Yeast Gene Family Research Articles

Unique trajectory of gene family evolution from genomic analysis of nearly all known species in an ancient yeast lineage.

Gene gains and losses are a major driver of genome evolution; their precise characterization can provide insights into the origin and diversification of major lineages. Here, we examined gene family evolution of 1,154 genomes from nearly all known species in the medically and technologically important yeast subphylum Saccharomycotina. We found that yeast gene family and genome evolution are distinct from plants, animals, and filamentous ascomycetes and are characterized by small genome sizes and smaller gene numbers but larger gene family sizes. Faster-evolving lineages (FELs) in yeasts experienced significantly higher rates of gene losses-commensurate with a narrowing of metabolic niche breadth-but higher speciation rates than their slower-evolving sister lineages (SELs). Gene families most often lost are those involved in mRNA splicing, carbohydrate metabolism, and cell division and are likely associated with intron loss, metabolic breadth, and non-canonical cell cycle processes. Our results highlight the significant role of gene family contractions in the evolution of yeast metabolism, genome function, and speciation, and suggest that gene family evolutionary trajectories have differed markedly across major eukaryotic lineages.

Empirical evidence that complexity limits horizontal gene transfer.

Horizontal gene transfer (HGT) is a major contributor to bacterial genome evolution, generating phenotypic diversity, driving the expansion of protein families, and facilitating the evolution of new phenotypes, new metabolic pathways, and new species. Comparative studies of gene gain in bacteria suggest that the frequency with which individual genes successfully undergo HGT varies considerably and may be associated with the number of protein-protein interactions in which the gene participates, i.e., its connectivity. Two non-exclusive hypotheses have emerged to explain why transferability should decrease with connectivity: the Complexity Hypothesis (Jain, Rivera, & Lake, 1999) and the Balance Hypothesis (Papp, Pál, & Hurst, 2003). These hypotheses predict that the functional costs of HGT arise from a failure of divergent homologues to make normal protein-protein interactions or from gene mis-expression, respectively. Here we describe genome-wide assessments of these hypotheses in which we used 74 existing prokaryotic whole genome shotgun libraries to estimate rates of horizontal transfer of genes from taxonomically diverse prokaryotic donors into E. coli. We show that 1) transferability declines as connectivity increases, 2) transferability declines as the divergence between donor and recipient orthologs increases, and that 3) the magnitude of this negative effect of divergence on transferability increases with connectivity. These effects are particularly robust among the translational proteins, which span the widest range of connectivities. Whereas the Complexity Hypotheses explains all three of these observations, the Balance Hypothesis explains only the first one.

Predominant Gain of Promoter TATA Box after Gene Duplication Associated with Stress Responses

TATA box, the core promoter element, exists in a broad range of eukaryotes, and the expression of TATA-containing genes usually responds to various environmental stresses. Hence, the evolution of TATA-box in duplicate genes may provide some clues for the interrelationship among environmental stress, expression differentiation, and duplicate gene preservation. In the present study, we observed that the TATA box is significantly overrepresented in duplicate genes compared with singletons in human, worm, Arabidopsis, and yeast genomes. We then conducted an extensive functional genomic analysis to investigate the evolution of TATA box along over 700 yeast gene family phylogenies. After reconstructing the ancestral TATA-box states (presence or absence), we found that significantly higher numbers of TATA box gain events than loss events had occurred after yeast gene duplications-the overall gain-loss ratio is about 3-4 to 1. Interestingly, these TATA-gain duplicate genes on average have experienced greater expression divergence from the ancestral expression states than their most closely related TATA-less duplicate partners, but only under environmental stress conditions (asymmetric evolution); indeed, under normal physiological conditions, they have similar expression divergence (symmetric evolution). Moreover, we showed that TATA-gain duplicates are enriched in stress-associated functional categories but that is not the case for TATA-ancestral duplicates (those inherited from their ancestors prior to duplication). Together, we conclude that after the gene duplication, gain of the TATA box in duplicate promoters may have played an important role in yeast duplicate preservation by accelerating expression divergence that may facilitate the adaptive evolution of the organism in response to environmental changes.

