Gerald R. Fink

Ty elements transpose through an RNA intermediate

We have followed Ty transposition with a donor Ty element, TyH3, whose expression is under the control of the GAL1 promoter. Sequence analysis reveals dramatic structural differences in TyH3 before and after transposition. If the donor TyH3 is marked with an intron-containing fragment, the intron is correctly spliced out of the Ty during transposition, suggesting that the Ty RNA is the intermediate for transposition. Furthermore, the pattern of sequence inheritance in progeny Ty insertions derived from the marked Ty follows the predictions of the model of retroviral reverse transcription. Comparison of marked Ty elements before and after movement shows that transposition is highly mutagenic to the Ty element. These results demonstrate that during transposition, Ty sequence information flows from DNA to RNA to DNA.

Intragenic tandem repeats generate functional variability.

Tandemly repeated DNA sequences are highly dynamic components of genomes. Most repeats are in intergenic regions, but some are in coding sequences or pseudogenes. In humans, expansion of intragenic triplet repeats is associated with various diseases, including Huntington chorea and fragile X syndrome. The persistence of intragenic repeats in genomes suggests that there is a compensating benefit. Here we show that in the genome of Saccharomyces cerevisiae, most genes containing intragenic repeats encode cell-wall proteins. The repeats trigger frequent recombination events in the gene or between the gene and a pseudogene, causing expansion and contraction in the gene size. This size variation creates quantitative alterations in phenotypes (e.g., adhesion, flocculation or biofilm formation). We propose that variation in intragenic repeat number provides the functional diversity of cell surface antigens that, in fungi and other pathogens, allows rapid adaptation to the environment and elusion of the host immune system.

Antisense Transcription Controls Cell Fate in Saccharomyces cerevisiae

Entry into meiosis is a key developmental decision. We show here that meiotic entry in Saccharomyces cerevisiae is controlled by antisense-mediated regulation of IME4, a gene required for initiating meiosis. In MAT a/alpha diploids the antisense IME4 transcript is repressed by binding of the a1/alpha2 heterodimer at a conserved site located downstream of the IME4 coding sequence. MAT a/alpha diploids that produce IME4 antisense transcript have diminished sense transcription and fail to initiate meiosis. Haploids that produce the sense transcript have diminished antisense transcription and manifest several diploid phenotypes. Our data are consistent with transcription interference as a regulatory mechanism at the IME4 locus that determines cell fate.

Genotype to Phenotype: A Complex Problem

In yeast, the impact of gene knockouts depends on genetic background. We generated a high-resolution whole-genome sequence and individually deleted 5100 genes in Σ1278b, a Saccharomyces cerevisiae strain closely related to reference strain S288c. Similar to the variation between human individuals, Σ1278b and S288c average 3.2 single-nucleotide polymorphisms per kilobase. A genome-wide comparison of deletion mutant phenotypes identified a subset of genes that were conditionally essential by strain, including 44 essential genes unique to Σ1278b and 13 unique to S288c. Genetic analysis indicates the conditional phenotype was most often governed by complex genetic interactions, depending on multiple background-specific modifiers. Our comprehensive analysis suggests that the presence of a complex set of modifiers will often underlie the phenotypic differences between individuals.

Yeast: An Experimental Organism for 21st Century Biology

In this essay, we revisit the status of yeast as a model system for biology. We first summarize important contributions of yeast to eukaryotic biology that we anticipated in 1988 in our first article on the subject. We then describe transformative developments that we did not anticipate, most of which followed the publication of the complete genomic sequence of Saccharomyces cerevisiae in 1996. In the intervening 23 years it appears to us that yeast has graduated from a position as the premier model for eukaryotic cell biology to become the pioneer organism that has facilitated the establishment of the entirely new fields of study called “functional genomics” and “systems biology.” These new fields look beyond the functions of individual genes and proteins, focusing on how these interact and work together to determine the properties of living cells and organisms.

