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1.
Bioinformatics ; 38(Supplement_2): ii13-ii19, 2022 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-36124790

RESUMO

MOTIVATION: Detecting synthetic lethality (SL) is a promising strategy for identifying anti-cancer drug targets. Targeting SL partners of a primary gene mutated in cancer is selectively lethal to cancer cells. Due to high cost of wet-lab experiments and availability of gold standard SL data, supervised machine learning for SL prediction has been popular. However, most of the methods are based on binary classification and thus limited by the lack of reliable negative data. Contrastive learning can train models without any negative sample and is thus promising for finding novel SLs. RESULTS: We propose NSF4SL, a negative-sample-free SL prediction model based on a contrastive learning framework. It captures the characteristics of positive SL samples by using two branches of neural networks that interact with each other to learn SL-related gene representations. Moreover, a feature-wise data augmentation strategy is used to mitigate the sparsity of SL data. NSF4SL significantly outperforms all baselines which require negative samples, even in challenging experimental settings. To the best of our knowledge, this is the first time that SL prediction is formulated as a gene ranking problem, which is more practical than the current formulation as binary classification. NSF4SL is the first contrastive learning method for SL prediction and its success points to a new direction of machine-learning methods for identifying novel SLs. AVAILABILITY AND IMPLEMENTATION: Our source code is available at https://github.com/JieZheng-ShanghaiTech/NSF4SL. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.


Assuntos
Antineoplásicos , Neoplasias , Genes Letais , Humanos , Neoplasias/genética , Software , Mutações Sintéticas Letais
2.
STAR Protoc ; 3(3): 101556, 2022 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-36060092

RESUMO

Combinatorial CRISPR screening is useful for investigating synthetic lethality (SL) gene pairs. Here, we detail the steps for dual-gRNA library construction, with the introduction of two backbones, LentiGuide_DKO and LentiCRISPR_DKO. We describe steps for in vitro screening with 22Rv1-Cas9 and SaOS2-Cas9 cells followed by sequencing and data analysis. By introducing two backbones, we optimized the library construction process, facilitated standard pair-end sequencing, and provided options of screening on cells with or without modification of Cas9 expression.


Assuntos
Sistemas CRISPR-Cas , RNA Guia , Sistemas CRISPR-Cas/genética , Biblioteca Gênica , Genes Letais , RNA Guia/genética , Mutações Sintéticas Letais
3.
G3 (Bethesda) ; 12(9)2022 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-35876798

RESUMO

Crosses between Drosophila simulans females and Drosophila melanogaster males produce viable F1 sons and poorly viable F1 daughters. Unlike most hybrid incompatibilities, this hybrid incompatibility violates Haldane's rule, the observation that incompatibilities preferentially affect the heterogametic sex. Furthermore, it has a different genetic basis than hybrid lethality in the reciprocal cross, with the causal allele in Drosophila melanogaster being a large species-specific block of complex satellite DNA on its X chromosome known as the 359-bp satellite, rather than a protein-coding locus. The causal allele(s) in Drosophila simulans are unknown but likely involve maternally expressed genes or factors since the F1 females die during early embryogenesis. The maternal haploid (mh) gene is an intriguing candidate because it is expressed maternally and its protein product localizes to the 359-bp repeat. We found that this gene has diverged extensively between Drosophila melanogaster and Drosophila simulans. This observation led to the hypothesis that Drosophila melanogaster mh may have coevolved with the 359-bp repeat and that hybrid incompatibility thus results from the absence of a coevolved mh allele in Drosophila simulans. We tested for the functional divergence of mh by creating matched transformants of Drosophila melanogaster and Drosophila simulans orthologs in both Drosophila melanogaster and Drosophila simulans strains. Surprisingly, we find that Drosophila simulans mh fully complements the female sterile phenotype of Drosophila melanogaster mh mutations. Contrary to our hypothesis, we find no evidence that adding a Drosophila melanogaster mh gene to Drosophila simulans increases hybrid viability.


