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Chapter 41 : Future of Functional Genomics of

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Abstract:

This chapter describes (i) the previous use of functional genomics in , (ii) the current status of genome-sequencing projects for this organism, (iii) potential annotation of the genome by using tiling microarrays, and (iv) future uses of functional genomics to dissect biology. Researcher generated a shotgun genomic DNA microarray for even though very little sequence information was available for this organism at the time. Differentially expressed genes can be categorized based on the function of their BLAST homologs in other organisms. Future gene expression profiling experiments using mutants that are trapped in a particular morphology independent of temperature may distinguish morphology-regulated genes from temperature-regulated genes. Functional genomics, through the use of wholegenome microarrays and related technologies, will open up genome-wide experimental approaches for the study of many aspects of the biology of . Functional genomics can also be used to identify potential virulence factors by subjecting to environmental conditions that mimic those experienced during infection. Functional genomics identifies candidate genes and suggests function. To ascertain the true function of a particular gene, further examination and experimentation is necessary. A number of molecular genetic techniques are available for in order to explore the role of genes identified through functional genomic screens. In addition, comparative genomic analysis of and other fungi will contribute to our understanding of the diversity of the fungal kingdom.

Citation: Sil A, Hwang L. 2006. Future of Functional Genomics of , p 611-625. In Heitman J, Filler S, Edwards, Jr. J, Mitchell A (ed), Molecular Principles of Fungal Pathogenesis. ASM Press, Washington, DC. doi: 10.1128/9781555815776.ch41

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Figures

Image of Figure 1
Figure 1

Life cycle of . This figure illustrates the various stages of the life cycle in the soil and in the host. The mycelial form grows in the soil (or at 25°C in the laboratory). It can mate and undergo meiosis, forming ascospores. The mycelia can also produce vegetative conidiospores through the process of conidiation, generating at least two types of conidiospores, macroconidia and microconidia. The conidia can germinate into either the mycelial form or the yeast form. Infection occurs when the soil is disrupted and the host inhales aerosolized conidia or hyphal fragments. These cells convert to the yeast form inside the host. The yeast form is infectious if introduced into an animal in the laboratory, but there is normally no host-to-host transmission of the yeast form. Schematic courtesy of Davina Hocking Murray.

Citation: Sil A, Hwang L. 2006. Future of Functional Genomics of , p 611-625. In Heitman J, Filler S, Edwards, Jr. J, Mitchell A (ed), Molecular Principles of Fungal Pathogenesis. ASM Press, Washington, DC. doi: 10.1128/9781555815776.ch41
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Image of Figure 2
Figure 2

Construction of a shotgun genomic microarray. A schematic of genomic DNA is shown at the top of the figure. The genomic DNA was partially digested with Sau3AI, size selected, cloned into pBluescript, and transformed into . Approximately 10,000 independent colonies were inoculated into 96-well plates, and these cultures were subjected to colony PCR with common primers in the vector. The resultant PCR products were spotted on glass slides to generate shotgun genomic microarrays. Reprinted from reference with permission.

Citation: Sil A, Hwang L. 2006. Future of Functional Genomics of , p 611-625. In Heitman J, Filler S, Edwards, Jr. J, Mitchell A (ed), Molecular Principles of Fungal Pathogenesis. ASM Press, Washington, DC. doi: 10.1128/9781555815776.ch41
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Image of Figure 3
Figure 3

Yeast- or mycelial-specific gene expression. (A) The histogram depicts the number of spots on the microarray ( axis) versus the log of the ratio of the mycelial signal to the yeast signal ( axis). A log ratio of zero indicates spots that are equivalently expressed. The numbers of clones that show differential expression from 5- to 100-fold in one morphologic form compared to the other are labeled in the figure. (B) Northern blot analysis of gene expression in yeast and mycelia. Total RNA from yeast (Y) is in the left lane, and total RNA from mycelia (M) is in the right lane. (actin) is equivalently expressed between the two RNA samples. (calcium binding protein), 63G8 (unknown microarray clone), , and are yeast specific by microarray and Northern analysis. 94B7 (unknown microarray clone) and are mycelium specific by microarray and Northern analysis. Reprinted from reference with permission.