Global Gene Deletion Analysis Exploring Yeast Filamentous Growth

Infectious Phenotype The pathogenic yeast Candida albicans needs to adopt a filamentous form to invade tissues. The distantly related yeast species Saccharomyces cerevisiae also takes on a filamentous form for nutrient foraging. Comparing genome-wide deletion libraries between the two species, Ryan et al. (p. 1353) identified genes involved in three different filamentous yeast phenotypes and found unique genes for each of these phenotypes. However, in addition, core genes, including a previously unknown conserved regulator, appear to have homologous roles in regulating filamentous growth in these distantly related yeast species. Yeast genes involved in the dimorphic switch between cell budding and filamentous growth types are identified. The dimorphic switch from a single-cell budding yeast to a filamentous form enables Saccharomyces cerevisiae to forage for nutrients and the opportunistic pathogen Candida albicans to invade human tissues and evade the immune system. We constructed a genome-wide set of targeted deletion alleles and introduced them into a filamentous S. cerevisiae strain, Σ1278b. We identified genes involved in morphologically distinct forms of filamentation: haploid invasive growth, biofilm formation, and diploid pseudohyphal growth. Unique genes appear to underlie each program, but we also found core genes with general roles in filamentous growth, including MFG1 (YDL233w), whose product binds two morphogenetic transcription factors, Flo8 and Mss11, and functions as a critical transcriptional regulator of filamentous growth in both S. cerevisiae and C. albicans.

Control of Transcription by Cell Size

The demonstration of a causal, regulatory relationship between cell size and gene expression in yeast suggests that cells maintain size in order to maintain transcriptional homeostasis.

Feed-Forward Regulation of a Cell Fate Determinant by an RNA-Binding Protein Generates Asymmetry in Yeast

Saccharomyces cerevisiae can divide asymmetrically so that the mother and daughter cells have different fates. We show that the RNA-binding protein Khd1 regulates asymmetric expression of FLO11 to determine daughter cell fate during filamentous growth. Khd1 represses transcription of FLO11 indirectly through its regulation of ASH1 mRNA. Khd1 also represses FLO11 through a post-transcriptional mechanism independent of ASH1. Cross-linking immunoprecipitation (CLIP) coupled with high-throughput sequencing shows that Khd1 directly binds repetitive sequences in FLO11 mRNA. Khd1 inhibits translation through this interaction, establishing feed-forward repression of FLO11. This regulation enables changes in FLO11 expression between mother and daughter cells, which establishes the asymmetry required for the developmental transition between yeast form and filamentous growth.

Interactions between chromosomal and nonchromosomal elements reveal missing heritability

Significance We show that the phenotype of an organism can be affected both by chromosomal and nonchromosomal information. The influence of the cytoplasmic, nonchromosomal information can have a profound effect on the phenotype and can be great enough to mask the effect of a chromosomal mutation. Our ability to quantify the contribution of this cytoplasmic component shows that it could comprise a significant portion of the “missing heritability.” In addition, our findings highlight the possibility of mitochondrial/nuclear incompatibility, which could be an important consideration in evaluating the feasibility of mitochondrial replacement therapy in humans. The measurement of any nonchromosomal genetic contribution to the heritability of a trait is often confounded by the inability to control both the chromosomal and nonchromosomal information in a population. We have designed a unique system in yeast where we can control both sources of information so that the phenotype of a single chromosomal polymorphism can be measured in the presence of different cytoplasmic elements. With this system, we have shown that both the source of the mitochondrial genome and the presence or absence of a dsRNA virus influence the phenotype of chromosomal variants that affect the growth of yeast. Moreover, by considering this nonchromosomal information that is passed from parent to offspring and by allowing chromosomal and nonchromosomal information to exhibit nonadditive interactions, we are able to account for much of the heritability of growth traits. Taken together, our results highlight the importance of including all sources of heritable information in genetic studies and suggest a possible avenue of attack for finding additional missing heritability.

Discovering regulatory overlapping RNA transcripts.

STEREO is a novel algorithm that discovers cis-regulatory RNA interactions by assembling complete and potentially overlapping same-strand RNA transcripts from tiling expression data. STEREO first identifies coherent segments of transcription and then discovers individual transcripts that are consistent with the observed segments given intensity and shape constraints. We used STEREO to identify 1446 regions of overlapping transcription in two strains of yeast, including transcripts that comprise a new form of molecular toggle switch that controls gene variegation.