Assuntos
Drosophila melanogaster , Drosophila , Animais , Drosophila/genética , Drosophila melanogaster/genética , Drosophila simulans/genética , Feminino , Genes Letais , Haploidia , Hibridização Genética , Masculino
4.
Pestic Biochem Physiol ; 184: 105087, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35715034

RESUMO

Sex-lethal (Sxl) encodes an RNA-binding protein that acts as the switch of sex determination in Drosophila and influences the genitalia formation and gonadal development. However, its sex-determination roles are not conserved in all insects and its role in the gonadal development of Lepidoptera is not well documented. In this study, three splicing variants of Sxl mRNA were identified in Spodoptera litura and they highly expressed in gonads, particularly in the testis. The mRNA levels of SlSxl exhibited higher expression in the spermatid than the testis sheaths, and gradually increased with the spermiogenesis. Sex-lethal protein (SlSXL) is mainly distributed in the cytoplasm of spermatocytes and the head of spermatid. Knockout of SlSxl resulted in fewer eupyrene sperm bundles and apyrene sperm bundles in the testes of moth and a large number of undeveloped spermatocysts retained in the moth of mutant testis, and leading to the reduction of oviposition and hatch rate in the offsprings after mating with female. These results suggest that SlSxl is a critical player in the spermiogenesis of S. litura.


Assuntos
Fertilidade , Genes Letais , Animais , Feminino , Fertilidade/genética , Masculino , RNA Mensageiro/metabolismo , Reprodução/genética , Spodoptera/metabolismo
5.
Int J Mol Sci ; 23(10)2022 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-35628534

RESUMO

Lysyl oxidase-like 2 (LOXL2) and 3 (LOXL3) are members of the lysyl oxidase family of enzymes involved in the maturation of the extracellular matrix. Both enzymes share a highly conserved catalytic domain, but it is unclear whether they perform redundant functions in vivo. In this study, we show that mice lacking Loxl3 exhibit perinatal lethality and abnormal skeletal development. Additionally, analysis of the genotype of embryos carrying double knockout of Loxl2 and Loxl3 genes suggests that both enzymes have overlapping functions during mouse development. Furthermore, we also show that ubiquitous expression of Loxl2 suppresses the lethality associated with Loxl3 knockout mice.


Assuntos
Aminoácido Oxirredutases , Aminoácido Oxirredutases/genética , Aminoácido Oxirredutases/metabolismo , Animais , Desenvolvimento Embrionário , Matriz Extracelular/metabolismo , Feminino , Genes Letais , Camundongos , Camundongos Knockout , Gravidez
6.
PLoS One ; 17(4): e0265539, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35385533

RESUMO

Holocarboxylase synthetase (HLCS) catalyzes the biotinylation of five distinct biotin-dependent carboxylases and perhaps chromatin proteins. HLCS deficiency causes multiple carboxylase deficiency which results in fatal consequences unless patients are diagnosed early and treated with pharmacological doses of biotin. The objective of this study was to develop an HLCS conditional knockout (KO) mouse and assess effects of HLCS knockout on embryo survival. In the mouse, exon 8 is flanked by LoxP sites, thereby removing a catalytically important region upon recombination by Cre. HLCS conditional KO mice were backcrossed for 14 generations with C57BL/6J mice to yield Hlcstm1Jze. Fertility and weight gain were normal and no frank disease phenotypes and abnormal feeding behavior were observed in the absence of Cre. HLCS knockout was embryonic lethal when dams homozygous for both the floxed Hlcs gene and tamoxifen-inducible Cre recombinase (denoted Hlcstm1.1Jze) were injected with tamoxifen on gestational days 2.5 and 10.5. This is the first report of an HLCS conditional KO mouse, which enables studies of the roles of HLCS and biotin in intermediary metabolism.


Assuntos
Carbono-Nitrogênio Ligases , Genes Letais , Deficiência de Holocarboxilase Sintetase , Animais , Biotina/metabolismo , Biotinilação , Carbono-Nitrogênio Ligases/genética , Carbono-Nitrogênio Ligases/metabolismo , Deficiência de Holocarboxilase Sintetase/tratamento farmacológico , Deficiência de Holocarboxilase Sintetase/genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Tamoxifeno
7.
BMC Plant Biol ; 22(1): 20, 2022 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-34991480

