RESUMEN
Human noroviruses (NoVs) are the most common cause of acute gastroenteritis worldwide. One major obstacle in developing NoV vaccines is the lack of robust cell culture for efficacy evaluation. In this study, we successfully developed a NoV virus-like particle (VLP) entry assay based on split NanoLuc luciferase (LgBiT and HiBiT) complementation. HiBiT-tagged NoV GII.4 VLP (VLP-HiBiT) can be efficiently produced in Pichia pastoris and retain binding activity towards NoV receptor histo-blood group antigens (HBGAs). A 293T-FUT2-LgBiT cell line was established and was shown to stably express cell surface HBGAs and intracellular LgBiT. GII.4 VLP-HiBiT can bind and enter into the 293-FUT2-LgBiT cells, producing strong luminescence signals in live cells. Anti-GII.4 sera can inhibit VLP-HiBiT entry into the 293-FUT2-LgBiT cells in a dose-dependent manner, and neutralizing titers well correlate with their blocking titers measured by HBGAs-binding blockade assay. Moreover, such a surrogate infection/neutralization assay can be applied to other NoV genotypes such as GI.1 and GII.17. Together, the VLP-HiBiT entry assay can mimic both NoV attachment and internalization in live cells and thus facilitate reliable and comprehensive evaluation of NoV vaccine and antibodies.
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Anticuerpos Antivirales/metabolismo , Anticuerpos Antivirales/farmacología , Luciferasas/genética , Norovirus/genética , Norovirus/inmunología , Internalización del Virus , Anticuerpos Antivirales/inmunología , Infecciones por Caliciviridae/virología , Prueba de Complementación Genética/métodos , Prueba de Complementación Genética/normas , Genotipo , Células HEK293 , Humanos , Luciferasas/metabolismo , Mediciones Luminiscentes , Saccharomycetales/genética , Vacunas de Partículas Similares a Virus/inmunología , Vacunas Virales/inmunología , Acoplamiento ViralRESUMEN
Pathological mutations in homology-directed repair (HDR) genes impact both future cancer risk and therapeutic options for patients. HDR is a high-fidelity DNA repair pathway for resolving DNA double-strand breaks throughout the genome. BRCA2 is an essential protein that mediates the loading of RAD51 onto resected DNA breaks, a key step in HDR. Germline mutations in BRCA2 are associated with an increased risk for breast, ovarian, prostate, and pancreatic cancer. Clinical findings of germline or somatic BRCA2 mutations in tumors suggest treatment with platinum agents or PARP inhibitors. However, when genetic analysis reveals a variant of uncertain significance (VUS) in the BRCA2 gene, precision medicine-based decisions become complex. VUS are genetic changes with unknown pathological impact. Current statistics indicate that between 10-20% of BRCA sequencing results are VUS, and of these, more than 50% are missense mutations. Functional assays to determine the pathological outcome of VUS are urgently needed to provide clinical guidance regarding cancer risk and treatment options. In this review, we provide a brief overview of BRCA2 functions in HDR, describe how BRCA2 VUS are currently assessed in the clinic, and how genetic and biochemical functional assays could be integrated into the clinical decision process. We suggest a multi-step workflow composed of robust and accurate functional assays to correctly evaluate the potential pathogenic or benign nature of BRCA2 VUS. Success in this precision medicine endeavor will offer actionable information to patients and their physicians.
