RESUMO
Successful chromosome segregation into gametes depends on tightly regulated interactions between the parental chromosomes. During meiosis, chromosomes are aligned end-to-end by an interface called the synaptonemal complex, which also regulates exchanges between them. However, despite the functional and ultrastructural conservation of this essential interface, how protein-protein interactions within the synaptonemal complex regulate chromosomal interactions remains poorly understood. Here, we describe a genetic interaction in the C. elegans synaptonemal complex, comprised of short segments of three proteins, SYP-1, SYP-3, and SYP-4. We identified the interaction through a saturated suppressor screen of a mutant that destabilizes the synaptonemal complex. The specificity and tight distribution of suppressors suggest a charge-based interface that promotes interactions between synaptonemal complex subunits and, in turn, allows intimate interactions between chromosomes. Our work highlights the power of genetic studies to illuminate the mechanisms that underlie meiotic chromosome interactions.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animais , Caenorhabditis elegans/metabolismo , Complexo Sinaptonêmico/genética , Complexo Sinaptonêmico/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Meiose/genética , Pareamento Cromossômico , Proteínas Nucleares/metabolismoRESUMO
Successful chromosome segregation into gametes depends on tightly-regulated interactions between the parental chromosomes. During meiosis, chromosomes are aligned end-to-end by an interface called the synaptonemal complex, which also regulates exchanges between them. However, despite the functional and ultrastructural conservation of this essential interface, how protein-protein interactions within the synaptonemal complex regulate chromosomal interactions remains poorly understood. Here we describe a novel interaction interface in the C. elegans synaptonemal complex, comprised of short segments of three proteins, SYP-1, SYP-3 and SYP-4. We identified the interface through a saturated suppressor screen of a mutant that destabilizes the synaptonemal complex. The specificity and tight distribution of suppressors point to a charge-based interface that promotes interactions between synaptonemal complex subunits and, in turn, allows intimate interactions between chromosomes. Our work highlights the power of genetic studies to illuminate the mechanisms that underly meiotic chromosome interactions.
RESUMO
Yeast and humans share thousands of genes despite a billion years of evolutionary divergence. While many human genes can functionally replace their yeast counterparts, nearly half of the tested shared genes cannot. For example, most yeast proteasome subunits are "humanizable," except subunits comprising the ß-ring core, including ß2c (HsPSMB7, a constitutive proteasome subunit). We developed a high-throughput pipeline to humanize yeast proteasomes by generating a large library of Hsß2c mutants and screening them for complementation of a yeast ß2 (ScPup1) knockout. Variants capable of replacing ScPup1 included (1) those impacting local protein-protein interactions (PPIs), with most affecting interactions between the ß2c C-terminal tail and the adjacent ß3 subunit, and (2) those affecting ß2c proteolytic activity. Exchanging the full-length tail of human ß2c with that of ScPup1 enabled complementation. Moreover, wild-type human ß2c could replace yeast ß2 if human ß3 was also provided. Unexpectedly, yeast proteasomes bearing a catalytically inactive HsPSMB7-T44A variant that blocked precursor autoprocessing were viable, suggesting an intact propeptide stabilizes late assembly intermediates. In contrast, similar modifications in human ß2i (HsPSMB10), an immunoproteasome subunit and the co-ortholog of yeast ß2, do not enable complementation in yeast, suggesting distinct interactions are involved in human immunoproteasome core assembly. Broadly, our data reveal roles for specific PPIs governing functional replaceability across vast evolutionary distances.
Assuntos
Complexo de Endopeptidases do Proteassoma , Saccharomyces cerevisiae , Humanos , Complexo de Endopeptidases do Proteassoma/genética , Saccharomyces cerevisiae/genéticaRESUMO
The environmental pathogen Cryptococcus neoformans claims over 180,000 lives each year. Survival of this basidiomycete at host CO2 concentrations has only recently been considered an important virulence trait. Through screening gene knockout libraries constructed in a CO2-tolerant clinical strain, we found mutations leading to CO2 sensitivity are enriched in pathways activated by heat stress, including calcineurin, Ras1-Cdc24, cell wall integrity, and Regulator of Ace2 and Morphogenesis (RAM). Overexpression of Cbk1, the conserved terminal kinase of the RAM pathway, partially restored defects of these mutants at host CO2 or temperature levels. In ascomycetes such as Saccharomyces cerevisiae and Candida albicans, transcription factor Ace2 is an important target of Cbk1, activating genes responsible for cell separation. However, no Ace2 homolog or any downstream component of the RAM pathway has been identified in basidiomycetes. Through in vitro evolution and comparative genomics, we characterized mutations in suppressors of cbk1Δ in C. neoformans that partially rescued defects in CO2 tolerance, thermotolerance, and morphology. One suppressor is the RNA translation repressor Ssd1, which is highly conserved in ascomycetes and basidiomycetes. The other is a novel ribonuclease domain-containing protein, here named PSC1, which is present in basidiomycetes and humans but surprisingly absent in most ascomycetes. Loss of Ssd1 in cbk1Δ partially restored cryptococcal ability to survive and amplify in the inhalation and intravenous murine models of cryptococcosis. Our discoveries highlight the overlapping regulation of CO2 tolerance and thermotolerance, the essential role of the RAM pathway in cryptococcal adaptation to the host condition, and the potential importance of post-transcriptional control of virulence traits in this global pathogen.
Assuntos
Criptococose , Cryptococcus neoformans , Termotolerância , Humanos , Camundongos , Animais , Cryptococcus neoformans/genética , Dióxido de Carbono , Termotolerância/genética , Morfogênese , Transdução de SinaisRESUMO
The cohesin complex is required for sister chromatid cohesion and genome compaction. Cohesin coiled coils (CCs) can fold at break sites near midpoints to bring head and hinge domains, located at opposite ends of coiled coils, into proximity. Whether ATPase activities in the head play a role in this conformational change is yet to be known. Here, we dissected functions of cohesin ATPase activities in cohesin dynamics in Schizosaccharomyces pombe. Isolation and characterization of cohesin ATPase temperature-sensitive (ts) mutants indicate that both ATPase domains are required for proper chromosome segregation. Unbiased screening of spontaneous suppressor mutations rescuing the temperature lethality of cohesin ATPase mutants identified several suppressor hotspots in cohesin that located outside of ATPase domains. Then, we performed comprehensive saturation mutagenesis targeted to these suppressor hotspots. Large numbers of the identified suppressor mutations indicated several different ways to compensate for the ATPase mutants: 1) Substitutions to amino acids with smaller side chains in coiled coils at break sites around midpoints may enable folding and extension of coiled coils more easily; 2) substitutions to arginine in the DNA binding region of the head may enhance DNA binding; or 3) substitutions to hydrophobic amino acids in coiled coils, connecting the head and interacting with other subunits, may alter conformation of coiled coils close to the head. These results reflect serial structural changes in cohesin driven by its ATPase activities potentially for packaging DNAs.
Assuntos
Adenosina Trifosfatases , Proteínas de Ciclo Celular , Proteínas Cromossômicas não Histona , Segregação de Cromossomos , Schizosaccharomyces , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Substituição de Aminoácidos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , DNA/metabolismo , Mutação , Domínios Proteicos , Schizosaccharomyces/enzimologia , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , CoesinasRESUMO
Cohesin holds sister chromatids together and is cleaved by separase/Cut1 to release DNA during the transition from mitotic metaphase to anaphase. The cohesin complex consists of heterodimeric structural maintenance of chromosomes (SMC) subunits (Psm1 and Psm3), which possess a head and a hinge, separated by long coiled coils. Non-SMC subunits (Rad21, Psc3 and Mis4) bind to the SMC heads. Kleisin/Rad21's N-terminal domain (Rad21-NTD) interacts with Psm3's head-coiled coil junction (Psm3-HCJ). Spontaneous mutations that rescued the cleavage defects in temperature-sensitive (ts) separase mutants were identified in the interaction interface, but the underlying mechanism is yet to be understood. Here, we performed site-directed random mutagenesis to introduce single amino acid substitutions in Psm3-HCJ and Rad21-NTD, and then identified 300 mutations that rescued the cohesin-releasing defects in a separase ts mutant. Mutational analysis indicated that the amino acids involved in hydrophobic cores (which may be in close contact) in Psm3-HCJ and Rad21-NTD are hotspots, since 80 mutations (approx. 27%) were mapped in these locations. Properties of these substitutions indicate that they destabilize the interaction between the Psm3 head and Rad21-NTD. Thus, they may facilitate sister chromatid separation in a cleavage-independent way through cohesin structural re-arrangement.
Assuntos
Anáfase , Proteínas de Schizosaccharomyces pombe , Substituição de Aminoácidos , Proteínas de Ciclo Celular , Proteínas Cromossômicas não Histona , DNA , Proteínas de Schizosaccharomyces pombe/genética , Separase/genética , CoesinasRESUMO
Plant breeding relies on the meiotic recombination or crossing over to generate the new combinations of the alleles along and among the chromosomes. However, crossing over is constrained in the crops such as barley by a combination of the low frequency and biased distribution. In this study, we attempted to identify the genes that limit the recombination by performing a suppressor screen for the restoration of fertility to the semi-fertile barley mutant desynaptic10 (des10), carrying a mutation in the barley ortholog of MutL-Homolog 3 (HvMLH3), a member of the MutL-homolog (MLH) family of DNA mismatch repair genes. des10 mutants exhibit reduced recombination and fewer chiasmata, resulting in the loss of obligate crossovers (COs) leading to chromosome mis-segregation. We identified several candidate suppressor lines and confirmed their restored fertility in an Hvmlh3 background in the subsequent generations. We focus on one of the candidate suppressor lines, SuppLine2099, which showed the most complete restoration of fertility. We characterized this line by using a target-sequence enrichment and sequencing (TENSEQ) capture array representing barley orthologs of 46 meiotic genes. We found that SuppLine2099 contained a C/T change in the anti-CO gene RecQ-like helicase 4 (RECQL4) resulting in the substitution of a non-polar glycine to a polar aspartic acid (G700D) amino acid in the conserved helicase domain. Single nucleotide polymorphism (SNP) genotyping of F3 populations revealed a significant increase in the recombination frequency in lines with Hvrecql4 in the Hvmlh3 background that was associated with the restoration of fertility. The genotyping also indicated that there was nearly double the recombination levels in homozygous Hvrecql4 lines compared to the wild type (WT). However, we did not observe any significant change in the distribution of CO events. Our results confirm the anti-CO role of RECQL4 in a large genome cereal and establish the possibility of testing the utility of increasing recombination in the context of traditional crop improvement.
RESUMO
Proximal spinal muscular atrophy (SMA) is an autosomal recessive neurodegenerative disorder characterized by motor neuron loss and subsequent atrophy of skeletal muscle. SMA is caused by deficiency of the essential survival motor neuron (SMN) protein, canonically responsible for the assembly of the spliceosomal small nuclear ribonucleoproteins (snRNPs). Therapeutics aimed at increasing SMN protein levels are efficacious in treating SMA. However, it remains unknown how deficiency of SMN results in motor neuron loss, resulting in many reported cellular functions of SMN and pathways affected in SMA. Herein is a perspective detailing what genetics and biochemistry have told us about SMA and SMN, from identifying the SMA determinant region of the genome, to the development of therapeutics. Furthermore, we will discuss how genetics and biochemistry have been used to understand SMN function and how we can determine which of these are critical to SMA moving forward.
Assuntos
Atrofia Muscular Espinal/genética , Animais , Humanos , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Atrofia Muscular Espinal/metabolismo , Atrofia Muscular Espinal/patologia , Mutação , Transdução de Sinais , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismo , Proteína 2 de Sobrevivência do Neurônio Motor/genética , Proteína 2 de Sobrevivência do Neurônio Motor/metabolismoRESUMO
High-throughput phenotypic screening is a key driver for the identification of novel chemical matter in drug discovery for challenging targets, especially for those with an unclear mechanism of pathology. For toxic or gain-of-function proteins, small-molecule suppressors are a targeting/therapeutic strategy that has been successfully applied. As with other high-throughput screens, the screening strategy and proper assays are critical for successfully identifying selective suppressors of the target of interest. We executed a small-molecule suppressor screen to identify compounds that specifically reduce apolipoprotein L1 (APOL1) protein levels, a genetically validated target associated with increased risk of chronic kidney disease. To enable this study, we developed homogeneous time-resolved fluorescence (HTRF) assays to measure intracellular APOL1 and apolipoprotein L2 (APOL2) protein levels and miniaturized them to 1536-well format. The APOL1 HTRF assay served as the primary assay, and the APOL2 and a commercially available p53 HTRF assay were applied as counterscreens. Cell viability was also measured with CellTiter-Glo to assess the cytotoxicity of compounds. From a 310,000-compound screening library, we identified 1490 confirmed primary hits with 12 different profiles. One hundred fifty-three hits selectively reduced APOL1 in 786-O, a renal cell adenocarcinoma cell line. Thirty-one of these selective suppressors also reduced APOL1 levels in conditionally immortalized human podocytes. The activity and specificity of seven resynthesized compounds were validated in both 786-O and podocytes.
Assuntos
Apolipoproteína L1/antagonistas & inibidores , Descoberta de Drogas/métodos , Ensaios de Triagem em Larga Escala , Podócitos/efeitos dos fármacos , Podócitos/metabolismo , Humanos , Bibliotecas de Moléculas PequenasRESUMO
Forward genetics has been extremely powerful for dissecting biological pathways in various model organisms. However, it is limited by the fact that redundant gene families and essential genes cannot be readily uncovered through such methods. Chemical genetics, on the other hand, provides a valuable complementary approach to probe biological processes and is suitable for not only genetic model organisms but also genetically less tractable species. We describe here a high-throughput chemical genetic screening method simply based on plant growth and developmental phenotypes in Arabidopsis. It was successfully utilized to study plant immunity and can be easily adapted for dissecting other plant signal transduction pathways.
Assuntos
Arabidopsis/genética , Testes Genéticos , Plântula/genética , Bibliotecas de Moléculas Pequenas/análise , Arabidopsis/efeitos dos fármacos , Arabidopsis/crescimento & desenvolvimento , Meios de Cultura , Mutação/genética , Plântula/efeitos dos fármacos , Sementes/efeitos dos fármacos , Sementes/genética , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
Mutant screens remain among the most powerful unbiased methods for identifying key genes in a pathway or process of interest. However, mutants impacting pollen function pose special challenges due to their genetic behavior. Here we describe an approach for isolating pollen mutants based on screening for suppressors of a low pollen fertility starting genotype. By identifying suppressor mutants with improved pollen fertility, we are able to identify new genes which are functionally relevant to pathway(s) causing low seed set in the original background. With this method, the low fertility of the genetic background may be due to one or more mutations or transgenes disrupting any aspect of pollen development or function. Furthermore, screening for improved pollen fertility biases toward recovery of the desired mutants due to their enhanced male transmission. The causative mutation is cloned using next-generation sequencing. The procedure uses both genetic and bioinformatics approaches to ultimately yield a very small list of candidate causative mutations speeding the transition from mutant phenotype to underlying gene.
Assuntos
Clonagem Molecular/métodos , Infertilidade das Plantas/genética , Pólen/genética , Supressão Genética , Arabidopsis , Cruzamentos Genéticos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Pólen/fisiologia , Análise de Sequência de DNA/métodosRESUMO
This chapter presents methods for exploiting the powerful tools available in the nematode worm Caenorhabditis elegans to understand the in vivo functions of cerebral cavernous malformation (CCM) genes and the organization of their associated signaling pathways. Included are methods for assessing phenotypes caused by loss-of-function mutations in the worm CCM genes kri-1 and ccm-3, CRISPR-based gene editing techniques, and protocols for conducting high-throughput forward genetic and small molecule screens.
Assuntos
Hemangioma Cavernoso do Sistema Nervoso Central/etiologia , Hemangioma Cavernoso do Sistema Nervoso Central/metabolismo , Fenótipo , Alelos , Animais , Sistemas CRISPR-Cas , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Edição de Genes , Predisposição Genética para Doença , Hemangioma Cavernoso do Sistema Nervoso Central/diagnóstico , Mutagênese , Mutação , Ribonucleoproteínas/metabolismoRESUMO
Viral proteins must intimately interact with the host cell machinery during virus replication. Here, we used the yeast Saccharomyces cerevisiae as a system to identify novel functional interactions between viral proteins and eukaryotic cells. Our work demonstrates that when the Middle East respiratory syndrome coronavirus (MERS-CoV) ORF4a accessory gene is expressed in yeast it causes a slow-growth phenotype. ORF4a has been characterized as an interferon antagonist in mammalian cells, and yet yeast lack an interferon system, suggesting further interactions between ORF4a and eukaryotic cells. Using the slow-growth phenotype as a reporter of ORF4a function, we utilized the yeast knockout library collection to perform a suppressor screen where we identified the YDL042C/SIR2 yeast gene as a suppressor of ORF4a function. The mammalian homologue of SIR2 is SIRT1, an NAD-dependent histone deacetylase. We found that when SIRT1 was inhibited by either chemical or genetic manipulation, there was reduced MERS-CoV replication, suggesting that SIRT1 is a proviral factor for MERS-CoV. Moreover, ORF4a inhibited SIRT1-mediated modulation of NF-κB signaling, demonstrating a functional link between ORF4a and SIRT1 in mammalian cells. Overall, the data presented here demonstrate the utility of yeast studies for identifying genetic interactions between viral proteins and eukaryotic cells. We also demonstrate for the first time that SIRT1 is a proviral factor for MERS-CoV replication and that ORF4a has a role in modulating its activity in cells.IMPORTANCE Middle East respiratory syndrome coronavirus (MERS-CoV) initially emerged in 2012 and has since been responsible for over 2,300 infections, with a case fatality ratio of approximately 35%. We have used the highly characterized model system of Saccharomyces cerevisiae to investigate novel functional interactions between viral proteins and eukaryotic cells that may provide new avenues for antiviral intervention. We identify a functional link between the MERS-CoV ORF4a proteins and the YDL042C/SIR2 yeast gene. The mammalian homologue of SIR2 is SIRT1, an NAD-dependent histone deacetylase. We demonstrate for the first time that SIRT1 is a proviral factor for MERS-CoV replication and that ORF4a has a role in modulating its activity in mammalian cells.
Assuntos
Infecções por Coronavirus/metabolismo , Infecções por Coronavirus/virologia , Interações Hospedeiro-Patógeno , Coronavírus da Síndrome Respiratória do Oriente Médio/fisiologia , Sirtuína 1/metabolismo , Replicação Viral , Linhagem Celular , Células Cultivadas , Infecções por Coronavirus/genética , Inativação Gênica , Humanos , Fenótipo , Ligação Proteica , Interferência de RNA , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Sirtuína 1/genética , Proteínas Estruturais Virais/genética , Proteínas Estruturais Virais/metabolismo , Leveduras/genética , Leveduras/metabolismoRESUMO
The mitotic kinetochore forms at the centromere for proper chromosome segregation. Deposition of the centromere-specific histone H3 variant, spCENP-A/Cnp1, is vital for the formation of centromere-specific chromatin and the Mis17-Mis6 complex of the fission yeast Schizosaccharomyces pombe is required for this deposition. Here we identified extragenic suppressors for a Mis17-Mis6 complex temperature-sensitive (ts) mutant, mis17-S353P, using whole-genome sequencing. The large and small daughter nuclei phenotype observed in mis17-S353P was greatly rescued by these suppressors. Suppressor mutations in two ribonuclease genes involved in the mRNA decay pathway, exo2 and pan2, may affect Mis17 protein level, as mis17 mutant protein level was recovered in mis17-S353P exo2 double mutant cells. Suppressor mutations in EKC/KEOPS complex genes may not regulate Mis17 protein level, but restored centromeric localization of spCENP-A/Cnp1, Mis6 and Mis15 in mis17-S353P Therefore, the EKC/KEOPS complex may inhibit Mis17-Mis6 complex formation or centromeric localization. Mutational analysis in protein structure indicated that suppressor mutations in the EKC/KEOPS complex may interfere with its kinase activity or complex formation. Our results suggest that the mRNA decay pathway and the EKC/KEOPS complex negatively regulate Mis17-Mis6 complex-mediated centromere formation by distinct and unexpected mechanisms.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Centrômero/genética , Centrômero/metabolismo , Estabilidade de RNA , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Regulação Fúngica da Expressão Gênica , Genes Reporter , Complexos Multiproteicos/metabolismo , Mutação , FenótipoRESUMO
Cohesin is a fundamental protein complex that holds sister chromatids together. Separase protease cleaves a cohesin subunit Rad21/SCC1, causing the release of cohesin from DNA to allow chromosome segregation. To understand the functional organization of cohesin, we employed next-generation whole-genome sequencing and identified numerous extragenic suppressors that overcome either inactive separase/Cut1 or defective cohesin in the fission yeast Schizosaccharomyces pombe Unexpectedly, Cut1 is dispensable if suppressor mutations cause disorders of interfaces among essential cohesin subunits Psm1/SMC1, Psm3/SMC3, Rad21/SCC1, and Mis4/SCC2, the crystal structures of which suggest physical and functional impairment at the interfaces of Psm1/3 hinge, Psm1 head-Rad21, or Psm3 coiled coil-Rad21. Molecular-dynamics analysis indicates that the intermolecular ß-sheets in the cohesin hinge of cut1 suppressor mutants remain intact, but a large mobility change occurs at the coiled coil bound to the hinge. In contrast, suppressors of rad21-K1 occur in either the head ATPase domains or the Psm3 coiled coil that interacts with Rad21. Suppressors of mis4-G1326E reside in the head of Psm3/1 or the intragenic domain of Mis4. These may restore the binding of cohesin to DNA. Evidence is provided that the head and hinge of SMC subunits are proximal, and that they coordinate to form arched coils that can hold or release DNA by altering the angles made by the arched coiled coils. By combining molecular modeling with suppressor sequence analysis, we propose a cohesin structure designated the "hold-and-release" model, which may be considered as an alternative to the prevailing "ring" model.
Assuntos
Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/metabolismo , DNA Fúngico/metabolismo , Mutação , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Proteínas de Ciclo Celular/genética , Cromátides/fisiologia , Proteínas Cromossômicas não Histona/genética , Segregação de Cromossomos , DNA Fúngico/genética , Modelos Moleculares , Proteínas Nucleares/genética , Fosfoproteínas/genética , Fosforilação , Conformação Proteica , Subunidades Proteicas , Schizosaccharomyces/crescimento & desenvolvimento , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Supressão Genética , CoesinasRESUMO
Suppressor screening is a powerful method to identify genes that, when mutated, rescue the temperature sensitivity of the original mutation. Previously, however, identification of suppressor mutations has been technically difficult. Due to the small genome size of Schizosaccharomyces pombe, we developed a spontaneous suppressor screening technique, followed by a cost-effective sequencing method. Genomic DNAs of 10 revertants that survived at the restrictive temperature of the original temperature sensitive (ts) mutant were mixed together as one sample before constructing a library for sequencing. Responsible suppressor mutations were identified bioinformatically based on allele frequency. Then, we isolated a large number of spontaneous extragenic suppressors for three ts mutants that exhibited defects in chromosome segregation at their restrictive temperature. Screening provided new insight into mechanisms of chromosome segregation: loss of Ufd2 E4 multi-ubiquitination activity suppresses defects of an AAA ATPase, Cdc48. Loss of Wpl1, a releaser of cohesin, compensates for the Eso1 mutation, which may destabilize sister chromatid cohesion. The segregation defect of a ts histone H2B mutant is rescued if it fails to be deubiquitinated by the SAGA complex, because H2B is stabilized by monoubiquitination.
Assuntos
Segregação de Cromossomos/genética , Genoma Fúngico , Estudo de Associação Genômica Ampla , Genômica , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/genética , Sequência de Aminoácidos , Genômica/métodos , Mutação , Estrutura Secundária de Proteína , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/metabolismo , Sequenciamento Completo do GenomaRESUMO
The comprehensive understanding of cellular signaling pathways remains a challenge due to multiple layers of regulation that may become evident only when the pathway is probed at different levels or critical nodes are eliminated. To discover regulatory mechanisms in canonical WNT signaling, we conducted a systematic forward genetic analysis through reporter-based screens in haploid human cells. Comparison of screens for negative, attenuating and positive regulators of WNT signaling, mediators of R-spondin-dependent signaling and suppressors of constitutive signaling induced by loss of the tumor suppressor adenomatous polyposis coli or casein kinase 1α uncovered new regulatory features at most levels of the pathway. These include a requirement for the transcription factor AP-4, a role for the DAX domain of AXIN2 in controlling ß-catenin transcriptional activity, a contribution of glycophosphatidylinositol anchor biosynthesis and glypicans to R-spondin-potentiated WNT signaling, and two different mechanisms that regulate signaling when distinct components of the ß-catenin destruction complex are lost. The conceptual and methodological framework we describe should enable the comprehensive understanding of other signaling systems.
Assuntos
Regulação da Expressão Gênica , Redes Reguladoras de Genes , Testes Genéticos/métodos , Via de Sinalização Wnt , Caseína Quinase I/deficiência , Proteínas do Citoesqueleto/deficiência , Genes Reporter , Haploidia , Humanos , Proteínas Wnt/genética , Proteínas Wnt/metabolismoRESUMO
The conditional control of protein expression is useful to characterize the function of proteins, especially of those that are essential for cell viability. Two degron-based systems, temperature-sensitive and auxin-inducible degrons, can be used to generate conditional mutants of budding yeast, simply by transforming appropriate cells with PCR-amplified DNA. We describe a protocol for the generation of temperature-sensitive and auxin-inducible degron mutants. We also show that a conditional mutant with few spontaneous revertants was generated by combining two degron systems for the Inn1 protein. Finally, we describe a suppressor screening method that uses the dual degron-Inn1 mutant to identify mutant proteins that suppress Inn1-K31A, which has a defect in cytokinesis.
Assuntos
Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Genes Fúngicos , Genes Supressores , Ácidos Indolacéticos/farmacologia , Mutação , Saccharomycetales/genética , Temperatura , Clonagem Molecular , Biblioteca Gênica , Plasmídeos/genética , Transformação GenéticaRESUMO
Genetic screens have proven to be a useful tool in the dissection of biological processes in plants. Specifically, suppressor screens have been widely used to study signal transduction pathways. Here we provide a detailed protocol for ethyl methanesulfonate (EMS) mutagenesis used in our suppressor screens in Arabidopsis and discuss the basic principles behind suppressor screen design and downstream analyses.
Assuntos
Arabidopsis/genética , Regulação da Expressão Gênica , Testes Genéticos , Proteínas de Plantas/genética , Arabidopsis/efeitos dos fármacos , Arabidopsis/metabolismo , Clonagem Molecular , Regulação para Baixo , Metanossulfonato de Etila , Regulação da Expressão Gênica/efeitos dos fármacos , Estudos de Associação Genética , Testes Genéticos/métodos , Mutagênese/efeitos dos fármacos , Mutação , Proteínas de Plantas/metabolismoRESUMO
The mushroom body (MB) of Drosophila melanogaster is an organized collection of interneurons that is required for learning and memory. Each of the three subtypes of MB neurons, γ, α'/ß', and α/ß, branch at some point during their development, providing an excellent model in which to study the genetic regulation of axon branching. Given the sequential birth order and the unique patterning of MB neurons, it is likely that specific gene cascades are required for the different guidance events that form the characteristic lobes of the MB. The nuclear receptor UNFULFILLED (UNF), a transcription factor, is required for the differentiation of all MB neurons. We have developed and used a classical genetic suppressor screen that takes advantage of the fact that ectopic expression of unf causes lethality to identify candidate genes that act downstream of UNF. We hypothesized that reducing the copy number of unf-interacting genes will suppress the unf-induced lethality. We have identified 19 candidate genes that when mutated suppress the unf-induced lethality. To test whether candidate genes impact MB development, we performed a secondary phenotypic screen in which the morphologies of the MBs in animals heterozygous for unf and a specific candidate gene were analyzed. Medial MB lobes were thin, missing, or misguided dorsally in five double heterozygote combinations (;unf/+;axin/+, unf/+;Fps85D/+, ;unf/+;Tsc1/+, ;unf/+;Rheb/+, ;unf/+;msn/+). Dorsal MB lobes were missing in ;unf/+;DopR2/+ or misprojecting beyond the termination point in ;unf/+;Sytß double heterozygotes. These data suggest that unf and unf-interacting genes play specific roles in axon development in a branch-specific manner.