RESUMO

BACKGROUND: Mitogen-activated protein kinase (MAPK) cascades are conserved signaling modules in eukaryotic organisms and play essential roles in immunity and stress responses. However, the role of MAPKs in chloroplast development remains to be evidently established. RESULTS: In this study, a rice chlorosis seedling lethality 1 (csl1) mutant with a Zhonghua11 (ZH11, japonica) background was isolated. Seedlings of the mutant were characterized by chlorotic leaves and death after the trefoil stage, and chloroplasts were observed to contain accumulated starch granules. Molecular cloning revealed that OsCSL1 encoded a MAPK kinase kinase22 (MKKK22) targeted to the endoplasmic reticulum (ER), and functional complementation of OsCSL1 was found to restore the normal phenotype in csl1 plants. The CRISPR/Cas9 technology was used for targeted disruption of OsCSL1, and the OsCSL1-Cas9 lines obtained therein exhibited yellow seedlings which phenocopied the csl1 mutant. CSL1/MKKK22 was observed to establish direct interaction with MKK4, and altered expression of MKK1 and MKK4 was detected in the csl1 mutant. Additionally, disruption of OsCSL1 led to reduced expression of chloroplast-associated genes, including chlorophyll biosynthetic genes, plastid-encoded RNA polymerases, nuclear-encoded RNA polymerase, and nuclear-encoded chloroplast genes. CONCLUSIONS: The findings of this study revealed that OsCSL1 played roles in regulating the expression of multiple chloroplast synthesis-related genes, thereby affecting their functions, and leading to wide-ranging defects, including chlorotic seedlings and severely disrupted chloroplasts containing accumulated starch granules.


Assuntos
Cloroplastos/fisiologia , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Biogênese de Organelas , Oryza/crescimento & desenvolvimento , Proteínas de Plantas/fisiologia , Clorofila/genética , Retículo Endoplasmático/metabolismo , Genes de Cloroplastos , Genes Letais , Proteínas Quinases Ativadas por Mitógeno/genética , Mutação , Oryza/genética , Oryza/ultraestrutura , Proteínas de Plantas/genética
8.
Methods Mol Biol ; 2377: 345-362, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34709626

RESUMO

Genetic balancer systems, which allow effective capture and maintenance of lethal mutations stably, play an important role in identifying essential genes. Whole-genome sequencing (WGS) followed by bioinformatics analysis, combined with genetic mapping data analysis, allows for an efficient and economical means of identifying genomic mutations in essential genes. Using this approach, we successfully identified 104 essential genes on ChrI, ChrIII, and ChrV in C. elegans. In this report, we described a protocol that sequences the genome of prebalanced Caenorhabditis elegans (C. elegans) strains to carry lethal mutations and identifies candidate causal mutations and candidate essential genes using a robust bioinformatics procedure.


Assuntos
Caenorhabditis elegans/genética , Animais , Caenorhabditis , Proteínas de Caenorhabditis elegans/genética , Mapeamento Cromossômico , Genes Essenciais , Genes Letais , Mutação
9.
Life Sci Alliance ; 5(3)2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-34969816

RESUMO

The RNA-sensing signaling pathway has been well studied as an essential antiviral mechanism of innate immunity. However, its role in non-infected cells is yet to be thoroughly characterized. Here, we demonstrated that the RNA sensing signaling pathway also reacts to the endogenous cellular RNAs in endothelial cells (ECs), and this reaction is regulated by the RNA-editing enzyme ADAR1. Cellular RNA sequencing analysis showed that EC RNAs endure extensive RNA editing, especially in the RNA transcripts of short interspersed nuclear elements. The EC-specific deletion of ADAR1 dramatically reduced the editing level on short interspersed nuclear element RNAs, resulting in newborn death in mice with damage evident in multiple organs. Genome-wide gene expression analysis revealed a prominent innate immune activation with a dramatically elevated expression of interferon-stimulated genes. However, blocking the RNA sensing signaling pathway by deletion of the cellular RNA receptor MDA-5 prevented interferon-stimulated gene expression and rescued the newborn mice from death. This evidence demonstrated that the RNA-editing/RNA-sensing signaling pathway dramatically modulates EC function, representing a novel molecular mechanism for the regulation of EC functions.


