1. Eukaryotic genomes: fungi
Chapter 18:
Eukaryotic genomes: fungi
Jonathan Pevsner, Ph.D.
Bioinformatics and Functional Genomics
(Wiley-Liss, 3rd edition, 2015)
You may use this PowerPoint for teaching purposes

2. Learning objectives After reading this chapter you should be able to:
■ provide an overview of how fungi are classified;
■ describe the features of the Saccharomyces cerevisiae genome;
■ discuss genome duplication in S. cerevisiae;
■ describe comparative genomics of the genus Saccharomyces; and
■ describe comparative genomics of other fungal phyla.

3. Outline Introduction Description and classification of fungi
Introduction to budding yeast Saccharomyces cerevisiae
Gene duplication and genome duplication
Comparative analyses of hemiascomycetes
Analysis of fungal genomes
Perspective

4. Introduction to fungi: phylogeny
Fungi are eukaryotic organisms that can be filamentous
(e.g. molds) or unicellular (e.g. the yeast Saccharomyces
cerevisiae).
Most fungi are aerobic (but S. cerevisiae can grow
anaerobically). Fungi have major roles in the ecosystem
in degrading organic waste. They have important roles
in fermentation, including the manufacture of steroids
and penicillin.
Several hundred fungal species are known to cause
disease in humans.

5. Fungi are a sister group with the metazoans (animals)
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We return to this phylogenetic tree in Chapter 19

6. Outline Introduction Description and classification of fungi
Introduction to budding yeast Saccharomyces cerevisiae
Gene duplication and genome duplication
Comparative analyses of hemiascomycetes
Analysis of fungal genomes
Perspective

7. Classification of fungi
About 70,000 fungal species have been described (as of 1995), but 1.5 million species may exist.
Four phyla:
Ascomycota yeasts, truffles, lichens
Basidiomycota rusts, smuts, mushrooms
Chytridiomycota Allomyces
Zygomycota feed on decaying vegetation

8. Classification of fungi
About 70,000 fungal species have been described (as of 1995), but 1.5 million species may exist.
Four phyla:
Ascomycota yeasts, truffles, lichens
Hemiascomycetae Génolevure project
Euascomycetae Neurospora
Loculoascomycetae
Laboulbeniomycetae parasites of insects
Basidiomycota rusts, smuts, mushrooms
Chytridiomycota Allomyces
Zygomycota feed on decaying vegetation

9. Alternate classification of fungi

10. Outline Introduction Description and classification of fungi
Introduction to budding yeast Saccharomyces cerevisiae
Gene duplication and genome duplication
Comparative analyses of hemiascomycetes
Analysis of fungal genomes
Perspective

11. Introduction to Saccharomyces cerevisiae
First species domesticated by humans
Called baker’s yeast (or brewer’s yeast)
Ferments glucose to ethanol and carbon dioxide
Model organism for studies of biochemistry,
genetics, molecular and cell biology
…rapid growth rate
…easy to modify genetically
…features typical of eukaryotes
…relatively simple (unicellular)
…relatively small genome

12. Sequencing the S. cerevisiae genome
The genome was sequenced by a highly cooperative
consortium in the early 1990s, chromosome by chromosome
(the whole genome shotgun approach was not used).
This involved 600 researchers in > 100 laboratories.
--Physical map created for all XVI chromosomes
--Library of 10 kb inserts constructed in phage
--The inserts were assembled into contigs
The sequence released in 1996, and published in 1997
(Goffeau et al., 1996; Mewes et al., 1997)

13. Features of the S. cerevisiae genome
Sequenced length: 12,068 kb = 12,068,000 base pairs
Length of repeats: 1,321 kb
Total length: 13,389 kb (~ 13 Mb)
Open reading frames (ORFs): 6,275
Questionable ORFs (qORFs): 390
Hypothetical proteins: 5,885
Introns in ORFs: 220
Introns in UTRs: 15
Intact Ty elements: 52
tRNA genes: 275
snRNA genes: 40

14. Features of the S. cerevisiae genome
A notable feature of the genome is its high gene density
(about one gene every 2 kilobases). Most bacteria have
about one gene per kb, but most eukaryotes have a
much sparser gene density.
Also, only 4% of S. cerevisiae genes are interrupted
by introns. By contrast, 40% of genes from the fungus
Schizosaccharomyces pombe have introns.