Citation: Sil A, Hwang L. 2006. Future of Functional Genomics of , p 611-625. In Heitman J, Filler S, Edwards, Jr. J, Mitchell A (ed), Molecular Principles of Fungal Pathogenesis. ASM Press, Washington, DC. doi: 10.1128/9781555815776.ch41
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Figure 4

Putative functions for differentially expressed genes identified by functional genomics. The figure shows cells growing in either the yeast form at 37°C or the mycelial form at 25°C. Genes were annotated based on homology and categorized based on the function of their ortholog in other organisms. A number of yeast-specific genes were annotated as potentially being involved in sulfur metabolism and growth rate/host survival. Mycelial-specific genes were implicated in polarized cell growth, melanin production, soil survival, and conidiation. Adapted from reference with permission.

Citation: Sil A, Hwang L. 2006. Future of Functional Genomics of , p 611-625. In Heitman J, Filler S, Edwards, Jr. J, Mitchell A (ed), Molecular Principles of Fungal Pathogenesis. ASM Press, Washington, DC. doi: 10.1128/9781555815776.ch41
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Figure 5

Schematic of tiling array. Spots from a shotgun genomic microarray that were induced in nitrosative stress and did not have a simple correspondence to a putative gene were selected for tiling. In the example shown, the spot overlaps two putative hits from the National Center for Biotechnology Information nonredundant database (nr hit 1 and nr hit 2). A region that extends 1 kb beyond the boundaries of the nr hit homologies was tiled as end-to-end 50-mer oligonucleotides.

Citation: Sil A, Hwang L. 2006. Future of Functional Genomics of , p 611-625. In Heitman J, Filler S, Edwards, Jr. J, Mitchell A (ed), Molecular Principles of Fungal Pathogenesis. ASM Press, Washington, DC. doi: 10.1128/9781555815776.ch41
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Figure 6

Gene expression analysis using tiling arrays allows definition of gene boundaries. Microarray spot C3F12 is a spot on the shotgun genomic array that is upregulated consistently in nitrosative stress. It does not overlap with any BLAST hits or sequenced cDNAs. The locations of (i) C3F12, (ii) an nr hit from (ubiquitin [Ubi4p]), and (iii) an unknown cDNA from the genome project are all mapped onto MiniContig 24. The relevant region of MiniContig 24 was tiled on a Combimatrix array as end-to-end 50-mers. This array was subjected to a competitive hybridization with differentially labeled probes (Cy3 [gray] was used to label cDNA generated from control cells, whereas Cy5 [black] was used to label cDNA generated from wild-type cells treated with reactive nitrogen intermediates [RNI]). Each vertical bar represents signal intensity obtained for the tile at that position. Both the plus and minus strands were tiled. The data indicate that the induced gene of interest is present on the plus strand and that the 5’ gene boundary extends significantly upstream of the homology with Ubi4p. The location of putative introns is revealed by transient interruptions in the signal intensity.

Citation: Sil A, Hwang L. 2006. Future of Functional Genomics of , p 611-625. In Heitman J, Filler S, Edwards, Jr. J, Mitchell A (ed), Molecular Principles of Fungal Pathogenesis. ASM Press, Washington, DC. doi: 10.1128/9781555815776.ch41
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Image of Figure 7
Figure 7

infection of RAW264.7 cells. Monolayers of macrophages were infected with . After 34 h, the monolayer was fixed and fungal cells were stained with periodic acid–Schiff base. The two macrophages shown are filled with cells. Image courtesy of Dervla Isaac.

Citation: Sil A, Hwang L. 2006. Future of Functional Genomics of , p 611-625. In Heitman J, Filler S, Edwards, Jr. J, Mitchell A (ed), Molecular Principles of Fungal Pathogenesis. ASM Press, Washington, DC. doi: 10.1128/9781555815776.ch41
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Image of Figure 8
Figure 8

G186AR macroconidia. The G186AR strain was grown under conidiating conditions. The image shows hyphae and tuberculate macroconidia. Image courtesy of Diane Inglis.

Citation: Sil A, Hwang L. 2006. Future of Functional Genomics of , p 611-625. In Heitman J, Filler S, Edwards, Jr. J, Mitchell A (ed), Molecular Principles of Fungal Pathogenesis. ASM Press, Washington, DC. doi: 10.1128/9781555815776.ch41
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