Dosage sensitivity and the evolution of gene families in yeast

According to what we term the balance hypothesis, an imbalance in the concentration of the subcomponents of a protein-protein complex can be deleterious. If so, there are two consequences: first, both underexpression and overexpression of protein complex subunits should lower fitness, and second, the accuracy of transcriptional co-regulation of subunits should reflect the deleterious consequences of imbalance. Here we show that all these predictions are upheld in yeast (Saccharomyces cerevisiae). This supports the hypothesis that dominance is a by-product of physiology and metabolism rather than the result of selection to mask the deleterious effects of mutations. Beyond this, single-gene duplication of protein subunits is expected to be harmful, as this, too, leads to imbalance. As then expected, we find that members of large gene families are rarely involved in complexes. The balance hypothesis therefore provides a single theoretical framework for understanding components both of dominance and of gene family size.

Genomic and proteomic analysis of mitochondrial carrier proteins in Arabidopsis.

Plant mitochondria maintain metabolic communication with the cytosol through a family of carrier proteins. In Arabidopsis, a subset of 45 putative genes encoding members of this family have been identified based on generalized mitochondrial carrier features. No gene clusters are apparent and few of the predicted protein products have mitochondrial targeting sequences recognized by bioinformatic predictors. Only nine genes are currently represented by more than 10 expressed sequence tags at The Institute for Genomic Research. Analyses of public microarray experiments reveal differential expression profiles of the more highly expressed members of this gene family in different plant organs and in response to plant hormone application and environmental stresses. A comparison of this Arabidopsis carrier subset (45) to the yeast gene family (35) reveals 10 orthologous groups between the two species. Recent surveys of the Arabidopsis mitochondrial proteome by two-dimensional gel separations have not identified any of these carrier proteins, presumably because of their hydrophobicity and basicity. Isolating integral membrane proteins from Arabidopsis mitochondria, using one-dimensional electrophoresis for protein separation and tandem mass spectrometry-based sequencing of doubly charged peptides, we have unequivocally identified specific carrier gene products located in mitochondria. This approach has identified six of the nine carriers represented highly in expressed sequence tag databases: adenine nucleotide translocator (At3g8580 and At5g13490), dicarboxylate/tricarboxylate carrier (At5g19760), phosphate carrier (At5g14040), uncoupling protein (At3g54110), and a carrier gene of unknown function (At4g01100). Overall, the combined transcript and protein expression data indicates that only a small subset of the carrier family of genes provide the majority of carrier proteins of Arabidopsis mitochondria.

Identification and Characterization of a Low Oxygen Response Element Involved in the Hypoxic Induction of a Family ofSaccharomyces cerevisiae Genes: IMPLICATIONS FOR THE CONSERVATION OF OXYGEN SENSING IN EUKARYOTES

An organism's ability to respond to changes in oxygen tension depends in large part on alterations in gene expression. The oxygen sensing and signaling mechanisms in eukaryotic cells are not fully understood. To further define these processes, we have studied the Delta9 fatty acid desaturase gene OLE1 in Saccharomyces cerevisiae. We have confirmed previous data showing that the expression of OLE1 mRNA is increased in hypoxia and in the presence of certain transition metals. OLE1 expression was also increased in the presence of the iron chelator 1,10-phenanthroline. A 142-base pair (bp) region 3' to the previously identified fatty acid response element was identified as critical for the induction of OLE1 in response to these stimuli using OLE1 promoter-lacZ reporter constructs. Electromobility shift assays confirmed the presence of an inducible band shift in response to hypoxia and cobalt. Mutational analysis defined the nonameric sequence ACTCAACAA as necessary for transactivation. A 20-base pair oligonucleotide containing this nonamer confers up-regulation by hypoxia and inhibition by unsaturated fatty acids when placed upstream of a heterologous promoter in a lacZ reporter construct. Additional yeast genes were identified which respond to hypoxia and cobalt in a manner similar to OLE1. A number of mammalian genes are also up-regulated by hypoxia, cobalt, nickel, and iron chelators. Hence, the identification of a family of yeast genes regulated in a similar manner has implications for understanding oxygen sensing and signaling in eukaryotes.

Functional Characterization of the YUR1, KTR1, and KTR2 Genes as Members of the Yeast KRE2/MNT1 Mannosyltransferase Gene Family

