RESUMO
To contain the spread of the SARS-CoV-2 pandemic, rapid development of vaccines was required in 2020. Rational design, international efforts, and a lot of hard work yielded the market approval of novel SARS-CoV-2 vaccines based on diverse platforms such as mRNA or adenovirus vectors. The great success of these technologies, in fact, contributed significantly to control the pandemic. Consequently, most scientific literature available in the public domain discloses the results of clinical trials and reveals data of efficaciousness. However, a description of processes and rationales that led to specific vaccine design is only partially available, in particular for adenovirus vectors, even though it could prove helpful for future developments. Here, we disclose our insights from the endeavors to design compatible functional adenoviral vector platform expression cassettes for the SARS-CoV-2 spike protein. We observed that contextualizing genes from an ssRNA virus into a DNA virus provides significant challenges. Besides affecting physical titers, expression cassette design of adenoviral vaccine candidates can affect viral propagation and spike protein expression. Splicing of mRNAs was affected, and fusogenicity of the spike protein in ACE2-overexpressing cells was enhanced when the ER retention signal was deleted.
Assuntos
Vacinas contra Adenovirus , COVID-19 , Humanos , COVID-19/prevenção & controle , Vacinas contra COVID-19 , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , RNA Mensageiro , Adenoviridae/genéticaRESUMO
As the MHC-I-pathway is key to antigen presentation to cytotoxic T-cells and, therefore, recognition by the host adaptive immune system, we hypothesized that SARS-CoV-2 including its Variants of Concern (VOCs), influences MHC-I expression on epithelial cell surfaces as an immune evasion strategy. We conducted an in vitro time course experiment with the human airway epithelial cell line Calu-3 and the human colorectal adenocarcinoma cell line Caco-2. Cells were infected with SARS-CoV-2 strains non-VOC/B.1.1, Alpha/B.1.1.7, Beta/B.1.351, Gamma/P.1, and Delta/B.1.617.2. At 2, 24, 48 and 72 h post-infection we performed RT-qPCR to track viral replication. Simultaneously, we performed intracellular staining with a serum of a double-vaccinated healthy adult containing a high amount of spike protein antibody. In flow cytometry experiments, we differentiated between infected (spike protein positive) and bystander (spike protein negative) cells. To compare their HLA expression levels, cells were stained extracellularly with anti-HLA-A-IgG and anti-HLA-B,C-IgG. While HLA-A expression was stable on infected Calu-3 cells for all variants, it increased to different degrees on bystander cells in samples infected with VOCs Beta, Gamma, Delta, or non-VOC over the time course analyzed. In contrast, HLA-A levels were stable in bystander Calu-3 cells in samples infected with the Alpha variant. The upregulation of MHC-I on spike protein negative bystander cells in Calu-3 cell cultures infected with Beta, Gamma, Delta, and partly non-VOC might suggest that infected cells are still capable of secreting inflammatory cytokines like type-I interferons stimulating the MHC-I expression on bystander cells. In comparison, there was no distinct effect on HLA expression level on Caco-2 cells of any of the VOCs or non-VOC. Further investigations of the full range of immune evasion strategies of SARS-CoV-2 variants are warranted.
