RESUMO
The fractal characteristics of DNA sequences are studied using the frequency chaos game representation (FCGR) and small-angle scattering (SAS) technique. The FCGR allows representation of the frequencies of occurrence of k-mers (oligonucleotides of length k) in the form of images. The numerically encoded data are then used in a SAS analysis to enhance hidden features in DNA sequences. It is shown that the simulated SAS intensity allows us to obtain the fractal dimensions and scaling factors at various scales. These structural parameters can be used to distinguish unambiguously between the scaling properties of complex hierarchical DNA sequences. The validity of this approach is illustrated on several sequences from: Escherichia coli, Mouse mitochondrion, Homo sapiens mitochondrion and Human cosmid.
Assuntos
Cosmídeos/genética , Escherichia coli/genética , Mitocôndrias/genética , Análise de Sequência de DNA/métodos , Algoritmos , Animais , DNA Bacteriano/genética , DNA Mitocondrial/genética , Fractais , Humanos , Camundongos , Dinâmica não Linear , Espalhamento a Baixo Ângulo , Fatores de TempoRESUMO
Cosmid libraries constructed from environmental metagenome samples are powerful tools for capturing the genomic diversity of complex microbial communities. The large insert size (â¼35 kb) of such libraries means they are compatible with downstream expression of large biosynthetic gene clusters (BGCs). This allows the discovery of previously undescribed natural products that would be inaccessible using traditional culture-based discovery pipelines. Here we describe methods for the construction of a cosmid metagenome library from a soil sample, and the process of screening that library for individual cosmid clones containing aromatic polyketide BGCs using degenerate primers that target the ketosynthase alpha (KSα) gene.
Assuntos
Metagenoma , Solo , Cosmídeos , Biblioteca Gênica , Microbiologia do SoloRESUMO
Simultaneous expression of multiplex guide RNAs (gRNAs) is valuable for knockout of multiple genes and also for effective disruption of a gene by introducing multiple deletions. We developed a method of Tetraplex-guide Tandem for construction of cosmids containing four and eight multiplex gRNA-expressing units in one step utilizing lambda in vitro packaging. Using this method, we produced an adenovirus vector (AdV) containing four multiplex-gRNA units for two double-nicking sets. Unexpectedly, the AdV could stably be amplified to the scale sufficient for animal experiments with no detectable lack of the multiplex units. When the AdV containing gRNAs targeting the H2-Aa gene and an AdV expressing Cas9 nickase were mixed and doubly infected to mouse embryonic fibroblast cells, deletions were observed in more than 80% of the target gene even using double-nicking strategy. Indels were also detected in about 20% of the target gene at two sites in newborn mouse liver cells by intravenous injection. Interestingly, when one double-nicking site was disrupted, the other was simultaneously disrupted, implying that two genes in the same cell may simultaneously be disrupted in the AdV system. The AdVs expressing four multiplex gRNAs could offer simultaneous knockout of four genes or two genes by double-nicking cleavages with low off-target effect.