Eukaryotic glycan structures are progressively elaborated in the secretory pathway. Following the addition of a core N-linked carbohydrate in the endoplasmic reticulum, glycoproteins move to the Golgi complex where the elongation of O-linked sugar chains and processing of complex N-linked oligosaccharide structures take place. In order to better define how such post-translational modifications occur, we have been studying a yeast gene family in which at least one member, KRE2/MNT1, is involved in protein glycosylation. The family currently contains five other members: YUR1, KTR1, KTR2, KTR3 and KTR4 (Mallet, L., Bussereau, F., and Jacquet, M. (1994) Yeast 10, 819-831). All encode putative type II membrane proteins with a short cytoplasmic N terminus, a membrane-spanning region, and a highly conserved catalytic lumenal domain. Kre2p/Mnt1p is a alpha 1,2-mannosyltransferase involved in O- and N-linked glycosylation (Häusler, A., Ballou, L., Ballou, C.E., and Robbins, P.W. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 6846-6850); however, the role of the other proteins has not yet been established. We have carried out a functional analysis of Ktr1p, Ktr2p, and Yur1p. By in vitro assays, Ktr1p, Ktr2p, and Yur1p have been shown to be mannosyltransferase but, in vivo, do not appear to be involved in O-glycosylation. Examination of the electrophoretic mobility of the N-linked modified protein invertase in null mutant strains indicates that Ktr1p, Ktr2p, and Yur1p are involved in N-linked glycosylation, possibly as redundant enzymes. As found with Kre2p (Hill, K., Boone, C., Goebl, M., Puccia, R., Sdicu, A.-M., and Bussey, H. (1992) Genetics 130, 273-283), Ktr1p, Ktr2p, and Yur1p also seem to be implicated in the glycosylation of cell wall mannoproteins, since yeast cells containing different gene disruptions become K1 killer toxin-resistant. Immunofluorescence microscopy reveals that like Kre2p; Ktr1p, Ktr2p and Yur1p are localized in the Golgi complex.

Gene trap tagging of PROLIFERA, an essential MCM2-3-5-like gene in Arabidopsis.

Gene trap transposon mutagenesis can identify essential genes whose functions in later development are obscured by an early lethal phenotype. In higher plants, many genes are required for haploid gametophyte viability, so that the phenotypic effects of their disruption cannot be readily observed in the diploid plant body. The PROLIFERA (PRL) gene, identified by gene trap transposon mutagenesis in Arabidopsis, is required for megaga-metophyte and embryo development. Reporter gene expression patterns revealed that PRL was expressed in dividing cells throughout the plant. PRL is related to the MCM2-3-5 family of yeast genes that are required for the initiation of DNA replication.

A new family of yeast genes implicated in ergosterol synthesis is related to the human oxysterol binding protein

We have identified three yeast genes, KES1, HES1 and OSH1, whose products show homology to the human oxysterol binding protein (OSBP). Mutations in these genes resulted in pleiotropic sterol-related phenotypes. These include tryptophan-transport defects and nystatin resistance, shown by double and triple mutants. In addition, mutant combinations showed small but apparently cumulative reductions in membrane ergosterol levels. The three yeast genes are also functionally related as overexpression of HES1 or KES1 alleviated the tryptophan-transport defect in kes1 delta or osh1 delta mutants, respectively. Our study implicates this new yeast gene family in ergosterol synthesis and provides comparative evidence of a role for human OSBP in cholesterol synthesis.

Transcriptional control of AAC3 gene encoding mitochondrial ADP/ATP translocator in Saccharomyces cerevisiae by oxygen, heme and ROX1 factor

The AAC3 gene of Saccharomyces cerevisiae encodes a mitochondrial ADP/ATP translocator which is subject to oxygen repression. Evidence is presented here, that the repression of AAC3 expression is dependent upon heme and the ROX1 factor. The promoter region of the AAC3 gene was isolated, sequenced, and deletion analysis was performed using lacZ as a reporter gene to determine the cis-acting regions responsible for the regulation of AAC3 expression. The results of the deletion analysis show that the negative control of the AAC3 gene by oxygen and ROX1 factor is mediated by an upstream repression sequence consisting of a T-rich segment adjacent to the consensus elements that are present in the 5' flanking regions of several other yeast genes. An additional upstream repressor site was located within the AAC3 promoter which, however, is not related either to oxygen or to ROX1 factor. The data presented here delineate the main cellular factors and DNA sequences involved in the regulatory mechanism by which an essential function for anaerobic cells growth, ADP/ATP translocation, is ensured. In addition, they show that the AAC3 gene belongs to the family of yeast genes including TIF51B, COX5b, HEM13 and CYC7 that are negatively regulated by oxygen and heme.

Yeast KRE2 defines a new gene family encoding probable secretory proteins, and is required for the correct N-glycosylation of proteins.