Assuntos
Adenosina Desaminase/genética , Células Endoteliais/metabolismo , Regulação da Expressão Gênica , Helicase IFIH1 Induzida por Interferon/genética , Edição de RNA , Transdução de Sinais , Animais , Biomarcadores , Deleção de Genes , Genes Letais , Imunidade Inata , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Sequências Repetitivas de Ácido Nucleico , Transcrição Genética
10.
Sci Rep ; 11(1): 23732, 2021 12 09.
Artigo em Inglês | MEDLINE | ID: mdl-34887431

RESUMO

Mendelian genetics poses practical limitations on the number of mutant genes that can be investigated simultaneously for their roles in embryonic development in the mouse. While CRISPR-based gene editing of multiple genes at once offers an attractive alternative strategy, subsequent breeding or establishment of permanent mouse lines will rapidly segregate the different mutant loci again. Direct phenotypic analysis of genomic edits in an embryonic lethal gene in F0 generation mice, or F0 mouse embryos, circumvents the need for breeding or establishment of mutant mouse lines. In the course of genotyping a large cohort of F0 CRISPants, where the embryonic lethal gene T/brachyury was targeted, we noted the presence of multiple CRISPR-induced modifications in individual embryos. Using long-read single-molecule Nanopore sequencing, we identified a wide variety of deletions, ranging up to 3 kb, that would not have been detected or scored as wildtype with commonly used genotyping methods that rely on subcloning and short-read or Sanger sequencing. Long-read sequencing results were crucial for accurate genotype-phenotype correlation in our F0 CRISPants. We thus demonstrate feasibility of screening manipulated F0 embryos for mid-gestation phenotypic consequences of CRISPR-induced mutations without requiring derivation of permanent mouse lines.


Assuntos
Sistemas CRISPR-Cas , Desenvolvimento Embrionário/genética , Edição de Genes , Genes Letais , Alelos , Animais , Sequência de Bases , Engenharia Genética , Genótipo , Mutação INDEL , Camundongos , Mutagênese , Fenótipo , RNA Guia
11.
Int J Mol Sci ; 22(24)2021 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-34948034

RESUMO

Substitution of the conserved Histidine 448 present in one of the three consensus elements characterizing the guanosine nucleotide binding domain (IF2 G2) of Escherichia coli translation initiation factor IF2 resulted in impaired ribosome-dependent GTPase activity which prevented IF2 dissociation from the ribosome, caused a severe protein synthesis inhibition, and yielded a dominant lethal phenotype. A reduced IF2 affinity for the ribosome was previously shown to suppress this lethality. Here, we demonstrate that also a reduced IF2 affinity for fMet-tRNA can suppress this dominant lethal phenotype and allows IF2 to support faithful translation in the complete absence of GTP hydrolysis. These results strengthen the premise that the conformational changes of ribosome, IF2, and fMet-tRNA occurring during the late stages of translation initiation are thermally driven and that the energy generated by IF2-dependent GTP hydrolysis is not required for successful translation initiation and that the dissociation of the interaction between IF2 C2 and the acceptor end of fMet-tRNA, which represents the last tie anchoring the factor to the ribosome before the formation of an elongation-competent 70S complex, is rate limiting for both the adjustment of fMet-tRNA in a productive P site and the IF2 release from the ribosome.


Assuntos
Escherichia coli/crescimento & desenvolvimento , GTP Fosfo-Hidrolases/metabolismo , Genes Letais , Fator de Iniciação 2 em Procariotos/química , Fator de Iniciação 2 em Procariotos/metabolismo , RNA de Transferência de Metionina/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Guanosina Trifosfato/química , Hidrólise , Modelos Moleculares , Fenótipo , Fator de Iniciação 2 em Procariotos/genética , Conformação Proteica , Domínios Proteicos , Ribossomos/química , Ribossomos/metabolismo
12.
J Vis Exp ; (174)2021 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-34515675

RESUMO

The development of new drugs that precisely target key proteins in human cancers is fundamentally altering cancer therapeutics. However, before these drugs can be used, their target proteins must be validated as therapeutic targets in specific cancer types. This validation is often performed by knocking out the gene encoding the candidate therapeutic target in a genetically engineered mouse (GEM) model of cancer and determining what effect this has on tumor growth. Unfortunately, technical issues such as embryonic lethality in conventional knockouts and mosaicism in conditional knockouts often limit this approach. To overcome these limitations, an approach to ablating a floxed embryonic lethal gene of interest in short-term cultures of malignant peripheral nerve sheath tumors (MPNSTs) generated in a GEM model was developed. This paper describes how to establish a mouse model with the appropriate genotype, derive short-term tumor cultures from these animals, and then ablate the floxed embryonic lethal gene using an adenoviral vector that expresses Cre recombinase and enhanced green fluorescent protein (eGFP). Purification of cells transduced with adenovirus using fluorescence-activated cell sorting (FACS) and the quantification of the effects that gene ablation exerts on cellular proliferation, viability, the transcriptome, and orthotopic allograft growth is then detailed. These methodologies provide an effective and generalizable approach to identifying and validating therapeutic targets in vitro and in vivo. These approaches also provide a renewable source of low-passage tumor-derived cells with reduced in vitro growth artifacts. This allows the biological role of the targeted gene to be studied in diverse biologic processes such as migration, invasion, metastasis, and intercellular communication mediated by the secretome.


Assuntos
Neoplasias de Bainha Neural , Neurofibrossarcoma , Alelos , Animais , Proliferação de Células , Transformação Celular Neoplásica , Genes Letais , Camundongos
13.
Genet Sel Evol ; 53(1): 75, 2021 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-34551728

RESUMO

BACKGROUND: We tested the hypothesis that breeding schemes with a pre-selection step, in which carriers of a lethal recessive allele (LRA) were culled, and with optimum-contribution selection (OCS) reduce the frequency of a LRA, control rate of inbreeding, and realise as much genetic gain as breeding schemes without a pre-selection step. METHODS: We used stochastic simulation to estimate true genetic gain realised at a 0.01 rate of true inbreeding (ΔFtrue) by breeding schemes that combined one of four pre-selection strategies with one of three selection strategies. The four pre-selection strategies were: (1) no carriers culled, (2) male carriers culled, (3) female carriers culled, and (4) all carriers culled. Carrier-status was known prior to selection. The three selection strategies were: (1) OCS in which [Formula: see text] was predicted and controlled using pedigree relationships (POCS), (2) OCS in which [Formula: see text] was predicted and controlled using genomic relationships (GOCS), and (3) truncation selection of parents. All combinations of pre-selection strategies and selection strategies were tested for three starting frequencies of the LRA (0.05, 0.10, and 0.15) and two linkage statuses with the locus that has the LRA being on a chromosome with or without loci affecting the breeding goal trait. The breeding schemes were simulated for 10 discrete generations (t = 1, …, 10). In all breeding schemes, ΔFtrue was calibrated to be 0.01 per generation in generations t = 4, …, 10. Each breeding scheme was replicated 100 times. RESULTS: We found no significant difference in true genetic gain from generations t = 4, …, 10 between breeding schemes with or without pre-selection within selection strategy. POCS and GOCS schemes realised similar true genetic gains from generations t = 4, …, 10. POCS and GOCS schemes realised 12% more true genetic gain from generations t = 4, …, 10 than truncation selection schemes. CONCLUSIONS: We advocate for OCS schemes with pre-selection against the LRA that cause animal suffering and high costs. At LRA frequencies of 0.10 or lower, OCS schemes in which male carriers are culled reduce the frequency of LRA, control rate of inbreeding, and realise no significant reduction in true genetic gain compared to OCS schemes without pre-selection against LRA.


Assuntos
Alelos , Cruzamento , Genes Letais , Genes Recessivos , Modelos Genéticos , Seleção Genética , Abate de Animais , Animais , Feminino , Frequência do Gene , Endogamia , Masculino , Linhagem , Processos Estocásticos
14.
Mol Neurodegener ; 16(1): 61, 2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34488813

RESUMO

Mutations in FUS, an RNA-binding protein involved in multiple steps of RNA metabolism, are associated with the most severe forms of amyotrophic lateral sclerosis (ALS). Accumulation of cytoplasmic FUS is likely to be a major culprit in the toxicity of FUS mutations. Thus, preventing cytoplasmic mislocalization of the FUS protein may represent a valuable therapeutic strategy. FUS binds to its own pre-mRNA creating an autoregulatory loop efficiently buffering FUS excess through multiple proposed mechanisms including retention of introns 6 and/or 7. Here, we introduced a wild-type FUS gene allele, retaining all intronic sequences, in mice whose heterozygous or homozygous expression of a cytoplasmically retained FUS protein (Fus∆NLS) was previously shown to provoke ALS-like disease or postnatal lethality, respectively. Wild-type FUS completely rescued the early lethality caused by the two Fus∆NLS alleles, and improved the age-dependent motor deficits and reduced lifespan caused by heterozygous expression of mutant FUS∆NLS. Mechanistically, wild-type FUS decreased the load of cytoplasmic FUS, increased retention of introns 6 and 7 in the endogenous mouse Fus mRNA, and decreased expression of the mutant mRNA. Thus, the wild-type FUS allele activates the homeostatic autoregulatory loop, maintaining constant FUS levels and decreasing the mutant protein in the cytoplasm. These results provide proof of concept that an autoregulatory competent wild-type FUS expression could protect against this devastating, currently intractable, neurodegenerative disease.


Assuntos
Esclerose Amiotrófica Lateral/terapia , Modelos Animais de Doenças , Regulação da Expressão Gênica/genética , Proteína FUS de Ligação a RNA/fisiologia , Alelos , Esclerose Amiotrófica Lateral/genética , Animais , Citoplasma/metabolismo , Demência Frontotemporal/genética , Genes Letais , Teste de Complementação Genética , Humanos , Íntrons/genética , Camundongos , Camundongos Transgênicos , Mutação , Ligação Proteica , Precursores de RNA/metabolismo , Proteína FUS de Ligação a RNA/deficiência , Proteína FUS de Ligação a RNA/genética , Proteínas Recombinantes/metabolismo , Transgenes
16.
Cell Rep ; 36(9): 109597, 2021 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-34469736

RESUMO

CRISPR screens have accelerated the discovery of important cancer vulnerabilities. However, single-gene knockout phenotypes can be masked by redundancy among related genes. Paralogs constitute two-thirds of the human protein-coding genome, so existing methods are likely inadequate for assaying a large portion of gene function. Here, we develop paired guide RNAs for paralog genetic interaction mapping (pgPEN), a pooled CRISPR-Cas9 single- and double-knockout approach targeting more than 2,000 human paralogs. We apply pgPEN to two cell types and discover that 12% of human paralogs exhibit synthetic lethality in at least one context. We recover known synthetic lethal paralogs MEK1/MEK2, important drug targets CDK4/CDK6, and other synthetic lethal pairs including CCNL1/CCNL2. Additionally, we identify ten tumor suppressor paralog pairs whose compound loss promotes cell proliferation. These findings nominate drug targets and suggest that paralog genetic interactions could shape the landscape of positive and negative selection in cancer.


Assuntos
Duplicação Gênica , Genes Letais , Genes Sintéticos , Genes Supressores de Tumor , Genoma Humano , Neoplasias/genética , Adulto , Antineoplásicos/farmacologia , Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas , Proliferação de Células , Feminino , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Masculino , Pessoa de Meia-Idade , Terapia de Alvo Molecular , Neoplasias/tratamento farmacológico , Neoplasias/patologia , RNA Guia/genética , RNA Guia/metabolismo
17.
Methods Mol Biol ; 2381: 39-56, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34590269

RESUMO

Synthetic dosage lethality (SDL) is a type of genetic interaction that occurs when increasing the expression of a gene causes a fitness defect, such as lethality, in a specific mutant background but has little effect on fitness in a wild-type background. SDL genetic interactions discovered in model organisms such as the budding yeast, Saccharomyces cerevisiae , represent candidate genetic interactions that may be conserved in human cells. In some cases, SDL genetic interactions can be applied to study the biological implications of genes overexpressed in cancer and to discover potential anticancer therapeutic drug targets. Here, we provide a protocol for screening a query overexpression gene against ordered arrays of yeast mutant strains to identify mutations that sensitize yeast to increased dosage of a specific gene product. We outline applications and procedures for screening with an inducibly overexpressed wild-type gene, a common feature of cancer cells, or with an inducibly overexpressed gene carrying a dominant-negative missense mutation as a model of protein-inhibitor interactions. This high-throughput screening platform is adapted from synthetic genetic array (SGA) technology and enables the generation of large-scale SDL genetic interaction networks that can be applied to study gene/pathway function and to identify cross-species cancer-relevant processes.