15. Features of S. cerevisiae genome
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Table 18.1
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ORF: open reading frame

16. ORFs in the S. cerevisiae genome
How are ORFs defined? In the initial genome analysis,
an ORF was defined as >100 codons (thus specifying
a protein of ~11 kilodaltons).
390 ORFs were listed as “questionable”, because they
were considered unlikely to be authentic genes.
For example, they were short, or exhibited unlikely
preferences for codon usage.
How many ORFs are there in the yeast genome?
There are 40,000 ORFs > 20 amino acids; how many
of these are authentic?

17. ORFs in the S. cerevisiae genome
Several criteria may be applied to decide if ORFs are
authentic protein-coding genes:
[1] evidence of conservation in other organisms
[2] experimental evidence of gene expression
(microarrays, SAGE, functional genomics)
The groups of Elizabeth Winzeler and Michael Snyder each described hundreds of previously unannotated
genes that are transcribed and translated.

18. Revising the S. cerevisiae gene count through comparative genomics
By sequencing three additional yeast species (Saccharomyces paradoxus, S. bayanus, S. mikatae), Kellis et al. showed that 503 genes should be deleted from the set of yeast genes (leaving 5,726 including 43 newly discovered genes).
See Kellis et al. (2003) Nature 423:241.

19. Proteins in S. cerevisiae 288c
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Visit NCBI Genome

20. Ten most common protein domains in S. cerevisiae
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From InterPro

21. S. cerevisiae has very few introns
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22. Exploring a typical S. cerevisiae chromosome (XII)
We will next familiarize ourselves with the S. cerevisiae
genome by exploring a typical chromosome, XII.
This chromosome features
38% GC content
very little repetitive DNA
few introns
six Ty elements (transposable elements)
a high ORF density: 534 ORFs > 100aa, and 72% of the
chromosome has protein-coding genes
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23. NCBI Map Viewer site: S. cerevisiae
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24. Saccharomyces Genome Database (SGD)
SGD is the main community web resource for studying Saccharomyces. We introduced it in Chapter 14.

25. Genome Workbench
Genome Workbench is a versatile tool from NCBI that includes a browser, a sophisticated query interface, and the opportunity to load files such as BAM and VCF.
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Search view of Genome Workbench: query Saccharomyces cerevisiae

26. Genome Workbench Genome Workbench view of genes on chromosome XII
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Genome Workbench view of genes on chromosome XII

27. Genome Workbench Additional tracks available on graphical view B&FG 3e
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Additional tracks available on graphical view

28. Genome Workbench display for a single gene on chromosome XII (VPS33/YLR396C)
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29. S. cerevisiae gene nomenclature
YKL159c
Y = yeast
K = 11th chromosome
L = left (or right) arm
159 = 159th ORF
c = Crick (bottom) strand
w = Watson (top) strand
RCN1 = wildtype gene
Rcn1p = protein
rcn1 = mutant allele
The research community studying S. cerevisiae established these nomenclature rules which are among the simplest and most informative in biology. From the name “YKL159c” you know the organism, chromosome and arm, ORF, strand, and molecule type.
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30. Ensembl includes resources for S. cerevisiae
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Resources include genome assembly data, comparative genomics, regulation, annotation, and variation

31. Ensembl includes resources for S. cerevisiae
In addition to yeast, Ensembl offers resources for a vast number of other genomes. The centralized resources of Ensembl are among the very most important in the field of genomics.
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Resources include genome assembly data, comparative genomics, regulation, annotation, and variation

32. Exploring chromosome (XII) on the command line
Visit ensembl.org to obtain a Genome Variation Format (GVF) file. Unpack it, then inspect it. There are ~260,000 rows.
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33. Exploring chromosome (XII) on the command line
The GVF file lists single nucleotide variants (SNVs) in the genome.
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34. Exploring chromosome (XII) on the command line
Send the data from chromosome XII to a new file called yeast_chrXII_SNVs.gvf. There are ~22,000 variants.
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35. Exploring chromosome (XII) on the command line
Next we’ll explore a file from ensembl.org that includes a variety of non-coding RNAs. You can download this as well. There are 1977 lines, and 413 separate entries.
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36. Exploring chromosome (XII) on the command line
Determine how many types of noncoding RNAs are present:
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37. Exploring chromosome (XII) on the command line
Restrict the output to chromosome XII, and view them:
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38. Outline Introduction Description and classification of fungi
Introduction to budding yeast Saccharomyces cerevisiae
Gene duplication and genome duplication
Comparative analyses of hemiascomycetes
Analysis of fungal genomes
Perspective

