P€oggeler,1999). The N. discreta genome would therefore be an

obvious candidate to be included in a comparative ge-

nomics approach, but it might be advisable to obtain the

sequence from at least one additional fungus from theSordaria/Neurospora group which is less similar to N.

crassa, in order to further increase the signal-to-noise

ratio that enables the distinction between functional and

non-functional sites. Candidates might be Neurospora

pannonica or Neurospora terricola as well as Sordaria

brevicollis or Sordaria sclerogenia, all of which are less

closely related to N. crassa than N. discreta and are well

distanced from S. macrospora also (Dettman et al., 2001;P€oggeler, 1999). Both N. pannonica and N. terricola are

homothallic species, as is S. macrospora; whereas

S. sclerogenia and S. brevicollis are two heterothallic

Sordaria species. Sequencing both homo- as well as het-

erothallic species might give insights into whether the

mode of sexual differentiation shapes genome structure

and evolution outside of the mating type loci. Sequence

information from more genes will undoubtedly be nec-essary to reach a conclusion about which species might be

the most useful to sequence for a comparative genomics

approach.

6. Conclusions

Our comparison demonstrates how closely the ge-nome organization of S. macrospora resembles that of

N. crassa not only within coding but also within non-

coding regions. Obtaining additional sequence infor-

mation of S. macrospora and other closely related

species might therefore facilitate the annotation of all of

their genomes including that of N. crassa, similarly to

what was described for yeasts (Cliften et al., 2003; Kellis

et al., 2003). It will also create the possibility to studyevolution of genomes that have been separated only

recently, and therefore, in turn, might provide insight

into the molecular basis of speciation.

Acknowledgments

The authors thank Swenja Ellßel, Ingeborg Gode-hardt, and Silke Nimtz for excellent technical assistance

and Dr. Giles Duffield for critical reading of the man-

uscript. We also thank our reviewers for helpful sug-

gestions to improve the manuscript. This work was

supported by grants from the Deutsche Forschungs-

gemeinschaft (DFG) to U.K. and S.P. (SFB 480, Pro-

jects A1 and A2), and the Ruhr-Universit€at Bochum to

M. Nowrousian et al. / Fungal Genetics and Biology 41 (2004) 285–292 291

M.N. (Programm zur F€orderung des wissenschaftlichenNachwuchses).

References

Cliften, P., Sudarsanam, P., Desikan, A., Fulton, L., Fulton, B.,

Majors, J., Waterston, R., Cohen, B.A., Johnston, M., 2003.

Finding functional features in Saccharomyces genomes by phylo-

genetic footprinting. Science 301, 71–76.

Dettman, J.R., Harbinski, F.M., Taylor, J.W., 2001. Ascospore

morphology is a poor predictor of the phylogenetic relationships

of Neurospora and Gelasinospora. Fung. Genet. Biol. 34, 49–61.

Dettman, J.R., Jacobson, D.J., Taylor, J.W., 2003. A multilocus

genealogical approach to phylogenetic species recognition in the

model eukaryote Neurospora. Evolution (in press).

Galagan, J., Calvo, S., Borkovich, K., Selker, E., Read, N., Jaffe, D.,

FitzHugh, W., Ma, L., Smirnov, S., Purcell, S., Rehman, B.,

Elkins, T., Engels, R., Wang, S., Nielsen, C., Butler, J., Endrizzi,

M., Qui, D., Ianakiev, P., Bell-Pedersen, D., Nelson, M., Werner-

Washburne, M., Selitrennikoff, C., Kinsey, J., Braun, E., Zelter, A.,

Schulte, U., Kothe, G., Jedd, G., Mewes, W., Staben, C., Marcotte,

E., Greenberg, D., Roy, A., Foley, K., Naylor, J., Stange-

Thomann, N., Barrett, R., Gnerre, S., Kamal, M., Kamvysselis,

M., Mauceli, E., Bielke, C., Rudd, S., Frishman, D., Krystofova,

S., Rasmussen, C., Metzenberg, R., Perkins, D., Kroken, S.,

Cogoni, C., Macino, G., Catcheside, D., Li, W., Pratt, R., Osmani,

S., DeSouza, C., Glass, L., Orbach, M., Berglund, J., Voelker, R.,

Yarden, O., Plamann, M., Seiler, S., Dunlap, J., Radford, A.,

Aramayo, R., Natvig, D., Alex, L., Mannhaupt, G., Ebbole, D.,

Freitag, M., Paulsen, I., Sachs, M., Lander, E., Nusbaum,, C.,

Birren, B., 2003. The genome sequence of the filamentous fungus

Neurospora crassa. Nature 422, 859–868.