Asunto(s)
Proteína BRCA2/genética , Toma de Decisiones Clínicas/métodos , Pruebas Genéticas/métodos , Síndrome de Cáncer de Mama y Ovario Hereditario/genética , Proteína BRCA2/metabolismo , Femenino , Prueba de Complementación Genética/métodos , Síndrome de Cáncer de Mama y Ovario Hereditario/diagnóstico , Síndrome de Cáncer de Mama y Ovario Hereditario/terapia , Humanos , Mutación , Flujo de TrabajoRESUMEN
The biosafety level 3 (BSL-3) requirement to culture severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a bottleneck for research. Here, we report a trans-complementation system that produces single-round infectious SARS-CoV-2 that recapitulates authentic viral replication. We demonstrate that the single-round infectious SARS-CoV-2 can be used at BSL-2 laboratories for high-throughput neutralization and antiviral testing. The trans-complementation system consists of two components: a genomic viral RNA containing ORF3 and envelope gene deletions, as well as mutated transcriptional regulator sequences, and a producer cell line expressing the two deleted genes. Trans-complementation of the two components generates virions that can infect naive cells for only one round but does not produce wild-type SARS-CoV-2. Hamsters and K18-hACE2 transgenic mice inoculated with the complementation-derived virions exhibited no detectable disease, even after intracranial inoculation with the highest possible dose. Thus, the trans-complementation platform can be safely used at BSL-2 laboratories for research and countermeasure development.
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COVID-19/virología , Contención de Riesgos Biológicos/métodos , SARS-CoV-2 , Células A549 , Animales , Chlorocebus aethiops , Cricetinae , Prueba de Complementación Genética/métodos , Genoma Viral , Células HEK293 , Humanos , Masculino , Ratones , Ratones Transgénicos , ARN Viral , SARS-CoV-2/genética , SARS-CoV-2/patogenicidad , SARS-CoV-2/fisiología , Células Vero , Virulencia , Replicación ViralRESUMEN
Cell cycle progression, or its arrest upon checkpoint activation, is directed by a complex array of cellular processes dependent on the diffusion of chemical signals. These signals regulate the onset of each cell cycle phase and prevent undesired phase transitions. Functional complementation is a robust strategy to identify such signals, by which mutant phenotypes are rescued through complementation with candidate factors. Here we describe a method that reclaims a five-decade old mammalian cell-cell fusion strategy of functional complementation to study the molecular control of cell cycle progression. The generation of cell-cell fusions (heterokaryons) allows for the analysis, via immunofluorescence, of cell cycle regulator dynamics and evaluating the effective rescue of cell cycle progression in specific genetic settings.
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Ciclo Celular , Prueba de Complementación Genética/métodos , Animales , Fusión Celular , Línea Celular , Células HeLa , Humanos , RatonesRESUMEN
The FabG 3-ketoacyl-acyl carrier protein (ACP) reductase of Escherichia coli has long been thought to be a classical member of the short-chain alcohol dehydrogenase/reductase (SDR) family. FabG catalyzes the essential 3-ketoacyl-ACP reduction step in the FAS II fatty acid synthesis pathway. Site-directed mutagenesis studies of several other SDR enzymes has identified three highly conserved amino acid residues, Ser, Tyr, and Lys, as the catalytic triad. Structural analyses of E. coli FabG suggested the triad S138-Y151-K155 to form a catalytically competent active site. To test this hypothesis, we constructed a series of E. coli FabG mutants and tested their 3-ketoacyl-ACP reductase activities both in vivo and in vitro. Our data show that plasmid-borne FabG mutants, including the double and triple mutants, restored growth of E. coli and Salmonella enterica fabG temperature-sensitive mutant strains under nonpermissive conditions. In vitro assays demonstrated that all of the purified FabG mutant proteins maintained fatty acid synthetic ability, although the activities of the single mutant proteins were 20% to 50% lower than that of wildtype FabG. The S138A, Y151F, and K155A residue substitutions were confirmed by tandem mass spectral sequencing of peptides that spanned all three residues. We conclude that FabG is not a classical short-chain alcohol dehydrogenase/reductase, suggesting that an alternative mode of 3-ketoacyl-ACP reduction awaits discovery.
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3-Oxoacil-(Proteína Transportadora de Acil) Reductasa/metabolismo , Oxidorreductasas de Alcohol/metabolismo , 3-Oxoacil-(Proteína Transportadora de Acil) Reductasa/fisiología , Oxidorreductasas de Alcohol/fisiología , Secuencia de Aminoácidos/genética , Sitios de Unión/fisiología , Dominio Catalítico/fisiología , Cristalografía por Rayos X/métodos , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Ácidos Grasos/metabolismo , Prueba de Complementación Genética/métodos , Modelos Moleculares , Oxidorreductasas/metabolismo , Unión Proteica/genéticaRESUMEN
This article describes several established approaches for genetic manipulation of Corynebacterium diphtheriae, the causative agent of diphtheria that is known to have provided key evidence for Koch's postulates on the germ theory. First, it includes a detailed gene deletion method that generates nonpolar, in-frame, markerless deletion mutants, utilizing the levansucrase SacB as a counter-selectable marker. Second, it provides a thorough protocol for rescuing deletion mutants using Escherichia coli-Corynebacterium shuttle vectors. Finally, a Tn5 transposon mutagenesis procedure is described. In principle, these protocols can be used for other Corynebacterium species, including Corynebacterium glutamicum and Corynebacterium matruchotii. © 2020 Wiley Periodicals LLC Basic Protocol 1: Gene deletion in Corynebacterium diphtheriae Basic Protocol 2: Complementation of a mutant strain Basic Protocol 3: Tn5 transposon mutagenesis of Corynebacterium diphtheriae.
Asunto(s)
Corynebacterium diphtheriae/genética , Corynebacterium/genética , Prueba de Complementación Genética/métodos , Mutagénesis Insercional/métodos , Elementos Transponibles de ADN , ADN Bacteriano , Difteria/microbiología , Escherichia coli/genética , Eliminación de Gen , Vectores Genéticos/genética , Hexosiltransferasas/genéticaRESUMEN
PCR-based amplification of annotated genes has allowed construction of expression clones at genome-scale using classical and recombination-based cloning technologies. However, genome-scale expression and purification of proteins for down-stream applications is often limited by challenges such as poor expression, low solubility, large size of multi-domain proteins, etc. Alternatively, DNA fragment libraries in expression vectors can serve as the source of protein fragments with each fragment encompassing a function of its whole protein counterpart. However, the random DNA fragmentation and cloning result in only 1 out of 18 clones being in the correct open-reading frame (ORF), thus, reducing the overall efficiency of the system. This necessitates the selection of correct ORF before expressing the protein fragments. This paper describes a highly efficient ORF selection system for DNA fragment libraries, which is based on split beta-lactamase protein fragment complementation. The system has been designed to allow seamless transfer of selected DNA fragment libraries into any downstream vector systems using a restriction enzyme-free cloning strategy. The strategy has been applied for the selection of ORF using model constructs to show near 100% selection of the clone encoding correct ORF. The system has been further validated by construction of an ORF-selected DNA fragment library of 30 genes of M. tuberculosis. Further, we have successfully demonstrated the cytosolic expression of ORF-selected protein fragments in E. coli.
Asunto(s)
Proteínas Bacterianas/genética , Clonación Molecular/métodos , Prueba de Complementación Genética/métodos , Sistemas de Lectura Abierta , beta-Lactamasas/genética , Proteínas Bacterianas/metabolismo , Escherichia coli , Biblioteca de Genes , Mycobacterium tuberculosis , beta-Lactamasas/metabolismoRESUMEN
Considered a commensal, the Gram-negative anaerobe Fusobacterium nucleatum is a key member of the oral microbiome due to its wide range of interactions with many oral microbes. While the periodontal pathogenic properties of this organism have widely been examined, its connotation with extra-oral infections, including preterm birth and colorectal cancer, has now become apparent. Nonetheless, little is known about the mechanisms of pathogenicity and the associated virulence factors of F. nucleatum, most likely due to limited genetic tools and facile methodology. Here, we describe molecular techniques for the genetic manipulation of F. nucleatum, including markerless, nonpolar gene deletion, complementation, and Tn5 transposon mutagenesis. Further, we provide methodology to assess virulence potential of F. nucleatum using a mouse model of preterm birth. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Generation of a galK mutant strain Basic Protocol 2: Complementation of a mutant strain Basic Protocol 3: Tn5 transposon mutagenesis of F. nucleatum Basic Protocol 4: Mouse model of preterm birth.
Asunto(s)
Infecciones por Fusobacterium/microbiología , Fusobacterium nucleatum/genética , Fusobacterium nucleatum/patogenicidad , Prueba de Complementación Genética/métodos , Técnicas Genéticas , Mutagénesis Insercional/métodos , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Elementos Transponibles de ADN , Modelos Animales de Enfermedad , Femenino , Fusobacterium nucleatum/fisiología , Eliminación de Gen , Humanos , Ratones , VirulenciaRESUMEN
BACKGROUND: For the majority of rare clinical missense variants, pathogenicity status cannot currently be classified. Classical homocystinuria, characterized by elevated homocysteine in plasma and urine, is caused by variants in the cystathionine beta-synthase (CBS) gene, most of which are rare. With early detection, existing therapies are highly effective. METHODS: Damaging CBS variants can be detected based on their failure to restore growth in yeast cells lacking the yeast ortholog CYS4. This assay has only been applied reactively, after first observing a variant in patients. Using saturation codon-mutagenesis, en masse growth selection, and sequencing, we generated a comprehensive, proactive map of CBS missense variant function. RESULTS: Our CBS variant effect map far exceeds the performance of computational predictors of disease variants. Map scores correlated strongly with both disease severity (Spearman's ϱ = 0.9) and human clinical response to vitamin B6 (ϱ = 0.93). CONCLUSIONS: We demonstrate that highly multiplexed cell-based assays can yield proactive maps of variant function and patient response to therapy, even for rare variants not previously seen in the clinic.
Asunto(s)
Cistationina betasintasa/genética , Prueba de Complementación Genética/métodos , Pruebas Genéticas/métodos , Homocistinuria/genética , Mutación Missense , Cistationina betasintasa/metabolismo , Genotipo , Humanos , Fenotipo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Recent large cancer studies have measured somatic alterations in an unprecedented number of tumours. These large datasets allow the identification of cancer-related sets of genetic alterations by identifying relevant combinatorial patterns. Among such patterns, mutual exclusivity has been employed by several recent methods that have shown its effectiveness in characterizing gene sets associated to cancer. Mutual exclusivity arises because of the complementarity, at the functional level, of alterations in genes which are part of a group (e.g., a pathway) performing a given function. The availability of quantitative target profiles, from genetic perturbations or from clinical phenotypes, provides additional information that can be leveraged to improve the identification of cancer related gene sets by discovering groups with complementary functional associations with such targets. In this work we study the problem of finding groups of mutually exclusive alterations associated with a quantitative (functional) target. We propose a combinatorial formulation for the problem, and prove that the associated computational problem is computationally hard. We design two algorithms to solve the problem and implement them in our tool UNCOVER. We provide analytic evidence of the effectiveness of UNCOVER in finding high-quality solutions and show experimentally that UNCOVER finds sets of alterations significantly associated with functional targets in a variety of scenarios. In particular, we show that our algorithms find sets which are better than the ones obtained by the state-of-the-art method, even when sets are evaluated using the statistical score employed by the latter. In addition, our algorithms are much faster than the state-of-the-art, allowing the analysis of large datasets of thousands of target profiles from cancer cell lines. We show that on two such datasets, one from project Achilles and one from the Genomics of Drug Sensitivity in Cancer project, UNCOVER identifies several significant gene sets with complementary functional associations with targets. Software available at: https://github.com/VandinLab/UNCOVER.
Asunto(s)
Biología Computacional/métodos , Neoplasias/genética , Análisis de Secuencia de ADN/métodos , Algoritmos , Regulación Neoplásica de la Expresión Génica/genética , Redes Reguladoras de Genes/genética , Prueba de Complementación Genética/métodos , Genómica/métodos , Humanos , Mutación , Programas InformáticosRESUMEN
Biological signaling networks use feedback control to dynamically adjust their operation in real time. Traditional static genetic methods such as gene knockouts or rescue experiments can often identify the existence of feedback interactions but are unable to determine what feedback dynamics are required. Here, we implement a new strategy, closed-loop optogenetic compensation (CLOC), to address this problem. Using a custom-built hardware and software infrastructure, CLOC monitors, in real time, the output of a pathway deleted for a feedback regulator. A minimal model uses these measurements to calculate and deliver-on the fly-an optogenetically enabled transcriptional input designed to compensate for the effects of the feedback deletion. Application of CLOC to the yeast pheromone response pathway revealed surprisingly distinct dynamic requirements for three well-studied feedback regulators. CLOC, a marriage of control theory and traditional genetics, presents a broadly applicable methodology for defining the dynamic function of biological feedback regulators.
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Retroalimentación Fisiológica , Regulación Fúngica de la Expresión Génica , Optogenética/métodos , Prueba de Complementación Genética/métodos , Factor de Apareamiento/genética , Factor de Apareamiento/metabolismo , Saccharomyces cerevisiae/genética , Programas Informáticos , Activación TranscripcionalRESUMEN
Cryptococcus neoformans is an opportunistic fungal pathogen, which causes life-threatening meningoencephalitis in immunocompromised individuals and is responsible for more than 1,000,000 infections and 600,000 deaths annually worldwide. Nevertheless, anti-cryptococcal therapeutic options are limited, mainly because of the similarity between fungal and human cellular structures. Owing to advances in genetic and molecular techniques and bioinformatics in the past decade, C. neoformans, belonging to the phylum basidiomycota, is now a major pathogenic fungal model system. In particular, genetic manipulation is the first step in the identification and characterization of the function of genes for understanding the mechanisms underlying the pathogenicity of C. neoformans. This unit describes protocols for constructing target gene deletion mutants using double-joint (DJ) PCR, constitutive overexpression strains using the histone H3 gene promoter, and epitope/fluorescence protein-tagged strains in C. neoformans. © 2018 by John Wiley & Sons, Inc.
Asunto(s)
Southern Blotting/métodos , Cryptococcus neoformans/genética , Prueba de Complementación Genética/métodos , Ingeniería Genética/métodos , Reacción en Cadena de la Polimerasa/métodos , Criptococosis/microbiología , Cryptococcus neoformans/fisiología , Humanos , Transformación BacterianaRESUMEN
Natural proteins represent a minuscule fraction of possible sequence space. These very rare sequences display remarkable properties: They fold into many different stable structures, and perform a wide range of complex biological functions. These two considerations-rarity and functionality-may suggest that natural proteins are somehow special. Is this true? We address this question by exploring attempts to recapitulate the special structures and functions of natural proteins into sequences designed de novo.
Asunto(s)
Proteínas de Escherichia coli/genética , Escherichia coli/genética , Prueba de Complementación Genética/métodos , Ingeniería de Proteínas/métodos , Secuencia de Aminoácidos , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Eliminación de Gen , Biblioteca de Genes , Modelos Moleculares , Unión Proteica , Conformación Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Pliegue de ProteínaRESUMEN
Human papillomavirus (HPV) genomes are replicated and maintained as extrachromosomal plasmids during persistent infection. The viral E2 proteins are thought to promote stable maintenance replication by tethering the viral DNA to host chromatin. However, this has been very difficult to prove genetically, as the E2 protein is involved in transcriptional regulation and initiation of replication, as well as its assumed role in genome maintenance. This makes mutational analysis of viral trans factors and cis elements in the background of the viral genome problematic and difficult to interpret. To circumvent this problem, we have developed a complementation assay in which the complete wild-type HPV18 genome is transfected into primary human keratinocytes along with subgenomic or mutated replicons that contain the minimal replication origin. The wild-type genome provides the E1 and E2 proteins in trans, allowing us to determine additional cis elements that are required for long-term replication and partitioning of the replicon. We found that, in addition to the core replication origin (and the three E2 binding sites located therein), additional sequences from the transcriptional enhancer portion of the URR (upstream regulatory region) are required in cis for long-term genome replication.IMPORTANCE Human papillomaviruses infect cutaneous and mucosal epithelial cells of the host, and this results in very-long-lived, persistent infection. The viral genomes are small, circular, double-stranded DNA molecules that replicate extrachromosomally in concert with cellular DNA. This replication strategy requires that the virus has a robust mechanism to partition and retain the viral genomes in dividing cells. This has been difficult to study, because viral transcription, replication, and partitioning are regulated by the same viral proteins and involve overlapping elements in the viral genome. We developed a complementation assay that allows us to separate these functions and define the elements required for long-term replication and stable maintenance replication of the HPV genome. This has important implications, as disruption of viral maintenance replication can eliminate viral genomes from infected cells, thus curing persistent HPV infection.
Asunto(s)
ADN Viral/genética , Genoma Viral , Papillomaviridae/genética , Factores de Transcripción/genética , Replicación Viral/genética , Sitios de Unión , Replicación del ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación Viral de la Expresión Génica , Prueba de Complementación Genética/métodos , Humanos , Queratinocitos/virología , Mutación , Proteínas Oncogénicas Virales/genética , Secuencias Reguladoras de Ácidos Nucleicos , Proteínas Virales/metabolismoRESUMEN
Dynamic protein-protein interactions (PPIs) play crucial roles in cell physiological processes. The protein-fragment complementation (PFC) assay has been developed as a powerful approach for the detection of PPIs, but its potential for identifying protein interacting regions is not optimized. Recently, an ascorbate peroxidase (APEX2)-based proximity-tagging method combined with mass spectrometry was developed to identify potential protein interactions in live cells. In this study, we tested whether APEX2 could be employed for PFC. By screening split APEX2 pairs attached to FK506-binding protein 12 (FKBP) and the FKBP12-rapamycin binding (FRB) domain, which interact with each other only in the presence of rapamycin, we successfully obtained an optimized pair for visualizing the interaction between FRB and FKBP12 with high specificity and sensitivity in live cells. The robustness of this APEX2 pair was confirmed by its application toward detecting the STIM1 and Orial1 homodimers in HEK-293 cells. With a subsequent mass spectrometry analysis, we obtained five different biotinylated sites that were localized to the known interaction region on STIM1 and were only detected when the homodimer formed. These results suggest that our PFC pair of APEX2 provides a potential tool for detecting PPIs and identifying binding regions with high specificity in live cells.
Asunto(s)
ADN-(Sitio Apurínico o Apirimidínico) Liasa/genética , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Prueba de Complementación Genética/métodos , Endonucleasas , Células HEK293 , Humanos , Enzimas Multifuncionales , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Molécula de Interacción Estromal 1/genética , Molécula de Interacción Estromal 1/metabolismo , Proteínas de Unión a Tacrolimus/genética , Proteínas de Unión a Tacrolimus/metabolismoRESUMEN
Two defective bovine parainfluenza virus type 3 (BPIV3) strains were generated, one lacking the membrane (M) protein gene and expressing EGFP (ΔM-EGFP) and the other lacking the fusion (F) protein gene and expressing mStrawberry (ΔF-mSB), by supplying deficient proteins in trans. When Madin-Darby bovine kidney (MDBK) cells were co-infected with ΔM-EGFP and ΔF-mSB at a multiplicity of infection (MOI) of 0.1, complemented viruses were easily obtained. Complemented viruses grew as efficiently as wild-type BPIV3 and could be passaged in MDBK cell cultures even at an MOI of 0.01, possibly due to multiploid virus particles containing genomes of both ΔM-EGFP and ΔF-mSB. This reciprocal complementation method using two defective viruses would be useful to express large or multiple proteins in cell cultures using paramyxovirus vectors.
Asunto(s)
Virus Defectuosos/genética , Prueba de Complementación Genética , Virus de la Parainfluenza 3 Bovina/genética , Animales , Bovinos , Técnicas de Cultivo de Célula , Línea Celular , Prueba de Complementación Genética/instrumentación , Prueba de Complementación Genética/métodos , Vectores Genéticos , Virus de la Parainfluenza 3 Bovina/crecimiento & desarrollo , Proteínas Virales de Fusión/genética , Proteínas de la Matriz Viral/genética , Replicación ViralRESUMEN
Ammonium is an important nitrogen sources for plant growth. In this study, we report on the gene characterization of the ammonium transporter AMT1 subfamily in the apple rootstock Malus robusta Rehd. Thirteen AMT genes were comprehensively evaluated from the apple genome (version 1.0). Then the gene features and expression patterns of five AMT1 members from M. robusta were analyzed. These genes fell into four clusters in the AMT phylogenetic tree: clade I (MrAMT1;1 and MrAMT1;3), clade II (MrAMT1;4), clade III (MrAMT1;2), and clade IV (MrAMT1;5). All the AMT1s, apart from MrAMT1;4, were expressed in vegetative organs and strongly responded to nitrogen concentration changes. For example, MrAMT1;2 and MrAMT1;3 had high transcript accumulation levels in the leaves and roots, respectively. Finally, the functions of these AMT1s were studied in detail by heterologous expression in yeast. These genes allowed strain 31019b to assimilate nitrogen, but their 15NH4+ uptake kinetics varied. These results revealed the functional roles of AMT1 during ammonium absorption in the AMT-defective mutant yeast system.
Asunto(s)
Proteínas de Transporte de Catión/genética , Malus/genética , Proteínas de Plantas/genética , Secuencia de Aminoácidos , Compuestos de Amonio/metabolismo , Transporte Biológico , Proteínas de Transporte de Catión/metabolismo , Regulación de la Expresión Génica de las Plantas , Prueba de Complementación Genética/métodos , Malus/metabolismo , Nitrógeno/metabolismo , Filogenia , Proteínas de Plantas/metabolismo , Raíces de Plantas/metabolismoRESUMEN
The synthesis of unsaturated fatty acids in Mycobacterium smegmatis is poorly characterized. Bioinformatic analysis revealed four putative fatty acid desaturases in its genome, one of which, MSMEG_1886, is highly homologous to desA3, the only palmitoyl/stearoyl desaturase present in the Mycobacterium tuberculosis genome. A MSMEG_1886 deletion mutant was partially auxotrophic for oleic acid and viable at 37°C and 25°C, although with a long lag phase in liquid medium. Fatty acid analysis suggested that MSMEG_1886 is a palmitoyl/stearoyl desaturase, as the synthesis of palmitoleic acid was abrogated, while oleic acid contents dropped by half in the mutant. Deletion of the operon MSMEG_1741-1743 (highly homologous to a Pseudomonas aeruginosa acyl-CoA desaturase) had little effect on growth of the parental strain; however the double mutant MSMEG_1886-MSMEG_1741-1743 strictly required oleic acid for growth. The ΔMSMEG_1886-ΔMSMEG_1741 double mutant was able to grow (poorly but better than the ΔMSMEG_1886 single mutant) in solid and liquid media devoid of oleic acid, suggesting a repressor role for ΔMSMEG_1741. Fatty acid analysis of the described mutants suggested that MSMEG_1742-43 desaturates C18:0 and C24:0 fatty acids. Thus, although the M. smegmatis desA3 homologue is the major player in unsaturated fatty acid synthesis, a second set of genes is also involved.
Asunto(s)
Ácido Graso Desaturasas/genética , Ácidos Grasos Insaturados/biosíntesis , Mycobacterium smegmatis/genética , Secuencia de Aminoácidos/genética , Proteínas Bacterianas/genética , Ácido Graso Desaturasas/metabolismo , Ácidos Grasos/biosíntesis , Ácidos Grasos/genética , Ácidos Grasos/metabolismo , Ácidos Grasos Monoinsaturados/metabolismo , Ácidos Grasos Insaturados/genética , Ácidos Grasos Insaturados/metabolismo , Prueba de Complementación Genética/métodos , Eliminación de Secuencia/genética , Homología de Secuencia de AminoácidoRESUMEN
The combination of drug resistance, lack of an effective vaccine, and ongoing conflict and poverty means that malaria remains a major global health crisis. Understanding metabolic pathways at all parasite life stages is important in prioritising and targeting novel anti-parasitic compounds. The unusual heme synthesis pathway of the rodent malaria parasite, Plasmodium berghei, requires eight enzymes distributed across the mitochondrion, apicoplast and cytoplasm. Deletion of the ferrochelatase (FC) gene, the final enzyme in the pathway, confirms that heme synthesis is not essential in the red blood cell stages of the life cycle but is required to complete oocyst development in mosquitoes. The lethality of FC deletions in the mosquito stage makes it difficult to study the impact of these mutations in the subsequent liver stage. To overcome this, we combined locus-specific fluorophore expression with a genetic complementation approach to generate viable, heterozygous oocysts able to produce a mix of FC expressing and FC deficient sporozoites. These sporozoites show normal motility and can invade liver cells, where FC deficient parasites can be distinguished by fluorescence and phenotyped. Parasites lacking FC exhibit a severe growth defect within liver cells, with development failure detectable in the early to mid stages of liver development in vitro. FC deficient parasites could not complete liver stage development in vitro nor infect naïve mice, confirming liver stage arrest. These results validate the heme pathway as a potential target for prophylactic drugs targeting liver stage parasites. In addition, we demonstrate that our simple genetic approach can extend the phenotyping window beyond the insect stages, opening considerable scope for straightforward reverse genetic analysis of genes that are dispensable in blood stages but essential for completing mosquito development.
Asunto(s)
Anopheles/parasitología , Prueba de Complementación Genética/métodos , Hemo/biosíntesis , Hígado/parasitología , Malaria/parasitología , Plasmodium berghei/crecimiento & desarrollo , Plasmodium berghei/metabolismo , Proteínas Protozoarias/metabolismo , Animales , Anopheles/crecimiento & desarrollo , Eritrocitos/metabolismo , Eritrocitos/parasitología , Femenino , Humanos , Estadios del Ciclo de Vida , Hígado/metabolismo , Masculino , Ratones , Plasmodium berghei/enzimología , Plasmodium berghei/genética , Proteínas Protozoarias/genéticaRESUMEN
Triggering receptor expressed on myeloid cells 2 (TREM2) is a single transmembrane molecule uniquely expressed in microglia. TREM2 mutations are genetically linked to Nasu-Hakola disease and associated with multiple neurodegenerative disorders, including Alzheimer's disease. TREM2 may regulate microglial inflammation and phagocytosis through coupling to the adaptor protein TYRO protein-tyrosine kinase-binding protein (TYROBP). However, there is no functional system for monitoring this protein-protein interaction. We developed a luciferase-based modality for real-time monitoring of TREM2-TYROBP coupling in live cells that utilizes split-luciferase complementation technology based on TREM2 and TYROBP fusion to the C- or N-terminal portion of the Renilla luciferase gene. Transient transfection of human embryonic kidney 293 cells with this reporter vector increased luciferase activity upon stimulation with an anti-TREM2 antibody, which induces their homodimerization. This was confirmed by ELISA-based analysis of the TREM2-TYROBP interaction. Antibody-mediated TREM2 stimulation enhanced spleen tyrosine kinase (SYK) activity and uptake of Staphylococcus aureus in microglial cell line BV-2 in a kinase-dependent manner. Interestingly, the TREM2 T66M mutation significantly enhanced luciferase activity without stimulation, indicating constitutive coupling to TYROBP. Finally, flow cytometry analyses indicated significantly lower surface expression of T66M TREM2 variant than wild type or other TREM2 variants. These results demonstrate that our TREM2 reporter vector is a novel tool for monitoring the TREM2-TYROBP interaction in real time.