39. Duplication of the S. cerevisiae genome
Analysis of the S. cerevisiae genome revealed that many
regions are duplicated, both intrachromosomally and
interchromosomally (within and between chromosomes). These duplicated regions include both genes and nongenic regions.
Such duplications reflect a fundamental aspect of
genome evolution.
What are the mechanisms by which regions of the genome duplicate?

40. Duplication of the S. cerevisiae genome
Mechanisms of gene duplication
tandem repeat
slippage
during
recombination
Gene
conversion
Segmental
duplication
Lateral
gene
transfer
polyploidy
e.g.
genome
tetraploidy

41. Whole genome duplication
hypothetical diploid genome
Autopolyploidy (as occurs in S. cerevisiae):
8 chromosomes became 16!
Allopolyploidy: hybridization between related species
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42. Duplication of the S. cerevisiae genome
Fate of duplicated genes
Both
copies
persist
One
copy is
deleted
One copy
becomes a
pseudogene
One copy
functionally
diverges

43. Duplication of the S. cerevisiae genome
What is the fate of duplicated genes? (see YGOB, below)
A duplicated gene (overall in eukaryotes) has a half life
of just several million years (Lynch and Conery, 2000).
50% to 92% of duplicated genes are lost (Wagner, 2001)
Consider four possible fates of a duplicated gene:
[1] Both copies persist (gene dosage effect)
[2] One copy is deleted (a common fate)
[3] One copy accumulates mutations and becomes
a pseudogene (no functional protein product)
[4] One copy (or both) diverges functionally. The
organism can perform a novel function.

44. Duplication of the S. cerevisiae genome
In 1970, Susumu Ohno published the book Evolution by
Gene Duplication.
He hypothesized that vertebrate genomes evolved by
two rounds of whole genome duplication. This provided
genomes with the “raw materials” (new genes) with which
to introduce various innovations.

45. Duplication of the S. cerevisiae genome
Ohno (1970):
“Had evolution been entirely dependent upon natural
selection, from a bacterium only numerous forms of
bacteria would have emerged. The creation of metazoans,
vertebrates, and finally mammals from unicellular
organisms would have been quite impossible, for such
big leaps in evolution required the creation of new gene
loci with previously nonexistent function. Only the
cistron that became redundant was able to escape from
the relentless pressure of natural selection. By escaping,
it accumulated formerly forbidden mutations to emerge
as a new gene locus.”

46. Duplication of the S. cerevisiae genome
Wolfe and Shields (1997, Nature) provided support for Ohno’s paradigm. They hypothesized that the yeast genome duplicated about 100 million years ago. There was a diploid yeast genome with about 5,000 genes. It doubled to a tetraploid number of 10,000 genes. Then there was massive gene loss and chromosomal rearrangement to yield the present day 6,000 genes.

47. BLASTP of yeast chromosomes shows 55 blocks of duplicated regions: evidence for S. cerevisiae genome
duplication.
Matches with scores >200 are shown. These are arranged in blocks of genes.
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48. Duplication of the S. cerevisiae genome
Evidence of genome duplication in yeast
-- Systematic BLAST searches show 55 blocks
of duplicated sequences.
-- There are 376 pairs of homologous genes.
You can see the results of chromosomal comparisons
on Ken Wolfe’s web site (YGOG; see below for examples) and at the SGD web site.

49. Duplication of the S. cerevisiae genome
Two models for the presence of duplication blocks
[1] Whole genome duplication (tetraploidy) followed by
gene loss and rearrangements
[2] Successive, independent duplication events

50. Duplication of the S. cerevisiae genome
Model [1] is favored for several reasons:
-- For 50 of 55 duplicated regions, the orientation of the
entire block is preserved with respect to the centromere.
The orientation is not random.
-- For model [2] we would expect 7 triplicated regions.
We observe only 0 or 1.
-- Gene order is maintained in 14 hemiascomycetes
(the Génolevures project)
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51. Duplication of the S. cerevisiae genome
Why are duplicated genes commonly lost? It might seem
highly advantageous to have a second copy of gene,
thus permitting functional divergence.
Ohno suggested two reasons:
[1] After duplication, a deleterious mutation in one of the two genes might now persist. Without duplication, the individual would have been selected against by such a mutation.
[2] The presence of a new paralogous sequence could lead to unequal crossing over of homologous chromosomes
during meiosis.