Assuntos
Adenoviridae/genética , Engenharia Genética/métodos , /genética , Animais , Sistemas CRISPR-Cas , Cosmídeos , Fibroblastos/metabolismo , Edição de Genes/métodos , Vetores Genéticos/genética , Células HEK293 , Células Hep G2 , Humanos , Mutação INDEL/genética , Camundongos Endogâmicos C57BL , Plasmídeos/genética , /metabolismoRESUMO
BACKGROUND: Chelocardin (CHD) exhibits a broad-spectrum antibiotic activity and showed promising results in a small phase II clinical study conducted on patients with urinary tract infections. Importantly, CHD was shown to be active also against tetracycline-resistant Gram-negative pathogens, which is gaining even more importance in today's antibiotic crisis. We have demonstrated that modifications of CHD through genetic engineering of its producer, the actinomycete Amycolatopsis sulphurea, are not only possible but yielded even more potent antibiotics than CHD itself, like 2-carboxamido-2-deacetyl-chelocardin (CD-CHD), which is currently in preclinical evaluation. A. sulphurea is difficult to genetically manipulate and therefore manipulation of the chd biosynthetic gene cluster in a genetically amenable heterologous host would be of high importance for further drug-discovery efforts. RESULTS: We report heterologous expression of the CHD biosynthetic gene cluster in the model organism Streptomyces albus del14 strain. Unexpectedly, we found that the originally defined CHD gene cluster fails to provide all genes required for CHD formation, including an additional cyclase and two regulatory genes. Overexpression of the putative pathway-specific streptomyces antibiotic regulatory protein chdB in A. sulphurea resulted in an increase of both, CHD and CD-CHD production. Applying a metabolic-engineering approach, it was also possible to generate the potent CHD analogue, CD-CHD in S. albus. Finally, an additional yield increase was achieved in S. albus del14 by in-trans overexpression of the chdR exporter gene, which provides resistance to CHD and CDCHD. CONCLUSIONS: We identified previously unknown genes in the CHD cluster, which were shown to be essential for chelocardin biosynthesis by expression of the full biosynthetic gene cluster in S. albus as heterologous host. When comparing to oxytetracycline biosynthesis, we observed that the CHD gene cluster contains additional enzymes not found in gene clusters encoding the biosynthesis of typical tetracyclines (such as oxytetracycline). This finding probably explains the different chemistries and modes of action, which make CHD/CD-CHD valuable lead structures for clinical candidates. Even though the CHD genes are derived from a rare actinomycete A. sulphurea, the yield of CHD in the heterologous host was very good. The corrected nucleotide sequence of the CHD gene cluster now contains all gene products required for the production of CHD in a genetically amenable heterologous host, thus opening new possibilities towards production of novel and potent tetracycline analogues with a new mode of action.
Assuntos
Genes Bacterianos , Família Multigênica , Streptomyces/genética , Tetraciclinas/biossíntese , Amycolatopsis/genética , Amycolatopsis/metabolismo , Antibacterianos/biossíntese , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Vias Biossintéticas , Clonagem Molecular , Cosmídeos , Engenharia Metabólica , Streptomyces/metabolismo , Tetraciclinas/farmacologiaRESUMO
BACKGROUND: Genome editing using the CRISPR/Cas9 system is now well documented in basic studies and is expected to be applied to gene therapy. Simultaneous expression of multiplex guide RNA (gRNA) and Cas9/Cas9 derivative is attractive for the efficient knockout of genes and a safe double-nicking strategy. However, such use is limited because highly multiplex gRNA-expressing units are difficult to maintain stably in plasmids as a result of deletion via homologous recombination. METHODS: Lambda in vitro packaging was used instead of transformation for the construction and preparation of large, cos-containing plasmid (cosmid). Polymerase chain reaction fragments containing multiplex gRNA units were obtained using the Four-guide Tandem method. Transfection was performed by lipofection. RESULTS: We constructed novel cosmids consisting of linearized plasmid-DNA fragments containing up to 16 copies of multiplex gRNA-expressing units as trimer or tetramer (polygonal cosmids). These cosmids behaved as if they were monomer plasmids, and multiplex units could stably be maintained and amplified with a lack of deletion. Surprisingly, the deleted cosmid was removed out simply by amplifying the cosmid stock using lambda packaging. The DNA fragments containing multiplex gRNA-units and Cas9 were transfected to 293 cells and were found to disrupt the X gene of hepatitis B virus by deleting a large region between the predicted sites. CONCLUSIONS: We present a simple method for overcoming the problem of constructing plasmids stably containing multiplex gRNA-expressing units. The method may enable the production of very large amounts of DNA fragments expressing intact, highly-multiplex gRNAs and Cas9/Cas9 derivatives for safe and efficient genome-editing therapy using non-viral vectors.