Assuntos
Genes Letais , Saccharomyces cerevisiae , Humanos , Mutação , Fenótipo , Saccharomyces cerevisiae/genética , Mutações Sintéticas Letais
18.
Sci Rep ; 11(1): 17093, 2021 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-34429461

RESUMO

Hybrid lethality, meaning the death of F1 hybrid seedlings, has been observed in many plant species, including Nicotiana. Previously, we have revealed that hybrids of the selected Nicotiana occidentalis accession and N. tabacum, an allotetraploid with S and T genomes, exhibited lethality characterized by the fading of shoot color. The lethality was suggested to be controlled by alleles of loci on the S and T genomes derived from N. sylvestris and N. tomentosiformis, respectively. Here, we extended the analysis of hybrid lethality using other two accessions of N. occidentalis identified from the five tested accessions. The two accessions were crossed with N. tabacum and its two progenitors, N. sylvestris and N. tomentosiformis. After crosses with N. tabacum, the two N. occidentalis accessions yielded inviable hybrid seedlings whose lethality was characterized by the fading of shoot color, but only the T genome of N. tabacum was responsible for hybrid lethality. Genetic analysis indicated that first-mentioned N. occidentalis accession carries a single gene causing hybrid lethality by allelic interaction with the S genome.


Assuntos
Genes Letais , Hibridização Genética , Tabaco/genética , Cruzamentos Genéticos , Aptidão Genética , Melhoramento Vegetal
19.
PLoS Genet ; 17(8): e1009744, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34424906

RESUMO

Postzygotic isolation by genomic conflict is a major cause for the formation of species. Despite its importance, the molecular mechanisms that result in the lethality of interspecies hybrids are still largely unclear. The genus Drosophila, which contains over 1600 different species, is one of the best characterized model systems to study these questions. We showed in the past that the expression levels of the two hybrid incompatibility factors Hmr and Lhr diverged in the two closely related Drosophila species, D. melanogaster and D. simulans, resulting in an increased level of both proteins in interspecies hybrids. The overexpression of the two proteins also leads to mitotic defects, a misregulation in the expression of transposable elements and decreased fertility in pure species. In this work, we describe a distinct six subunit protein complex containing HMR and LHR and analyse the effect of Hmr mutations on complex integrity and function. Our experiments suggest that HMR needs to bring together components of centromeric and pericentromeric chromatin to fulfil its physiological function and to cause hybrid male lethality.


Assuntos
Proteínas de Drosophila/genética , Isolamento Reprodutivo , Animais , Centrômero/metabolismo , Elementos de DNA Transponíveis/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Drosophila simulans/genética , Drosophila simulans/metabolismo , Genes Letais/genética , Especiação Genética , Hibridização Genética/genética , Reprodução/genética
20.
Hum Mutat ; 42(11): 1473-1487, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34405919

RESUMO

We aimed to identify incompletely penetrant (IP) variants and interallelic interactions in autosomal recessive disorders by a population-genetic approach. Genotype and clinical data were collected from 9038 patients of European origin with ASL, ATP7B, CAPN3, CFTR, CTNS, DHCR7, GAA, GALNS, GALT, IDUA, MUT, NPHS1, NPHS2, PAH, PKHD1, PMM2, or SLC26A4-related disorders. We calculated the relative allele frequency of each pathogenic variant (n = 1936) to the loss-of-function (LOF) variants of the corresponding gene in the patient ( A C p t V / A C p t L O F ) and the general population ( AC gnomAD V / AC gnomAD LOF ) and estimated the penetrance of each variant by calculating their ratio: ( A C p t V / A C p t L O F ) ( A C g n o m A D V / A C g n o m A D L O F ) (V/LOF ratio). We classified all variants as null or hypomorphic based on the associated clinical phenotype. We found 25 variants, 29% of the frequent 85 variants, to be underrepresented in the patient population (V/LOF ratio <30% with p < 7.22 × 10-5 ), including 22 novel ones in the ASL, CAPN3, CFTR, GAA, GALNS, PAH, and PKHD1 genes. In contrast to the completely penetrant variants (CP), the majority of the IP variants were hypomorphic (IP: 16/18, 88%; CP: 177/933, 19.0%; p = 5.12 × 10-10 ). Among them, only the NPHS2 R229Q variant was subject to interallelic interactions. The proposed algorithm identifies frequent IP variants and estimates their penetrance and interallelic interactions in large patient cohorts.


Assuntos
Alelos , Genes Recessivos , Doenças Genéticas Inatas/genética , Genética Populacional , Estudos de Coortes , Feminino , Genes Letais , Humanos , Masculino
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