52. Duplication of the S. cerevisiae genome
To consider the fate of duplicated genes, consider the example of genes involved in vesicle transport.
Vesicles carry cargo from one destination to another.
Proteins on vesicles (e.g. vesicle-associated membrane protein, VAMP; Snc1p in yeast) bind to proteins on target membranes (e.g. syntaxin in mammalian and other eukaryotic systems, or Sso1p in yeast).
In S. cerevisiae, genome duplication appears to be
responsible for the presence of two syntaxins
(SSO1 and SSO2) and two VAMPs (SNC1 and SNC2).

53. Duplication of the S. cerevisiae genome
Snc1p
Snc2p
Sso1p
Sso2p

54. Search for
information
on SSO1 (or any
yeast gene) at the
SGD website

55. The SGD record for SSO1 provides information on function

56. Duplication of the S. cerevisiae genome
The SGD website reveals that the SSO1 gene is nonessential (i.e. the null mutant is viable), but the double knockout of SSO1 and SSO1 is lethal. Thus, these paralogs may offer functional redundancy to the organism.
Also, these proteins could participate in distinct (but complementary) intracellular trafficking steps.

57. Outline Introduction Description and classification of fungi
Introduction to budding yeast Saccharomyces cerevisiae
Gene duplication and genome duplication
Comparative analyses of hemiascomycetes
Analysis of fungal genomes
Perspective

58. Phylogeny of yeasts showing whole genome duplication (red circle)
We can align fungal genomes and see evidence for whole genome duplication. We can date the occurrence of this duplication event.
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59. Comparative analyses of hemiascomycetes: Whole genome duplication
You can explore duplicated genome regions using the Yeast Gene Order Browser (YGOB) at:

60. Yeast Gene Order Browser
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61. Yeast Gene Order Browser
post-WGD tracks
pre-WGD tracks: species that did not undergo WGD
post-WGD tracks: mirror
those at top
patterns reflect of duplicated genes reflect whole genome duplication
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62. Yeast Gene Order Browser: patterns of gene loss after WGD

63. Patterns of gene loss after whole-genome duplication in three species
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64. Patterns of gene loss after whole-genome duplication in three species
For three species that underwent whole-geneome duplication (C. glabrata, S. cerevisiae, and S. castellii) there are 14 possible fates including
loss of no genes (class 0),
loss of one gene from any one of the three lineages (class 1A, 1B, 1C),
loss of two genes (class 2),
loss of three genes from different loci (class 3), or
loss of three genes in a convergent manner (class 4; loss of duplicated orthologs)(most common fate of duplicated genes).
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65. Comparative analyses of hemiascomycetes:
Identification of functional elements
Kellis et al. (2003) compared S. paradoxus, S. mikatae, and S. bayanus to S. cerevisiae (divergence dates: 5 to 20 MYA). There were clear orthologous matches, except at the telomeres.
For the Gal4 transcription factor and other functional elements, comparative analyses have helped delineate regulatory regions.

66. Yeast Gal4 transcription factor binding site (between GAL10, GAL1)
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67. Outline Introduction Description and classification of fungi
Introduction to budding yeast Saccharomyces cerevisiae
Gene duplication and genome duplication
Comparative analyses of hemiascomycetes
Analysis of fungal genomes
Perspective

68. Fungal genome projects: representative examples of the Ascomycetes
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ID refers to the NCBI genome project identifier

69. Fungal genome projects: representative examples of the Basidiomycetes
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ID refers to the NCBI genome project identifier

70. Fungal genome projects:
representative examples of fungi other than Ascomycetes and Basiciomycetes
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ID refers to the NCBI genome project identifier

71. Fungal genomes
We now briefly consider a set of prominent fungal genomes, arranged alphabetically:
Aspergillus nidulans
Candida albicans
Cryptococcus neoformans: model fungal pathogen
Encephalitozoon cuniculi: atypical fungus
Neurospora crassa
Phanerochaete chrysosporium: first Basidiomycete
Schizosaccharomyces pombe: fission yeast

72. Fungal pathogen: Aspergillus nidulans
--Of 185 Aspergillus species, 20 are human pathogens
--A. nidulans has a sexual life cycle (in contrast to A. fumigatus and A. oryzae [sake, miso, soy]).
--A. nidulans has animal-like peroxisomal enzymes

73. TaxPlot tool (NCBI) compares proteins in two proteomes

74. Fungal pathogen: Candida albicans
--Diploid sexually reproducing fungus
--Causes opportunistic infections in humans
--Genome: 14.8 Mb with 8 chromosome pairs. Seven of these are constant, and the 8th varies from 3 to 4 Mb.
--No known haploid state; the heterozygous diploid state was sequenced.
--Over 7600 open reading frames
--CUG is translated as serine (rather than leucine)

75. An atypical fungus: Encephalitozoon cuniculi
Microsporidia are single-celled eukaryotes that lack
mitochondria and peroxisomes. Consistent with their
roles as parasites, the E. cuniculi genome is severely
reduced in size (2000 proteins, only 2.9 Mb). They were
thought to represent deep-branching protozoans, but
recent phylogenetic studies place them as an outgroup
to fungi.

76. Fungal origin for the microsporidial parasite Encephalitozoon cuniculi (arrow)
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Phylogenetic analysis of vacuolar ATPase subunit A from animals, plants, fungi, protists, bacteria, and archaea

77. Mechanisms of reduction of genome size in microsporidia
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An ancestral genome is shown schematically with seven genes (blue, red) and large intergenic regions (black line).

78. Orange bread mold: Neurospora crassa
Beadle and Tatum chose N. crassa as a model organism
to study gene-protein relationships. The genome sequence
was reported: 39 Mb, 7 chromosomes, 10,082 ORFs
(Galagan et al., 2003).
N. crassa has only 10% repetitive DNA, and incredibly,
only 8 pairs of duplicated genes that encode proteins
>100 amino acids. This is because Neurospora uses
“repeat-induced point mutation” (RIP), a mechanism by
which the genome is scanned for duplicated (repeated)
sequences. This appears to serve as a genomic defense
system, inactivating potentially harmful transposons.

79. Phanerochaete chrysosporium: first Basidiomycete
Basidiomycota diverged from the better-characterized Ascomycota over 500 million years ago.
Genome: 30 Mb of DNA, 10 chromosomes.
11,777 genes predicted.
White rot fungi (such as P. chrysosporium) degrade the major components of plant cell walls, including cellulose and lignins, using a series of oxidases and peroxidases. The genome encodes hundreds of enzymes that are able to cleave carbohydrates.
Comparative analyses of 31 fungal genomes suggests the emergence of white rot with wood-decaying capabilities about 295 MYA.

80. Schizosaccharomyces pombe
The S. pombe genome is 13.8 Mb and encodes ~4900
predicted proteins. Some bacterial genomes encode
more proteins (e.g. Mesorhizobium loti with 6752,
and Streptomyces coelicolor with 7825 genes).
Chromosome Genes Coding
Mb 2, %
Mb 1, %
Mb %
Total Mb 4, %

81. Schizosaccharomyces pombe
S. pombe diverged from S. cerevisiae about
330 to 420 million years ago.
Many genes are as divergent between these
two fungi as they are diverged from humans.

82. Outline Introduction Description and classification of fungi
Introduction to budding yeast Saccharomyces cerevisiae
Gene duplication and genome duplication
Comparative analyses of hemiascomycetes
Analysis of fungal genomes
Perspective

83. Perspectives
The budding yeast S. cerevisiae is one of the most significant
organisms in biology:
Its genome is the first of a eukaryote to be sequenced
Its biology is simple relative to metazoans
Through yeast genetics, powerful functional genomics
approaches have been applied to study all yeast genes
It is important to note that even for yeast, our knowledge
of basic biological questions is highly incomplete. For humans and other eukaryotes we still understand relatively little about how the genotype of an organism leads to its characteristic phenotype. Studies in S. cerevisiae help elucidate these relationships.

84. Features of S. pombe genome
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