Assuntos
Sistemas CRISPR-Cas , Cosmídeos/genética , Amplificação de Genes , Edição de Genes , Expressão Gênica , Bacteriófago lambda/genética , Ordem dos Genes , Marcação de Genes , Vírus da Hepatite B/genética , Humanos , Deleção de Sequência , TransfecçãoRESUMO
Cosmid libraries can represent an entire genome in a library of circular DNA molecules, allowing for the faithful amplification, cloning and isolation of large genomic DNA fragments. Moreover, using the so-called shuttle cosmid vectors, genomic DNA may be propagated in bacteria and in eukaryotic cells, which is a prerequisite for classic functional cloning and for the newly described Cos-Seq strategies.
Assuntos
Clonagem Molecular , Cosmídeos/genética , Biblioteca Gênica , Leishmania/genéticaRESUMO
We apply matrix completion methods for haplotype assembly from NGS reads to develop the new HapSVT, HapNuc, and HapOPT algorithms. This is performed by applying a mathematical model to convert the reads to an incomplete matrix and estimating unknown components. This process is followed by quantizing and decoding the completed matrix in order to estimate haplotypes. These algorithms are compared to the state-of-the-art algorithms using simulated data as well as the real fosmid data. It is shown that the SNP missing rate and the haplotype block length of the proposed HapOPT are better than those of HapCUT2 with comparable accuracy in terms of reconstruction rate and switch error rate. A program implementing the proposed algorithms in MATLAB is freely available at https://github.com/smajidian/HapMC.
Assuntos
Haplótipos/genética , Sequenciamento de Nucleotídeos em Larga Escala , Algoritmos , Benchmarking , Cosmídeos/genética , Modelos Genéticos , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Visceral leishmaniasis (VL), caused by the protozoan parasites Leishmania donovani and L. infantum, is responsible for â¼30â¯000 deaths annually. Available treatments are inadequate, and there is a pressing need for new therapeutics. N-Myristoyltransferase (NMT) remains one of the few genetically validated drug targets in these parasites. Here, we sought to pharmacologically validate this enzyme in Leishmania. A focused set of 1600 pyrazolyl sulfonamide compounds was screened against L. major NMT in a robust high-throughput biochemical assay. Several potent inhibitors were identified with marginal selectivity over the human enzyme. There was little correlation between the enzyme potency of these inhibitors and their cellular activity against L. donovani axenic amastigotes, and this discrepancy could be due to poor cellular uptake due to the basicity of these compounds. Thus, a series of analogues were synthesized with less basic centers. Although most of these compounds continued to suffer from relatively poor antileishmanial activity, our most potent inhibitor of LmNMT (DDD100097, K i of 0.34 nM) showed modest activity against L. donovani intracellular amastigotes (EC50 of 2.4 µM) and maintained a modest therapeutic window over the human enzyme. Two unbiased approaches, namely, screening against our cosmid-based overexpression library and thermal proteome profiling (TPP), confirm that DDD100097 (compound 2) acts on-target within parasites. Oral dosing with compound 2 resulted in a 52% reduction in parasite burden in our mouse model of VL. Thus, NMT is now a pharmacologically validated target in Leishmania. The challenge in finding drug candidates remains to identify alternative strategies to address the drop-off in activity between enzyme inhibition and in vitro activity while maintaining sufficient selectivity over the human enzyme, both issues that continue to plague studies in this area.