We have cloned, sequenced and disrupted the KRE2 gene of Saccharomyces cerevisiae, identified by killer-resistant mutants with a defective cell wall receptor for the toxin. The KRE2 gene is close to PHO8 on chromosome 4, and encodes a predicted 49-kD protein, Kre2p, that probably enters the secretory pathway. Haploid cells carrying a disruption of the KRE2 locus grow more slowly than wild-type cells at 30 degrees, and fail to grow at 37 degrees. At 30 degrees, kre2 mutants showed altered N-linked glycosylation of proteins, as the average size of N-linked outer chains was reduced. We identified two other genes, YUR1 on chromosome 10, and KTR1 on chromosome 15, whose predicted products share 36% identity with Kre2p over more than 300 amino acid residues. Yur1p has an N-terminal signal sequence like Kre2p, while Ktr1p has a predicted topology consistent with a type 2 membrane protein. In all cases the conserved regions of these proteins appear to be on the lumenal side of secretory compartments, suggesting related function. KRE2, KTR1 and YUR1 define a new yeast gene family.

The crooked neck gene of Drosophila contains a motif found in a family of yeast cell cycle genes.

The crooked neck (crn) gene of Drosophila encodes a protein of 702 amino acids and contains 16 tandemly arranged copies of a 34-amino-acid repeat that is similar to the tetratrico peptide repeat (TPR). Multiple copies of the TPR motif have also been found in a family of yeast genes, including several members that are necessary for cell division. TPR-containing proteins encoded by the yeast genes CDC16, CDC23, and nuc2+ are required for progression through the G2/M transition of the cell cycle. Loss of zygotic expression of crn causes defects in the proliferation of brain neuroblasts and results in the absence of identified neuronal lineages in the central and peripheral nervous systems. The sequence similarity and mutant phenotypes are consistent with a cell cycle requirement for the crn gene product.

A homologue of the bacterial heat-shock gene DnaJ that alters protein sorting in yeast

Heat-shock proteins have been implicated in assembly of protein complexes, correct protein folding and uptake of proteins into organelles. In Escherichia coli, the heat-shock protein DnaJ and the Hsp70 homologue, DnaK, act together to disassemble a protein complex involved in bacteriophage lambda replication. We report the identification of SCJ1, a gene in the yeast Saccharomyces cerevisiae that encodes a homologue of the bacterial DnaJ protein. SCJ1 was identified by a genetic screen in which increased expression of candidate genes results in missorting of a nuclear-targeted test protein. The predicted amino-acid sequence of SCJ1 is 37% identical to the entire E. coli DnaJ protein. Hybridization experiments indicate that there is a family of yeast genes related to SCJ1. These findings suggest that the Hsp70 DnaK-DnaJ interaction is general to eukaryotes.

Comparison of the nucleotide sequences of a yeast gene family: II. Analysis of spontaneous deletions and insertions

We compared the nucleotide sequences of 3 yeast invertase genes in regions where the homology is better than 90%. In the noncoding region 40 gaps of 1–61 bases were found. This is about half as much as the nucleotide substitutions in the same sequences. We grouped the gaps into 5 categories by their length and the characteristics of their sequences. Group I gaps are about 20 nucleotides long and are flanked by repeated sequence of 6 bases which may trigger the deletion of one of the repeats and the sequence between the repeats. Group II gaps are characterized by a small repeated sequence which is missing in one of the invertase genes. Gaps which occur in sequences exclusively made up of one of the 4 bases are summarized in group III. The 4 gaps in group IV do not show any of these sequence characteristics and they are all just one base long. A 61 nucleotide sequence found in only one of the invertase genes seems to be of complex origin. We conclude that small repeated sequences or monotonous sequences are prone to deletion or insertion mutations.

Comparison of the nucleotide sequences of a yeast gene family: I. Distribution and spectrum of spontaneous base substitutions

The nucleotide sequences of closely related members of a gene family can be used to investigate spontaneous mutations. Here we analyse the sequences of different yeast invertase genes which are more than 93% identical in the coding region and share some very similar, but not identical sequences in the noncoding flanking regions. Since all except one of the invertase genes are active, most of the base substitutions are silent. Within the coding region the base substitutions are unevenly distributed, indicating that parts of the genes were homogenized, probably via gene conversion. Transitions occurred more frequently than transversions in both, coding and noncoding regions. In the coding region pyrimidine transitions were the most abundant event due to silent changes mainly in the third codon position. In the noncoding region pyrimidine and purine transitions were found at equal frequencies. Transversions inverting base pairs (A-T and G-C) outnumber transversions changing base pairs (A-C and G-T). While the spectrum of mutations in the coding region is influenced by selective pressure to maintain the amino acid sequence, the spectrum in the noncoding region may be much less affected by selective pressure.