Goffeau, A., Barrell, B.G., Bussey, H., Davis, R.W., Dujon, B.,

Feldmann, H., Galibert, F., Hoheisel, J.D., Jacq, C., Johnston, M.,

Louis, E.J., Mewes, H.W., Murakami, Y., Philippsen, P., Tettelin,

H., Oliver, S.G., 1996. Life with 6000 genes. Science 274, 546–567.

Graia, F., Lespinet, O., Rimbault, B., Dequard-Chablat, M., Coppin,

E., Picard, M., 2001. Genome quality control: RIP (repeat-induced

point mutation) comes to Podospora. Mol. Microbiol. 40, 586–595.

Guti�errez, S., D�ıez, B., Montenegro, E., Mart�ın, J.F., 1991. Charac-

terization of the Cephalosporium acremonium pcbAB gene encoding

alpha-aminoadipyl-cysteinyl-valine synthetase, a large multido-

main peptide synthetase: linkage to the pcbC gene and evidence

of multiple functional domains. J. Bacteriol. 173, 2354–2365.

Hamann, A., Feller, F., Osiewacz, H.D., 2000. The degenerate DNA

transposon Pat and repeat-induced point mutation (RIP) in

Podospora anserina. Mol. Gen. Genet. 263, 1061–1069.

Hamer, L., Pan, H., Adachi, K., Orbach, M.J., Page, A., Ramamur-

thy, L., Woessner, J.P., 2001. Regions of microsynteny in Magna-

porthe grisea and Neurospora crassa. Fung. Genet. Biol. 33,

137–143.

Huang, X., Miller, M., 1991. A time-efficient, linear-space local

similarity algorithm. Adv. Appl. Math. 12, 337–357.

Hughes, J.D., Estep, P.W., Tavazoie, S., Church, G.M., 2000.

Computational identification of cis-regulatory elements associated

with groups of functionally related genes in Saccharomyces

cerevisiae. J. Mol. Biol. 296, 1205–1214.

Ikeda, K., Nakayashiki, H., Kataoka, T., Tamba, H., Hashimoto, Y.,

Tosa, Y., Mayama, S., 2002. Repeat-induced point mutation (RIP)

in Magnaporthe grisea: implications for its sexual cycle in the

natural field context. Mol. Microbiol. 45, 1355–1364.

Kellis, M., Patterson, N., Endrizzi, M., Birren, B., Lander, E., 2003.

Sequencing and comparison of yeast species to identify genes and

regulatory elements. Nature 423, 241–254.

Le Chevanton, L., Leblon, G., Lebilcot, S., 1989. Duplications created

by transformation in Sordaria macrospora are not inactivated

during meiosis. Mol. Gen. Genet. 218, 390–396.

Le Chevanton, L., Zickler, D., 1991. Sordaria macrospora: the

transition to the age of gene manipulation. In: Bennett, J.W.,

Lasure, L.L. (Eds.), More gene manipulations in fungi. Academic

Press, San Diego, pp. 291–303.

Malpertuy, A., Tekaia, F., Casar�egola, S., Aigle, M., Artiguenave, F.,

Blandin, G., Bolotin-Fukuhara, M., Bon, E., Brottier, P., de

Montigny, J., Durrens, P., Gaillardin, C., L�epingle, A., Llorente,

B., Neuv�eglise, C., Ozier-Kalogeropoulos, O., Potier, S., Saurin,

W., Toffano-Nioche, C., W�esolowski-Louvel, M., Wincker, P.,

Weissenbach, J., Souciet,, J.L., Dujon, B., 2000. Genomic explo-

ration of the hemiascomycetous yeasts: 19. Ascomycetes-specific

genes.. FEBS Lett. 487, 113–121.

Masloff, S., P€oggeler, S., K€uck, U., 1999. The pro1+ gene from

Sordaria macrospora encodes a C6 zinc finger transcription factor

required for fruiting body development. Genetics 152, 191–199.

Nelson, M.A., Kang, S., Braun, E.L., Crawford, M.E., Dolan, P.L.,

Leonard, P.M., Mitchell, J., Armijo, A.M., Bean, L., Blueyes, E.,

Cushing, T., Errett, A., Fleharty, M., Gorman, M., Judson, K.,

Miller, R., Ortega, J., Pavlova, I., Perea, J., Todisco, S., Trujillo,

R., Valentine, J., Wells, A., Werner-Washburn, M., Yazzie, S.,

Natvig, D.O., 1997. Expressed sequences from conidial, mycelial,

and sexual stages of Neurospora crassa. Fung. Genet. Biol. 21, 348–

363.

Nowrousian, M., K€uck, U., Loser, K., Weltring, K., 2000. The fungal

acl1 and acl2 genes encode two polypeptides with homology to the

N- and C-terminal parts of the animal ATP citrate lyase polypep-

tide. Curr. Genet. 37, 189–193.

Nowrousian, M., Masloff, S., P€oggeler, S., K€uck, U., 1999. Cell

differentiation during sexual development of the fungus Sordaria

macrospora requires ATP citrate lyase activity. Mol. Cell. Biol. 19,

450–460.

P€oggeler, S., 1997. Sequence characteristics within nuclear genes from

Sordaria macrospora. Fung. Genet. Newslett. 44, 41–44.

P€oggeler, S., 1999. Phylogenetic relationships between mating-type

sequences from homothallic and heterothallic ascomycetes. Curr.

Genet. 36, 222–231.

P€oggeler, S., 2000. Two pheromone precursor genes are transcription-

ally expressed in the homothallic ascomycete Sordaria macrospora.

Curr. Genet. 37, 403–411.

P€oggeler, S., K€uck, U., 2000. Comparative analysis of the mating-type

loci from Neurospora crassa and Sordaria macrospora: identifica-

tion of novel transcribed ORFs. Mol. Gen. Genet. 263, 292–301.

P€oggeler, S., K€uck, U., 2001. Identification of transcriptionally

expressed pheromone receptor genes in filamentous ascomycetes.

Gene 280, 9–17.

P€oggeler, S., K€uck, U., 2003. A WD40-repeat protein regulates fungal

cell differentiation and can functionally be substituted by striatin, a

mammalian homologue. Eukaryot. Cell, in press.

P€oggeler, S., Risch, S., K€uck, U., Osiewacz, H.D., 1997. Mating-type

genes from the homothallic fungus Sordaria macrospora are

functionally expressed in a heterothallic ascomycete. Genetics

147, 567–580.

Prade, R.A., Ayoubi, P., Krishnan, S., Macwana, S., Russell, H., 2001.

Accumulation of stress and inducer-dependent plant-cell-wall-

degrading enzymes during asexual development in Aspergillus

nidulans. Genetics 157, 957–967.

Randall, T.A., Metzenberg, R.L., 1995. Species-specific and mating

type-specific DNA regions adjacent to mating type idiomorphs in

the genus Neurospora. Genetics 141, 119–136.

Ren, B., Robert, F., Wyrick, J.J., Aparicio, O., Jennings, E.G., Simon,

I., Zeitlinger, J., Schreiber, J., Hannett, N., Kanin, E., Volkert,

T.L., Wilson, C.J., Bell, S.P., Young, R.A., 2000. Genome-wide

location and function of DNA binding proteins. Science 290, 2306–

2309.

292 M. Nowrousian et al. / Fungal Genetics and Biology 41 (2004) 285–292

Roth, F.P., Hughes, J.D., Estep, P.W., Church, G.M., 1998. Finding

DNA regulatory motifs within unaligned noncoding sequences

clustered by whole-genome mRNA quantitation. Nat. Biotechnol.

16, 939–945.

Selker, E.U., Cambareri, E.B., Jensen, B.C., Haack, K.R., 1987.

Rearrangement of duplicated DNA in specialized cells of Neuros-

pora. Cell 51, 741–752.

Silar, P., Barreau, C., Debuchy, R., Kicka, S., Turcq, B., Sainsard-

Chanet, A., Sellem, C.H., Billault, A., Cattolico, L., Duprat, S.,

Weissenbach, J., 2003. Characterization of the genomic organiza-

tion of the region bordering the centromere of chromosome V of

Podospora anserina by direct sequencing. Fung. Genet. Biol. 39,

250–263.

Thompson-Coffe, C., Borioli, G., Zickler, D., Rosa, A., 1999. Pyruvate

decarboxylase filaments are associated with the cortical cytoskel-

eton of asci and spores over the sexual cycle of filamentous

ascomycetes. Fung. Genet. Biol. 26, 71–80.

van Heemst, D., James, F., P€oggeler, S., Berteaux-Lecellier, V.,

Zickler, D., 1999. Spo76p is a conserved chromosome protein that

links the mitotic and meiotic programs morphogenesis. Cell, 261–

271.

Zhu, H., Nowrousian, M., Kupfer, D., Colot, H.V., Berrocal-Tito, G.,

Lai, H., Bell-Pedersen, D., Roe, B.A., Loros, J.J., Dunlap, J.C.,

2001. Analysis of expressed sequence tags from two starvation,

time of day-specific libraries of Neurospora crassa reveals novel

clock-controlled genes. Genetics 157, 1057–1065.

Zickler, D., de Lares, L., Moreau, P., Leblon, G., 1985. Defective

pairing and synaptonemal complex formation in a Sordaria mutant

(spo44) with a translocated segment of the nucleolar organizer.

Chromosoma 92, 37–47.

Zickler, D., Moreau, P., Huynh, A., Slezec, A., 1992. Correlation

between pairing initiation sites, recombination nodules and

meiotic recombination in Sordaria macrospora. Genetics 132,

135–148.