Assuntos
Aciltransferases/antagonistas & inibidores , Antiprotozoários/farmacologia , Descoberta de Drogas , Leishmania donovani/efeitos dos fármacos , Pirazóis/química , Pirazóis/farmacologia , Animais , Cosmídeos , Feminino , Ensaios de Triagem em Larga Escala , Humanos , Leishmaniose Visceral/tratamento farmacológico , Camundongos , Camundongos Endogâmicos BALB C , Carga Parasitária , Proteoma/análise , ProteômicaRESUMO
The in vivo physiological role of the gene cobZ, which encodes precorrin-3B synthase, which catalyzes the initial porphyrin ring contraction step of cobalamin biosynthesis via the cob pathway, has been demonstrated here for the first time. Cobalamin is known to be essential for an early step of bacteriochlorophyll biosynthesis in anoxygenic purple bacteria. The cobZ (cobZRR) gene of the purple bacterium Rhodospirillum rubrum was localized to a 23.5 kb insert of chromosomal DNA contained on the cosmid pSC4. pSC4 complemented several mutants of bacteriochlorophyll and carotenoid biosynthesis, due to the presence of the bchCX and crtCDEF genes at one end of the cosmid insert, flanking cobZRR. A second gene, citB/tcuB, immediately downstream of cobZRR, shows homologies to both a tricarballylate oxidoreductase (tcuB) and a gene (citB) involved in signal transduction during citrate uptake. CobZRR shows extensive homology to the N-terminal domain of the bifunctional CobZ from Rhodobacter capsulatus, and the R. rubrum citB/tcuB gene is homologous to the CobZ C-terminal domain. A mutant, SERGK25, containing a terminatorless kanamycin interposon inserted into cobZRR, could not grow by anaerobic photosynthesis, but grew normally under dark, aerobic and microaerophilic conditions with succinate and fructose as carbon sources. The anaerobic in vivo activity of CobZ indicates that it does not require oxygen as a substrate. The mutant excreted large amounts of protoporphyrin IX-monomethylester, a brown precursor of bacteriochlorophyll biosynthesis. The mutant was complemented either by the cobZRR gene in trans, or when exogenous cobalamin was added to the medium. A deletion mutant of tcuB/citB did not exhibit the cob phenotype. Thus, a role for tcuB/citB in cobalamin biosynthesis could not be confirmed.
Assuntos
Fotossíntese/fisiologia , Rhodospirillum rubrum , Vitamina B 12/biossíntese , Sequência de Aminoácidos , Bacterioclorofilas/biossíntese , Carotenoides/biossíntese , Cosmídeos/genética , DNA Bacteriano/genética , Deleção de Genes , Metiltransferases/genética , Oxirredutases/genética , Oxigênio/metabolismo , Porfirinas/metabolismo , Rhodospirillum rubrum/enzimologia , Rhodospirillum rubrum/genética , Rhodospirillum rubrum/metabolismoRESUMO
Increasing drug resistance towards first line antimony-derived compounds has forced the introduction of novel therapies in leishmaniasis endemic areas including amphotericin B and miltefosine. However, their use is threatened by the emergence and spread of drug-resistant strains. In order to discover stage-dependent resistance genes, we have adapted the Cos-Seq approach through the introduction of macrophage infections in the pipeline. A L. infantum intracellular amastigote population complemented with a L. infantum cosmid library was submitted to increasing concentrations of miltefosine, amphotericin B and pentavalent antimonials in experimental infections of THP-1â¯cells. For each step of selection, amastigotes were extracted and cosmids were isolated and submitted to next-generation sequencing, followed by subsequent gene-enrichment analyses. Cos-Seq screen in amastigotes revealed four highly enriched loci for antimony, five for miltefosine and one for amphotericin B. Of these, a total of seven cosmids were recovered and tested for resistance in both promastigotes and amastigotes. Candidate genes within the pinpointed genomic regions were validated using single gene overexpression in wild-type parasites and/or gene disruption by means of a CRISPR-Cas9-based approach. This led to the identification and validation of a stage-independent antimony-resistance gene (LinJ.06.1010) coding for a putative leucine rich repeat protein and a novel amastigote-specific miltefosine-resistance gene (LinJ.32.0050) coding for a member of the SEC13 family of WD-repeat proteins. This study further reinforces the power of Cos-Seq approach to discover novel drug-resistance genes, some of which are life-stages specific.
Assuntos
Antiprotozoários/farmacologia , Resistência a Medicamentos/genética , Sequenciamento de Nucleotídeos em Larga Escala , Leishmania infantum/efeitos dos fármacos , Leishmania infantum/genética , Anfotericina B/farmacologia , Animais , Antimônio/farmacologia , Antimônio/uso terapêutico , Sistemas CRISPR-Cas , Cosmídeos , Biblioteca Gênica , Leishmaniose/tratamento farmacológico , Estágios do Ciclo de Vida/efeitos dos fármacos , Estágios do Ciclo de Vida/genética , Macrófagos/parasitologia , Fosforilcolina/análogos & derivados , Fosforilcolina/farmacologia , Fosforilcolina/uso terapêuticoRESUMO
The study of aflatoxin in Aspergillus spp. has garnered the attention of many researchers due to aflatoxin's carcinogenic properties and frequency as a food and feed contaminant. Significant progress has been made by utilizing the model organism Aspergillus nidulans to characterize the regulation of sterigmatocystin (ST), the penultimate precursor of aflatoxin. A previous forward genetic screen identified 23 A. nidulans mutants involved in regulating ST production. Six mutants were characterized from this screen using classical mapping (five mutations in mcsA) and complementation with a cosmid library (one mutation in laeA). The remaining mutants were backcrossed and sequenced using Illumina and Ion Torrent sequencing platforms. All but one mutant contained one or more sequence variants in predicted open reading frames. Deletion of these genes resulted in identification of mutant alleles responsible for the loss of ST production in 12 of the 17 remaining mutants. Eight of these mutations were in genes already known to affect ST synthesis (laeA, mcsA, fluG, and stcA), while the remaining four mutations (in laeB, sntB, and hamI) were in previously uncharacterized genes not known to be involved in ST production. Deletion of laeB, sntB, and hamI in A. flavus results in loss of aflatoxin production, confirming that these regulators are conserved in the aflatoxigenic aspergilli. This report highlights the multifaceted regulatory mechanisms governing secondary metabolism in Aspergillus Additionally, these data contribute to the increasing number of studies showing that forward genetic screens of fungi coupled with whole-genome resequencing is a robust and cost-effective technique.IMPORTANCE In a postgenomic world, reverse genetic approaches have displaced their forward genetic counterparts. The techniques used in forward genetics to identify loci of interest were typically very cumbersome and time-consuming, relying on Mendelian traits in model organisms. The current work was pursued not only to identify alleles involved in regulation of secondary metabolism but also to demonstrate a return to forward genetics to track phenotypes and to discover genetic pathways that could not be predicted through a reverse genetics approach. While identification of mutant alleles from whole-genome sequencing has been done before, here we illustrate the possibility of coupling this strategy with a genetic screen to identify multiple alleles of interest. Sequencing of classically derived mutants revealed several uncharacterized genes, which represent novel pathways to regulate and control the biosynthesis of sterigmatocystin and of aflatoxin, a societally and medically important mycotoxin.
Assuntos
Aspergillus nidulans/genética , Aspergillus nidulans/metabolismo , Regulação Fúngica da Expressão Gênica , Metabolismo Secundário/genética , Cosmídeos/genética , DNA Fúngico/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genes Fúngicos , Teste de Complementação Genética , Genoma Fúngico , Sequenciamento de Nucleotídeos em Larga Escala , Mutação , Esterigmatocistina/metabolismoRESUMO
The majority of environmental bacteria are not readily cultured in the lab, leaving the natural products they make inaccessible using culture-dependent discovery methods. Cloning and heterologous expression of DNA extracted from environmental samples (environmental DNA, eDNA) provides a means of circumventing this discovery bottleneck. To facilitate the identification of clones containing biosynthetic gene clusters, we developed a model heterologous expression reporter strain Streptomyces albus::bpsA ΔPPTase. This strain carries a 4Î-phosphopantetheinyl transferase (PPTase)-dependent blue pigment synthase A gene, bpsA, in a PPTase deletion background. eDNA clones that express a functional PPTase restore production of the blue pigment, indigoidine. As PPTase genes often occur in biosynthetic gene clusters (BGCs), indigoidine production can be used to identify eDNA clones containing BGCs. We screened a soil eDNA library hosted in S. albus::bpsA ΔPPTase and identified clones containing non-ribosomal peptide synthetase (NRPS), polyketide synthase (PKS) and mixed NRPS/PKS biosynthetic gene clusters. One NRPS gene cluster was shown to confer the production of myxochelin A to S. albus::bpsA ΔPPTase.