Assuntos
COVID-19 , SARS-CoV-2 , Adulto , Humanos , Células CACO-2 , Glicoproteína da Espícula de Coronavírus/genética , COVID-19/genética , Imunoglobulina GRESUMO
BACKGROUND: Human breast milk has a high microRNA (miRNA) content. It remains unknown whether and how milk miRNAs might affect intestinal gene regulation and homeostasis of the developing microbiome after initiating enteral nutrition. However, this requires that relevant milk miRNA amounts survive the gastrointestinal (GI) passage, are taken up by cells, and become available to the RNA interference machinery. It seems important to dissect the fate of these miRNAs after oral ingestion and GI passage. OBJECTIVES: Our goal was to analyze the potential transmissibility of milk miRNAs via the gastrointestinal system in neonate humans and a porcine model in vivo to contribute to the discussion of whether milk miRNAs could influence gene regulation in neonates and thus might vertically transmit developmental relevant signals. METHODS: We performed cross-species profiling of miRNAs via deep sequencing and utilized dietary xenobiotic taxon-specific milk miRNA (xenomiRs) as tracers in human and porcine neonates, followed by functional studies in primary human fetal intestinal epithelial cells using adenovirus-type 5-mediated miRNA gene transfer. RESULTS: Mammals share many milk miRNAs yet exhibit taxon-specific miRNA fingerprints. We traced bovine-specific miRNAs from formula nutrition in human preterm stool and 9 d after the onset of enteral feeding in intestinal cells (ICs) of preterm piglets. Thereafter, several xenomiRs accumulated in the ICs. Moreover, a few hours after introducing enteral feeding in preterm piglets with supplemented reporter miRNAs (cel-miR-39-5p/-3p), we observed their enrichment in blood serum and in argonaute RISC catalytic component 2 (AGO2)-immunocomplexes from intestinal biopsies. CONCLUSIONS: Milk-derived miRNAs survived GI passage in human and porcine neonates. Bovine-specific miRNAs accumulated in ICs of preterm piglets after enteral feeding with bovine colostrum/formula. In piglets, colostrum supplementation with cel-miR-39-5p/-3p resulted in increased blood concentrations of cel-miR-39-3p and argonaute RISC catalytic component 2 (AGO2) loading in ICs. This suggests the possibility of vertical transmission of miRNA signaling from milk through the neonatal digestive tract.
Assuntos
Enterocolite Necrosante , MicroRNAs , Animais , Bovinos , Feminino , Humanos , Animais Recém-Nascidos , Células Epiteliais/patologia , Trato Gastrointestinal , MicroRNAs/genética , Leite , Suínos , Leite HumanoRESUMO
Carcinomas are characterized by a widespread upregulation of intercellular junctions that create a barrier to immune response and drug therapy. Desmoglein 2 (DSG2) represents such a junction protein and serves as one adenovirus receptor. Importantly, the interaction between human adenovirus type 3 (Ad3) and DSG2 leads to the shedding of the binding domain followed by a decrease in the junction protein expression and transient tight junction opening. Junction opener 4 (JO-4), a small recombinant protein derived from the Ad3 fiber knob, was previously developed with a higher affinity to DSG2. JO-4 protein has been proven to enhance the effects of antibody therapy and chemotherapy and is now considered for clinical trials. However, the effect of the JO4 mutation in the context of a virus remains insufficiently studied. Therefore, we introduced the JO4 mutation to various adenoviral vectors to explore their infection properties. In the current experimental settings and investigated cell lines, the JO4-containing vectors showed no enhanced transduction compared with their parental vectors in DSG2-high cell lines. Moreover, in DSG2-low cell lines, the JO4 vectors presented a rather weakened effect. Interestingly, DSG2-negative cell line MIA PaCa-2 even showed resistance to JO4 vector infection, possibly due to the negative effect of JO4 mutation on the usage of another Ad3 receptor: CD46. Together, our observations suggest that the JO4 vectors may have an advantage to prevent CD46-mediated sequestration, thereby achieving DSG2-specific transduction.
Assuntos
Adenovírus Humanos , Vetores Genéticos , Adenovírus Humanos/fisiologia , Linhagem Celular , Desmogleína 2/genética , Desmogleína 2/metabolismo , Terapia Genética , Vetores Genéticos/genética , Humanos , Junções IntercelularesRESUMO
Adenovirus-based vectors are playing an important role as efficacious genetic vaccines to fight the current COVID-19 pandemic. Furthermore, they have an enormous potential as oncolytic vectors for virotherapy and as vectors for classic gene therapy. However, numerous vector-host interactions on a cellular and noncellular level, including specific components of the immune system, must be modulated in order to generate safe and efficacious vectors for virotherapy or classic gene therapy. Importantly, the current widespread use of Ad vectors as vaccines against COVID-19 will induce antivector immunity in many humans. This requires the development of strategies and techniques to enable Ad-based vectors to evade pre-existing immunity. In this review article, we discuss the current status of genetic and chemical capsid modifications as means to modulate the vector-host interactions of Ad-based vectors.
Assuntos
Adenoviridae/genética , COVID-19/prevenção & controle , Capsídeo/química , Adenoviridae/imunologia , COVID-19/imunologia , COVID-19/terapia , Vacinas contra COVID-19/administração & dosagem , Vacinas contra COVID-19/imunologia , Genes Virais , Vetores Genéticos , Humanos , Imunidade , Terapia Viral Oncolítica/métodos , Pandemias , SARS-CoV-2/imunologia , SARS-CoV-2/isolamento & purificaçãoRESUMO
Arming of oncolytic viruses with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been shown as a viable approach to increase the antitumor efficacy in melanoma. However, melanoma cells may be partially or completely resistant to TRAIL or develop TRAIL resistance, thus counteracting the antitumor efficiency of TRAIL-armed oncolytic viruses. Recently, we found that TRAIL resistance in melanoma cells can be overcome by inhibition of antiapoptotic Bcl-2 protein myeloid cell leukemia 1 (Mcl-1). Here, we investigated whether the cytotoxicity of AdV-TRAIL, an oncolytic adenovirus, which expresses TRAIL after induction by doxycycline (Dox), can be improved in melanoma cells by silencing of Mcl-1. Two melanoma cell lines, the TRAIL-resistant MeWo and the TRAIL-sensitive Mel-HO were investigated. Treatment of both cell lines with AdV-TRAIL resulted in a decrease of cell viability, which was caused by an increase of apoptosis and necrosis. The proapoptotic effects were dependent on induction of TRAIL by Dox and were more pronounced in Mel-HO than in MeWo cells. SiRNA-mediated silencing of Mcl-1 resulted in a further significant decrease of cell viability and a further increase of apoptosis and necrosis in AdV-TRAIL-infected MeWo and Mel-HO cells. However, while in absolute terms, the effects were more pronounced in Mel-HO cells, in relative terms, they were stronger in MeWo cells. These results show that silencing of Mcl-1 represents a suitable approach to increase the cytotoxicity of a TRAIL-armed oncolytic adenovirus in melanoma cells. KEY MESSAGES: ⢠Cytotoxicity of TRAIL-expressing adenovirus can be enhanced by silencing of Mcl-1. ⢠The effect occurs in TRAIL-sensitive and TRAIL-resistant melanoma cells. ⢠Increase of apoptosis is the main mechanism induced by Mcl-1 silencing.
Assuntos
Adenoviridae/genética , Apoptose , Inativação Gênica , Terapia Genética , Melanoma/terapia , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Terapia Viral Oncolítica , Vírus Oncolíticos/genética , Neoplasias Cutâneas/terapia , Ligante Indutor de Apoptose Relacionado a TNF/genética , Adenoviridae/metabolismo , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Melanoma/genética , Melanoma/metabolismo , Melanoma/virologia , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Necrose , Vírus Oncolíticos/metabolismo , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/virologia , Ligante Indutor de Apoptose Relacionado a TNF/metabolismoRESUMO
Adenovirus vectors are potent tools for genetic vaccination and oncolytic virotherapy. However, they are prone to multiple undesired vector-host interactions, especially after in vivo delivery. It is a consensus that the limitations imposed by undesired vector-host interactions can only be overcome if defined modifications of the vector surface are performed. These modifications include shielding of the particles from unwanted interactions and targeting by the introduction of new ligands. The goal of the protocol presented here is to enable the reader to generate shielded and, if desired, retargeted human adenovirus gene transfer vectors or oncolytic viruses. The protocol will enable researchers to modify the surface of adenovirus vector capsids by specific chemical attachment of synthetic polymers, carbohydrates, lipids, or other biological or chemical moieties. It describes the cutting-edge technology of combined genetic and chemical capsid modifications, which have been shown to facilitate the understanding and overcoming of barriers for in vivo delivery of adenovirus vectors. A detailed and commented description of the crucial steps for performing specific chemical reactions with biologically active viruses or virus-derived vectors is provided. The technology described in the protocol is based on the genetic introduction of (naturally absent) cysteine residues into solvent-exposed loops of adenovirus-derived vectors. These cysteine residues provide a specific chemical reactivity that can, after production of the vectors to high titers, be exploited for highly specific and efficient covalent chemical coupling of molecules from a wide variety of substance classes to the vector particles. Importantly, this protocol can easily be adapted to perform a broad variety of different (non-thiol-based) chemical modifications of adenovirus vector capsids. Finally, it is likely that non-enveloped virus-based gene transfer vectors other than adenovirus can be modified from the basis of this protocol.
Assuntos
Adenoviridae/genética , Proteínas do Capsídeo/genética , Vetores Genéticos/genética , Animais , Fenômenos Biofísicos , Capsídeo , Proteínas do Capsídeo/química , Cisteína , Técnicas de Transferência de Genes , Terapia Genética/métodos , Humanos , Ligantes , Terapia Viral Oncolítica/métodos , Vírus Oncolíticos/genética , Polímeros/análiseRESUMO
BACKGROUND: During sexual reproduction in the unicellular ciliate Stylonychia somatic macronuclei differentiate from germline micronuclei. Thereby, programmed sequence reduction takes place, leading to the elimination of > 95% of germline sequences, which priorly adopt heterochromatin structure via H3K27me3. Simultaneously, 27nt-ncRNAs become synthesized from parental transcripts and are bound by the Argonaute protein PIWI1. RESULTS: These 27nt-ncRNAs cover sequences destined to the developing macronucleus and are thought to protect them from degradation. We provide evidence and propose that RNA/DNA base-pairing guides PIWI1/27nt-RNA complexes to complementary macronucleus-destined DNA target sequences, hence transiently causing locally stalled replication during polytene chromosome formation. This spatiotemporal delay enables the selective deposition of temporarily available histone H3.4K27me3 nucleosomes at all other sequences being continuously replicated, thus dictating their prospective heterochromatin structure before becoming developmentally eliminated. Concomitantly, 27nt-RNA-covered sites remain protected. CONCLUSIONS: We introduce the concept of 'RNA-induced DNA replication interference' and explain how the parental functional genome partition could become transmitted to the progeny.
Assuntos
Cilióforos/fisiologia , Replicação do DNA , Histonas/genética , RNA Guia de Cinetoplastídeos/genética , Proteínas Argonautas/metabolismo , Cilióforos/genética , Variação Genética , Genoma de Protozoário , Micronúcleo Germinativo/genética , RNA de Protozoário/genéticaRESUMO
Currently, virus-based vectors, namely derivatives of the adenovirus, are frequently used in a wide variety of ex vivo or local gene therapeutic applications. However, the efficacy of virus-based vectors in systemic applications is presently still extremely limited. Complex interactions of the various vector types with the patient's organism hinder successful vector deployment. Exemplary, here we summarize barriers to systemic application of Adenovirus-based vectors leading either to acute toxic effects or rapid vector neutralization and discuss strategies to overcome these barriers aiming to develop more efficient vector types.
Assuntos
Adenoviridae/genética , Vetores Genéticos/genética , Animais , Terapia Genética/métodos , HumanosRESUMO
De novo addition of telomeric sequences can occur at broken chromosomes and must be well controlled, which is essential during programmed DNA reorganization processes. In ciliated protozoa an extreme form of DNA-reorganization is observed during macronuclear differentiation after sexual reproduction leading to the elimination of specific parts of the germline genome. Regulating these processes involves small noncoding RNAs, but in addition DNA-reordering, excision and amplification require RNA templates deriving from the parental macronucleus. We show that these putative RNA templates can carry telomeric repeats. Microinjection of RNA templates carrying modified telomeres into the developing macronucleus leads to modified telomeres in vegetative cells, providing strong evidence, that de novo addition of telomeres depends on a telomere-containing transcript from the parental macronucleus.
Assuntos
Replicação do DNA , RNA/genética , Telômero/genética , Moldes Genéticos , Nucléolo Celular/genética , Nucléolo Celular/metabolismo , Cilióforos/genética , Cilióforos/metabolismo , Amplificação de Genes , Variação Genética , Modelos Biológicos , RNA de Cadeia Dupla/genética , RNA não Traduzido/genética , Telômero/metabolismoRESUMO
Stylonychia lemnae is a classical model single-celled eukaryote, and a quintessential ciliate typified by dimorphic nuclei: A small, germline micronucleus and a massive, vegetative macronucleus. The genome within Stylonychia's macronucleus has a very unusual architecture, comprised variably and highly amplified "nanochromosomes," each usually encoding a single gene with a minimal amount of surrounding noncoding DNA. As only a tiny fraction of the Stylonychia genes has been sequenced, and to promote research using this organism, we sequenced its macronuclear genome. We report the analysis of the 50.2-Mb draft S. lemnae macronuclear genome assembly, containing in excess of 16,000 complete nanochromosomes, assembled as less than 20,000 contigs. We found considerable conservation of fundamental genomic properties between S. lemnae and its close relative, Oxytricha trifallax, including nanochromosomal gene synteny, alternative fragmentation, and copy number. Protein domain searches in Stylonychia revealed two new telomere-binding protein homologs and the presence of linker histones. Among the diverse histone variants of S. lemnae and O. trifallax, we found divergent, coexpressed variants corresponding to four of the five core nucleosomal proteins (H1.2, H2A.6, H2B.4, and H3.7) suggesting that these ciliates may possess specialized nucleosomes involved in genome processing during nuclear differentiation. The assembly of the S. lemnae macronuclear genome demonstrates that largely complete, well-assembled highly fragmented genomes of similar size and complexity may be produced from one library and lane of Illumina HiSeq 2000 shotgun sequencing. The provision of the S. lemnae macronuclear genome sets the stage for future detailed experimental studies of chromatin-mediated, RNA-guided developmental genome rearrangements.
Assuntos
Cilióforos/genética , Genoma de Protozoário/genética , Macronúcleo/genética , Biblioteca Gênica , Variação Genética , Histonas/genética , FilogeniaRESUMO
BACKGROUND: Regulation of chromatin structure involves deposition of selective histone variants into nucleosome arrays. Numerous histone H3 variants become differentially expressed by individual nanochromosomes in the course of macronuclear differentiation in the spirotrichous ciliate Stylonychia lemnae. Their biological relevance remains to be elucidated. RESULTS: We show that the differential assembly of H3 variants into chromatin is strongly correlated with the functional separation of chromatin structures in developing macronuclei during sexual reproduction in Stylonychia, thus probably determining the fate of specific sequences. Specific H3 variants approximately 15 kDa or 20 kDa in length are selectively targeted by post-translational modifications. We found that only the 15 kDa H3 variants including H3.3 and H3.5, accumulate in the early developing macronucleus, and these also occur in mature macronuclei. H3.7 is a 20 kDa variant that specifically becomes enriched in macronuclear anlagen during chromosome polytenization. H3.7, acetylated at lysine-32 (probably equivalent to lysine-36 of most H3 variants), is specifically associated with a sequence class that is retained in the mature macronucleus and therefore does not undergo developmental DNA elimination. H3.8 is another 20 kDa variant that is restricted to the micronucleus. H3.8 is selectively targeted by lysine methylation and by serine or threonine phosphorylation. Intriguingly, the expression and chromatin localization of the histone variant H3.3 was impaired during macronuclear differentiation after RNA interference knock-down of Piwi expression. CONCLUSIONS: Differential deposition of H3 variants into chromatin strongly correlates with the functional distinction of genomic sequence classes on the chromatin level, thus helping to determine the fate of specific DNA sequences during sexual reproduction in Stylonychia. Consequently, H3 variants are selectively targeted by post-translational modifications, possibly as a result of deviations within the recognition motifs, which allow binding of effector proteins. We propose that differential assembly of histone variants into chromatin of various nuclear types could contribute to nuclear identity, for example, during differential development of either new micronuclei or a macronuclear anlage from mitosis products of the zygote nucleus (synkaryon). The observation that the Piwi-non-coding RNA (ncRNA) pathway influences the expression and deposition of H3.3 in macronuclear anlagen indicates for the first time that selective histone variant assembly into chromatin might possibly depend on ncRNA.
RESUMO
BACKGROUND: DNA methylation and demethylation are important epigenetic regulatory mechanisms in eukaryotic cells and, so far, only partially understood. We exploit the minimalistic biological ciliate system to understand the crosstalk between DNA modification and chromatin structure. In the macronucleus of these cells, the DNA is fragmented into individual short DNA molecules, each representing a functional expression and replication unit. Therefore, long range epigenomic interaction can be excluded in this system. RESULTS: In the stichotrichous ciliate Stylonychia lemnae, cytosine methylation occurs in a small subset of macronuclear nanochromosomes expressed only during sexual reproduction. Methylation pattern shows similarity to that observed in fungi and Drosophila. Cytosine methylation correlates with gene activity and chromatin structure. Upon gene activation, cytosines become demethylated and a redistribution of histone post-translational modifications (PTMs) takes place. Evidence is presented that the formation of a permissive chromatin structure in the vicinity of the 5meCs precedes cytosine methylation and is probably a necessary prerequisite for their demethylation. Shortly after demethylation of cytosines occurs, the parental macronucleus degenerates, a new macronucleus is formed from a micronuclear derivative and the specific methylation pattern is transmitted from the germline micronucleus to the new macronucleus. CONCLUSIONS: We show that very few, or even only one, discrete methylated cytosines are required to assign regulatory functions at a specific locus. Furthermore, evidence is provided that a permissive chromatin structure is probably a necessary prerequisite for the demethylation of specific cytosines. Our results allow us to propose a mechanistic model for the biological function of cytosine methylation in the ciliate cell and its regulation during the cell cycle.
RESUMO
We exploit the unusual genome organization of the ciliate cell to analyze the control of specific gene amplification during a nuclear differentiation process. Ciliates contain two types of nuclei within one cell, the macronucleus and the micronucleus; and after sexual reproduction a new macronucleus is formed from a micronuclear derivative. During macronuclear differentiation, most extensive DNA reorganization, elimination, and fragmentation processes occur, resulting in a macronucleus containing short DNA molecules (nanochromosomes) representing individual genetic units and each being present in high copy number. It is believed that these processes are controlled by small nuclear RNAs but also by a template derived from the old macronucleus. We first describe the exact copy numbers of selected nanochromosomes in the macronucleus, and define the timing during nuclear differentiation at which copy number is determined. This led to the suggestion that DNA processing and copy number control may be closely related mechanisms. Degradation of an RNA template derived from the macronucleus leads to significant decrease in copy number, whereas injection of additional template molecules results in an increase in copy number and enhanced expression of the corresponding gene. These observations can be incorporated into a mechanistic model about an RNA-dependent epigenetic regulation of gene copy number during nuclear differentiation. This highlights that RNA, in addition to its well-known biological functions, can also be involved in the control of gene amplification.
Assuntos
Cilióforos/genética , DNA de Protozoário/metabolismo , Amplificação de Genes/fisiologia , Genes de Protozoários/fisiologia , Macronúcleo/metabolismo , Micronúcleo Germinativo/metabolismo , RNA de Protozoário/metabolismo , RNA Nuclear Pequeno/metabolismo , Animais , Cromossomos/genética , Cromossomos/metabolismo , Cilióforos/metabolismo , DNA de Protozoário/genética , Epigênese Genética/fisiologia , Dosagem de Genes/fisiologia , Macronúcleo/genética , Micronúcleo Germinativo/genética , Modelos Genéticos , RNA de Protozoário/genética , RNA Nuclear Pequeno/genéticaRESUMO
During macronuclear differentiation in ciliated protozoa, extensive DNA rearrangement and DNA excision processes occur, and these are most profound in stichotrichous ciliates, such as Stylonychia or Oxytricha. This review describes the morphological and molecular events taking place during macronuclear development in stichotrichous ciliates. Various models for the regulation of macronuclear differentiation have been proposed and will be discussed here. Finally, an attempt to speculate about the biological consequences of these rearrangement and excision processes will be made. Because specific elimination of DNA sequences not required in the differentiated nucleus can be regarded as the most extreme form of gene silencing, results obtained in these cells may also be relevant for our understanding of differentiation processes in higher eukaryotic organisms.
Assuntos
Cilióforos/genética , Macronúcleo/genética , Animais , Divisão Celular/genética , Cilióforos/citologia , Cilióforos/crescimento & desenvolvimento , Epigênese Genética/genética , Evolução Molecular , Rearranjo Gênico/genética , Micronúcleo Germinativo/genética , Modelos Genéticos , Deleção de Sequência/genéticaRESUMO
BACKGROUND: Programmed DNA elimination and reorganization frequently occur during cellular differentiation. Development of the somatic macronucleus in some ciliates presents an extreme case, involving excision of internal eliminated sequences (IESs) that interrupt coding DNA segments (macronuclear destined sequences, MDSs), as well as removal of transposon-like elements and extensive genome fragmentation, leading to 98% genome reduction in Stylonychia lemnae. Approximately 20-30% of the genes are estimated to be scrambled in the germline micronucleus, with coding segment order permuted and present in either orientation on micronuclear chromosomes. Massive genome rearrangements are therefore critical for development. METHODOLOGY/PRINCIPAL FINDINGS: To understand the process of DNA deletion and reorganization during macronuclear development, we examined the population of DNA molecules during assembly of different scrambled genes in two related organisms in a developmental time-course by PCR. The data suggest that removal of conventional IESs usually occurs first, accompanied by a surprising level of error at this step. The complex events of inversion and translocation seem to occur after repair and excision of all conventional IESs and via multiple pathways. CONCLUSIONS/SIGNIFICANCE: This study reveals a temporal order of DNA rearrangements during the processing of a scrambled gene, with simpler events usually preceding more complex ones. The surprising observation of a hidden layer of errors, absent from the mature macronucleus but present during development, also underscores the need for repair or screening of incorrectly-assembled DNA molecules.
Assuntos
DNA de Protozoário/genética , Rearranjo Gênico , Animais , Núcleo Celular/genética , Cilióforos/genética , Clonagem Molecular , Reação em Cadeia da Polimerase , Recombinação Genética , Deleção de SequênciaRESUMO
Ciliates have a somatic and a germline nucleus; after sexual conjugation a new somatic nucleus forms from the new zygotic germline nucleus. Formation of the somatic nucleus involves precise elimination of a large portion of DNA sequences from the germline. Here we compare the architecture of the germline and somatic versions of the actin I gene in two geographically isolated strains of Stylonychia lemnae. We show that the structure of the germline gene is surprisingly mercurial, with the distinction between germline-limited and somatic sequences variable over the course of evolution. This is, to our knowledge, the first example of evolutionary swapping of retained versus deleted sequences during ciliate development, with sequences deleted during development that are specifically retained in another strain.
Assuntos
Cilióforos/genética , Conversão Gênica , Genes de Protozoários , Genoma de Protozoário , Micronúcleo Germinativo/genética , Actinas/genética , Animais , