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Chapter 3 : Postgenomic Strategies for Genetic Analysis: Insight from and

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Postgenomic Strategies for Genetic Analysis: Insight from and , Page 1 of 2

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Abstract:

This chapter discusses the choice of genes for study in the postgenomic era. One major limitation of using a host organism as a model for the heterologous expression of a gene of interest in another organism is differences in genetic coding between the two organisms. This problem, for example, exists when using as a host for genes, where the codon CUG specifies leucine in but serine in ; thus, proteins may be nonfunctional when translated in . The idea of considering several different screens is an extrapolation from the well-accepted idea that many different phenotypes contribute to the virulence of a pathogen. When an insertion mutant is found with a phenotype of interest, it is verified that the insertion itself is the cause of the phenotype by three approaches. First, multiple independent insertion mutant isolates are tested, all of which should have the same phenotype. Second, the insertion mutation is complemented with a clone of the complete ORF and flanking sequences, which should restore a wild-type phenotype. Third, engineered Δorf/orf Δ deletion strains are created with two successive transformations rather than an allelic recombination step; the deletion mutant is expected to have a phenotype similar to the initial insertion mutant. A study revealed two different kinds of phenotypic defects, thus suggesting that genetic analysis might contribute to dissection of a biofilm developmental pathway.

Citation: Nobile C, Mitchell A. 2006. Postgenomic Strategies for Genetic Analysis: Insight from and , p 35-44. In Heitman J, Filler S, Edwards, Jr. J, Mitchell A (ed), Molecular Principles of Fungal Pathogenesis. ASM Press, Washington, DC. doi: 10.1128/9781555815776.ch3

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Figures

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Figure 1.

Frequencies of mutants with severe growth defects under various selection conditions. Mutant growth properties ( ) are grouped by gene categories. Selection conditions and the severity of defects are as described in Table 1 . Gene categories include a representative random group (Chr7+Chr3), a group about which little is known (UKUK), the entire set of nonessential genes (All), and those with biochemically informative sequence motifs (TxnF+MbP+PrK+CWP). Abbreviations are described in Table 1 .

Citation: Nobile C, Mitchell A. 2006. Postgenomic Strategies for Genetic Analysis: Insight from and , p 35-44. In Heitman J, Filler S, Edwards, Jr. J, Mitchell A (ed), Molecular Principles of Fungal Pathogenesis. ASM Press, Washington, DC. doi: 10.1128/9781555815776.ch3
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Image of Figure 2.
Figure 2.

(A) Properties of the cassette. is a functional gene flanked by nonfunctional Δ’ and Δ’ deletion alleles. The alleles are in direct repeat orientation and share 500 bp of homology, so they can undergo recombination. Recombination between the alleles yields a functional gene and excises (B) Double-disruption selection. Transformation of strain BWP17 with results in expression of and confers an Arg+ phenotype. If the insertion becomes homozygous through mitotic recombination or gene conversion, then a second recombination event within one cassette yields an Arg+ Ura+ segregant. These Arg+ Ura+ segregants are of two classes. One class carries a insertion allele and a insertion allele. We refer to these segregants as homozygotes. The other class carries three copies of the gene: a insertion allele, a insertion allele, and a wild-type allele. We refer to these segregants as allelic triplication derivatives. A trisomic genetic structure is diagrammed for simplicity, but their precise genetic structure has not been determined.

Citation: Nobile C, Mitchell A. 2006. Postgenomic Strategies for Genetic Analysis: Insight from and , p 35-44. In Heitman J, Filler S, Edwards, Jr. J, Mitchell A (ed), Molecular Principles of Fungal Pathogenesis. ASM Press, Washington, DC. doi: 10.1128/9781555815776.ch3
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Tables

Generic image for table
Table 1.

Distribution of growth-defective mutants under various selection conditions

Citation: Nobile C, Mitchell A. 2006. Postgenomic Strategies for Genetic Analysis: Insight from and , p 35-44. In Heitman J, Filler S, Edwards, Jr. J, Mitchell A (ed), Molecular Principles of Fungal Pathogenesis. ASM Press, Washington, DC. doi: 10.1128/9781555815776.ch3
Generic image for table
Table 2.

Effect of reduced selection stringency on mutant detection

Citation: Nobile C, Mitchell A. 2006. Postgenomic Strategies for Genetic Analysis: Insight from and , p 35-44. In Heitman J, Filler S, Edwards, Jr. J, Mitchell A (ed), Molecular Principles of Fungal Pathogenesis. ASM Press, Washington, DC. doi: 10.1128/9781555815776.ch3

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