Assuntos
Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Clonagem Molecular/métodos , Genes Bacterianos , Metagenoma/genética , Família Multigênica , Streptomyces/genética , Transferases (Outros Grupos de Fosfato Substituídos)/classificação , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Produtos Biológicos , Cosmídeos , Escherichia coli/genética , Teste de Complementação Genética , Biblioteca Genômica , Metagenômica , Peptídeo Sintases/genética , Filogenia , Piperidonas/metabolismo , Microbiologia do SoloRESUMO
Nonpathogenic fowl adenoviruses (FAdVs) are amenable for engineering multivalent vaccine platforms due to large stretches of nonessential DNA sequences in their genomes. We describe the generation of FAdV-9-based vaccine platforms by targeted homologous recombination in an infectious clone (pPacFAdV-9 or wild type FAdmid) containing the entire viral genome in a cosmid vector. The viral DNA is subsequently released from the cosmid by restriction enzyme digestion followed by transfection in a chicken hepatoma cell line (CH-SAH). Virus is harvested, propagated, and verified for foreign gene expression.
Assuntos
Aviadenovirus/genética , Cosmídeos/genética , Vacinas Virais/genética , Animais , Linhagem Celular Tumoral , Galinhas , Vetores Genéticos/genética , Genoma Viral , TransfecçãoRESUMO
Aristeromycin is a unique carbocyclic nucleoside antibiotic produced by Streptomyces citricolor. In order to elucidate its intriguing carbocyclic formation, we used a genome-mining approach to identify the responsible enzyme. In silico screening with known cyclitol synthases involved in primary metabolism, such as myo-inositol-1-phosphate synthase (MIPS) and dehydroqunate synthase (DHQS), identified a unique MIPS orthologue (Ari2) encoded in the genome of S.â citricolor. Heterologous expression of the gene cluster containing ari2 with a cosmid vector in Streptomyces albus resulted in the production of aristeromycin, thus indicating that the cloned DNA region (37.5â kb) with 33 open reading frames contains its biosynthetic gene cluster. We verified that Ari2 catalyzes the formation of a novel five-membered cyclitol phosphate from d-fructose 6-phosphate (F6P) with NAD+ as a cofactor. This provides insight into cyclitol phosphate synthase as a member of the MIPS family of enzymes. A biosynthetic pathway to aristeromycin is proposed based on bioinformatics analysis of the gene cluster.
Assuntos
Adenosina/análogos & derivados , Antibacterianos/biossíntese , Proteínas de Bactérias/metabolismo , Ciclitóis/metabolismo , Mio-Inositol-1-Fosfato Sintase/metabolismo , Fósforo-Oxigênio Liases/metabolismo , Adenosina/biossíntese , Adenosina/química , Antibacterianos/química , Proteínas de Bactérias/genética , Cosmídeos/genética , Cosmídeos/metabolismo , Ciclitóis/química , Espectroscopia de Ressonância Magnética , Família Multigênica , Mio-Inositol-1-Fosfato Sintase/genética , Nucleosídeos/química , Fósforo-Oxigênio Liases/genética , Espectrometria de Massas por Ionização por Electrospray , Streptomyces coelicolor/enzimologia , Streptomyces coelicolor/genéticaRESUMO
Bacterial lipoproteins are extracellular proteins tethered to cell membranes by covalently attached lipids. Deleting the lipoprotein signal peptidase (lsp) gene in Streptomyces coelicolor results in growth and developmental defects that cannot be restored by reintroducing lsp. This led us to hypothesise that lsp is essential and that the lsp mutant we isolated previously had acquired compensatory secondary mutations. Here we report resequencing of the genomes of wild-type M145 and the cis-complemented ∆lsp mutant (BJT1004) to map and identify these secondary mutations but we show that they do not increase the efficiency of disrupting lsp and are not lsp suppressors. We provide evidence that they are induced by introducing the cosmid St4A10∆lsp, as part of ReDirect PCR mutagenesis protocol, which transiently duplicates a number of important cell division genes. Disruption of lsp using a suicide vector (which does not result in gene duplication) still results in growth and developmental delays and we conclude that loss of Lsp function results in developmental defects due to the loss of all lipoproteins from the cell membrane. Significantly, our results also indicate the use of cosmid libraries for the genetic manipulation of bacteria can lead to phenotypes not necessarily linked to the gene(s) of interest.
Assuntos
Ácido Aspártico Endopeptidases/genética , Proteínas de Bactérias/genética , Cosmídeos/genética , Lipoproteínas/genética , Streptomyces coelicolor/genética , Escherichia coli/metabolismo , Deleção de Genes , Teste de Complementação Genética , Genoma Bacteriano , Mutagênese , Mutação , Fenótipo , Reação em Cadeia da PolimeraseRESUMO
As baculoviruses usually have a narrow insecticidal spectrum, knowing the mechanisms by which they control the host-range is prerequisite for improvement of their applications as pesticides. In this study, from supernatant of culture cells transfected with DNAs of an Autographa californica multiple nucleopolyhedrovirus (AcMNPV) mutant lacking the antiapoptotic gene p35 (vAc(∆P35)) and a cosmid representing a fragment of Spodoptera exigua nucleopolyhedrovirus (SeMNPV), a viral strain was plaque-purified and named vAcRev. vAcRev had a broader host range than either vAc(∆P35) or SeMNPV parental virus, being able to infect not only the permissive hosts of its parental viruses but also a nonpermissive host (Spodoptera litura). Genome sequencing indicated that vAcRev comprises a mixture of two viruses with different circular dsDNA genomes. One virus contains a genome similar to vAc(∆P35), while in the other viral genome, a 24.4 kbp-fragment containing 10 essential genesis replaced with a 4 kbp-fragment containing three SeMNPV genes including a truncated Se-iap3 gene. RNA interference and ectopic expression assays found that Se-iap3 is responsible for the host range expansion of vAcRev, suggesting that Se-iap3 inhibits the progression of apoptosis initiated by viral infection and promotes viral propagation in hosts both permissive and non-permissive for AcMNPV and SeMNPV.
Assuntos
Especificidade de Hospedeiro/fisiologia , Nucleopoliedrovírus/genética , Spodoptera/virologia , Animais , Cosmídeos/genética , Cosmídeos/metabolismo , DNA Viral/química , DNA Viral/metabolismo , Genoma Viral/genética , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismo , Lepidópteros/virologia , Nucleopoliedrovírus/crescimento & desenvolvimento , Nucleopoliedrovírus/fisiologia , Interferência de RNA , Análise de Sequência de DNA , Células Sf9/citologia , Células Sf9/virologia , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/genética , Proteínas Virais/metabolismo , Replicação ViralRESUMO
Innovative strategies are needed to accelerate the identification of antimicrobial drug targets and resistance mechanisms. Here we develop a sensitive method, which we term Cosmid Sequencing (or "Cos-Seq"), based on functional cloning coupled to next-generation sequencing. Cos-Seq identified >60 loci in the Leishmania genome that were enriched via drug selection with methotrexate and five major antileishmanials (antimony, miltefosine, paromomycin, amphotericin B, and pentamidine). Functional validation highlighted both known and previously unidentified drug targets and resistance genes, including novel roles for phosphatases in resistance to methotrexate and antimony, for ergosterol and phospholipid metabolism genes in resistance to miltefosine, and for hypothetical proteins in resistance to paromomycin, amphothericin B, and pentamidine. Several genes/loci were also found to confer resistance to two or more antileishmanials. This screening method will expedite the discovery of drug targets and resistance mechanisms and is easily adaptable to other microorganisms.
Assuntos
Resistência a Medicamentos/genética , Genes de Protozoários , Sequenciamento de Nucleotídeos em Larga Escala , Leishmania infantum/genética , Antiprotozoários/farmacologia , Cosmídeos/genética , Resistência a Medicamentos/efeitos dos fármacos , Fosfolipídeos/genéticaRESUMO
Dinoflagellates are one of the last major lineages of eukaryotes for which little is known about genome structure and organization. We report here the sequence and gene structure of a clone isolated from a cosmid library which, to our knowledge, represents the largest contiguously sequenced, dinoflagellate genomic, tandem gene array. These data, combined with information from a large transcriptomic library, allowed a high level of confidence of every base pair call. This degree of confidence is not possible with PCR-based contigs. The sequence contains an intron-rich set of five highly expressed gene repeats arranged in tandem. One of the tandem repeat gene members contains an intron 26,372 bp long. This study characterizes a splice site consensus sequence for dinoflagellate introns. Two to nine base pairs around the 3' splice site are repeated by an identical two to nine base pairs around the 5' splice site. The 5' and 3' splice sites are in the same locations within each repeat so that the repeat is found only once in the mature mRNA. This identically repeated intron boundary sequence might be useful in gene modeling and annotation of genomes.
Assuntos
Dinoflagelados/genética , Genoma de Protozoário , Genômica/métodos , Íntrons , Splicing de RNA , Sequência de Aminoácidos , Sequência de Bases , Cosmídeos , Biblioteca Gênica , Genoma de Protozoário/genética , Dados de Sequência Molecular , Sequências de Repetição em TandemRESUMO
In the course of our search for anti-dormant Mycobacterial substances, nybomycin (1) was re-discovered from the culture broth of a marine-derived Streptomyces sp. on the bioassay-guided separation. Compound 1 showed anti-microbial activity against Mycobacterium smegmatis and Mycobacterium bovis BCG with the MIC of 1.0µg/mL under both actively growing aerobic conditions and dormancy inducing hypoxic conditions. Compound 1 is also effective to Mycobacterium tuberculosis including the clinically isolated strains. The mechanistic analysis indicated that 1 bound to DNA and induces a unique morphological change to mycobacterial bacilli leading the bacterial cell death.
Assuntos
Antituberculosos/farmacologia , DNA Bacteriano/antagonistas & inibidores , Mycobacterium tuberculosis/efeitos dos fármacos , Streptomyces/química , Antituberculosos/química , Antituberculosos/isolamento & purificação , Organismos Aquáticos , Técnicas de Cultura de Células , Cosmídeos/química , Cosmídeos/metabolismo , DNA Bacteriano/química , Farmacorresistência Bacteriana/genética , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Mycobacterium bovis/efeitos dos fármacos , Mycobacterium bovis/genética , Mycobacterium bovis/metabolismo , Mycobacterium bovis/ultraestrutura , Mycobacterium smegmatis/efeitos dos fármacos , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/metabolismo , Mycobacterium smegmatis/ultraestrutura , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/ultraestrutura , Quinolonas/química , Quinolonas/isolamento & purificação , Quinolonas/farmacologia , Streptomyces/metabolismoRESUMO
A two-step method, i.e., the transfer acyl analysis and then the chiral HPLC analysis, was employed in the screening of the cosmid library of Microbacterium hydrocarbonoxydans genome. Two enantiocomplementary γ-lactamase clones were found. A 40-kb cosmid showed (-)-γ-lactamase activity, and the activity was from Mhg which was reported previously according to the results of PCR identifying experiment. The 37-kb (+)-γ-lactamase cosmid was further constructed into a pUC18 plasmid library and screened by the same two-step method. A plasmid clone harboring a 1.6-kb fragment showed (+)-γ-lactamase activity. A 555-bp ORF in the 1.6-kb fragment showed high (+)-γ-lactamase activity when it was expressed under the control of T7 promoter. The coding protein showed significant homology with bacterial isochorismatase. The (+)-γ-lactamase was characterized and compared with the (-)-γ-lactamase Mhg. This is another report that two enantiocomplementary γ-lactamases are present in the same strain.