Genetics最新文献

Azole resistance in the pathogenic yeast Candida glabrata is a serious clinical complication and increasing in frequency. The majority of resistant organisms have been found to contain a substitution mutation in the Zn2Cys6 zinc cluster-containing transcription factor Pdr1. These mutations typically lead to this factor driving high, constitutive expression of target genes like the ATP-binding cassette transporter-encoding gene CDR1. Overexpression of Cdr1 is required for the observed elevated fluconazole resistance exhibited by strains containing one of these hyperactive PDR1 alleles. While the identity of hyperactive PDR1 alleles has been extensively documented, the mechanisms underlying how these gain-of-function (GOF) forms of Pdr1 lead to elevated target gene transcription are not well understood. We have used a tandem affinity purification-tagged form of Pdr1 to identify coactivator proteins that biochemically purify with the wild-type and 2 different GOF forms of Pdr1. Three coactivator proteins were found to associate with Pdr1: the SWI/SNF complex Snf2 chromatin remodeling protein and 2 different components of the SAGA complex, Spt7 and Ngg1. We found that deletion mutants lacking either SNF2 or SPT7 exhibited growth defects, even in the absence of fluconazole challenge. To overcome these issues, we employed a conditional degradation system to acutely deplete these coactivators and determined that loss of either coactivator complex, SWI/SNF or SAGA, caused defects in Pdr1-dependent transcription. A double degron strain that could be depleted for both SWI/SNF and SAGA exhibited a profound defect in PDR1 autoregulation, revealing that these complexes work together to ensure high-level Pdr1-dependent gene transcription.

The conserved Rad2/XPG family 5'-3' exonuclease, exonuclease 1 (Exo1), plays many roles in DNA metabolism including during resolution of DNA double-strand breaks via homologous recombination. Prior studies provided evidence that the end resection activity of Exo1 is downregulated in yeast and mammals by Cdk1/2 family cyclin-dependent and checkpoint kinases, including budding yeast kinase Rad53 which functions in mitotic cells. Here, we provide evidence that the master meiotic kinase Mek1, a paralog of Rad53, limits 5'-3' single-strand resection at the sites of programmed meiotic DNA breaks. Mutational analysis suggests that the mechanism of Exo1 suppression by Mek1 differs from that of Rad53.

Interspecific F1 hybrids between Asian (Oryza sativa) and African rice (Oryza glaberrima) exhibit severe sterility caused by the accumulation of hybrid sterility genes/loci at 15 or more loci. The mechanisms underlying the hybrid sterility genes are largely unknown; however, a few genes associated with the killer-protector system, which is the system most frequently associated with hybrid sterility genes, have been identified. We previously produced fertile plants as tetraploids derived from diploid interspecific F1 hybrids through anther culture; therefore, it was suggested that hybrid sterility could be overcome following tetraploidization. We investigated whether tetraploid interspecific plants produced by crossing are fertile and tested the involvement of hybrid sterility genes in the process. Fertile tetraploid interspecific F1 hybrid plants were obtained by crossing 2 tetraploids of O. sativa and O. glaberrima. To elucidate the relationships between pollen fertility and the hybrid sterility loci in the tetraploid F1 microspores, we performed genetic analyses of the tetraploid F2 hybrids and diploid plants obtained from the microspores of tetraploid interspecific hybrids by anther culture. The result suggested that the tetraploid interspecific hybrids overcame pollen and seed infertility based on the proportion of loci with the killer-protector system present in the tetraploids. The heterozygous hybrid sterility loci with the killer-protector system in the tetraploid segregate the homozygous killed allele (16.7-21.4%), with more than three-quarters of the gametes surviving. We theoretically and experimentally demonstrated that fertile rice progenies can be grown from tetraploid interspecific hybrids.

The meiosis-specific kinase Mek1 regulates key steps in meiotic recombination in the budding yeast, Saccharomyces cerevisiae. MEK1 limits resection at double-strand break (DSB) ends and is required for preferential strand invasion into homologs, a process known as interhomolog bias. After strand invasion, MEK1 promotes phosphorylation of the synaptonemal complex protein Zip1 that is necessary for DSB repair mediated by a crossover-specific pathway that enables chromosome synapsis. In addition, Mek1 phosphorylation of the meiosis-specific transcription factor, Ndt80, regulates the meiotic recombination checkpoint that prevents exit from pachytene when DSBs are present. Mek1 interacts with Ndt80 through a 5-amino acid sequence, RPSKR, located between the DNA-binding and activation domains of Ndt80. AlphaFold Multimer modeling of a fragment of Ndt80 containing the RPSKR motif and full-length Mek1 indicated that RPSKR binds to an acidic loop located in the Mek1 FHA domain, a noncanonical interaction with this motif. A second protein, the 5'-3' helicase Rrm3, similarly interacts with Mek1 through an RPAKR motif and is an in vitro substrate of Mek1. Genetic analysis using various mutants in the MEK1 acidic loop validated the AlphaFold model, in that they specifically disrupt 2-hybrid interactions with Ndt80 and Rrm3. Phenotypic analyses further showed that the acidic loop mutants are defective in the meiotic recombination checkpoint and, in certain circumstances, exhibit more severe phenotypes compared to the NDT80 mutant with the RPSKR sequence deleted, suggesting that additional, as yet unknown, substrates of Mek1 also bind to Mek1 using an RPXKR motif.

Our knowledge of human evolutionary history has been greatly advanced by paleogenomics. Since the 2020s, the study of ancient DNA has increasingly focused on reconstructing the recent past. However, the accuracy of paleogenomic methods in resolving questions of historical and archaeological importance amidst the increased demographic complexity and decreased genetic differentiation remains an open question. We evaluated the performance and behavior of two commonly used methods, qpAdm and the f3-statistic, on admixture inference under a diversity of demographic models and data conditions. We performed two complementary simulation approaches-firstly exploring a wide demographic parameter space under four simple demographic models of varying complexities and configurations using branch-length data from two chromosomes-and secondly, we analyzed a model of Eurasian history composed of 59 populations using whole-genome data modified with ancient DNA conditions such as SNP ascertainment, data missingness, and pseudohaploidization. We observe that population differentiation is the primary factor driving qpAdm performance. Notably, while complex gene flow histories influence which models are classified as plausible, they do not reduce overall performance. Under conditions reflective of the historical period, qpAdm most frequently identifies the true model as plausible among a small candidate set of closely related populations. To increase the utility for resolving fine-scaled hypotheses, we provide a heuristic for further distinguishing between candidate models that incorporates qpAdm model P-values and f3-statistics. Finally, we demonstrate a significant performance increase for qpAdm using whole-genome branch-length f2-statistics, highlighting the potential for improved demographic inference that could be achieved with future advancements in f-statistic estimations.

The genes encoding ribosomal RNA are highly conserved across life and in almost all eukaryotes are present in large tandem repeat arrays called the rDNA. rDNA repeat unit size is conserved across most eukaryotes but has expanded dramatically in mammals, principally through the expansion of the intergenic spacer region that separates adjacent rRNA coding regions. Here, we used long-read sequence data from representatives of the major amniote lineages to determine where in amniote evolution rDNA unit size increased. We find that amniote rDNA unit sizes fall into two narrow size classes: "normal" (∼11-20 kb) in all amniotes except monotreme, marsupial, and eutherian mammals, which have "large" (∼35-45 kb) sizes. We confirm that increases in intergenic spacer length explain much of this mammalian size increase. However, in stark contrast to the uniformity of mammalian rDNA unit size, mammalian intergenic spacers differ greatly in sequence. These results suggest a large increase in intergenic spacer size occurred in a mammalian ancestor and has been maintained despite substantial sequence changes over the course of mammalian evolution. This points to a previously unrecognized constraint on the length of the intergenic spacer, a region that was thought to be largely neutral. We finish by speculating on possible causes of this constraint.

Centromeric localization of evolutionarily conserved CENP-A (Cse4 in Saccharomyces cerevisiae) is essential for chromosomal stability. Mislocalization of overexpressed CENP-A to noncentromeric regions contributes to chromosomal instability in yeasts, flies, and humans. Overexpression and mislocalization of CENP-A observed in many cancers are associated with poor prognosis. Previous studies have shown that F-box proteins, Cdc4 and Met30 of the Skp, Cullin, F-box ubiquitin ligase cooperatively regulate proteolysis of Cse4 to prevent Cse4 mislocalization and chromosomal instability under normal physiological conditions. Mck1-mediated phosphorylation of Skp, Cullin, F-box-Cdc4 substrates such as Cdc6 and Rcn1 enhances the interaction of the substrates with Cdc4. Here, we report that Mck1 interacts with Cse4, and Mck1-mediated proteolysis of Cse4 prevents Cse4 mislocalization for chromosomal stability. Our results showed that mck1Δ strain overexpressing CSE4 (GAL-CSE4) exhibits lethality, defects in ubiquitin-mediated proteolysis of Cse4, mislocalization of Cse4, and reduced Cse4-Cdc4 interaction. Strain expressing GAL-cse4-3A with mutations in three potential Mck1 phosphorylation consensus sites (S10, S16, and T166) also exhibits growth defects, increased stability with mislocalization of Cse4-3A, chromosomal instability, and reduced interaction with Cdc4. Constitutive expression of histone H3 (Δ16H3) suppresses the chromosomal instability phenotype of GAL-cse4-3A strain, suggesting that the chromosomal instability phenotype is linked to Cse4-3A mislocalization. We conclude that Mck1 and its three potential phosphorylation sites on Cse4 promote Cse4-Cdc4 interaction and this contributes to ubiquitin-mediated proteolysis of Cse4 preventing its mislocalization and chromosomal instability. These studies advance our understanding of pathways that regulate cellular levels of CENP-A to prevent mislocalization of CENP-A in human cancers.

The elongation of Caenorhabditis elegans embryos allows examination of mechanical interactions between adjacent tissues. Muscle contractions during late elongation induce the remodeling of epidermal circumferential actin filaments through mechanotransduction. Force inputs from the muscles deform circumferential epidermal actin filament, which causes them to be severed, eventually reformed, and shortened. This squeezing force drives embryonic elongation. We investigated the possible role of the nonmuscle myosins NMY-1 and NMY-2 in this process using nmy-1 and nmy-2 thermosensitive alleles. Our findings show these myosins act redundantly in late elongation, since double nmy-2(ts); nmy-1(ts) mutants immediately stop elongation when raised to 25°C. Their inactivation does not reduce muscle activity, as measured from epidermis deformation, suggesting that they are directly involved in the multistep process of epidermal remodeling. Furthermore, NMY-1 and NMY-2 inactivation is reversible when embryos are kept at the nonpermissive temperature for a few hours. However, after longer exposure to 25°C double mutant embryos fail to resume elongation, presumably because NMY-1 was seen to form protein aggregates. We propose that the two C. elegans nonmuscle myosin II act during actin remodeling either to bring severed ends or hold them.

Behavioral plasticity allows animals to modulate their behavior based on experience and environmental conditions. Caenorhabditis elegans exhibits experience-dependent changes in its behavioral responses to various modalities of sensory cues, including odorants, salts, temperature, and mechanical stimulations. Most of these forms of behavioral plasticity, such as adaptation, habituation, associative learning, and imprinting, are shared with other animals. The C. elegans nervous system is considerably tractable for experimental studies-its function can be characterized and manipulated with molecular genetic methods, its activity can be visualized and analyzed with imaging approaches, and the connectivity of its relatively small number of neurons are well described. Therefore, C. elegans provides an opportunity to study molecular, neuronal, and circuit mechanisms underlying behavioral plasticity that are either conserved in other animals or unique to this species. These findings reveal insights into how the nervous system interacts with the environmental cues to generate behavioral changes with adaptive values.

Observations of genetically repeated evolution (repeatability) in complex organisms are incongruent with the Fisher-Orr model, which implies that repeated use of the same gene should be rare when mutations are pleiotropic (i.e. affect multiple traits). When spatially divergent selection occurs in the presence of migration, mutations of large effect are more strongly favored, and hence, repeatability is more likely, but it is unclear whether this observation is limited by pleiotropy. Here, we explore this question using individual-based simulations of a two-patch model incorporating multiple quantitative traits governed by mutations with pleiotropic effects. We explore the relationship between fitness trade-offs and repeatability by varying the alignment between mutation effect and spatial variation in trait optima. While repeatability decreases with increasing trait dimensionality, trade-offs in mutation effects on traits do not strongly limit the contribution of a locus of large effect to repeated adaptation, particularly under increased migration. These results suggest that repeatability will be more pronounced for local rather than global adaptation. Whereas pleiotropy limits repeatability in a single-population model, when there is local adaptation with gene flow, repeatability can occur if some loci are able to produce alleles of large effect, even when there are pleiotropic trade-offs.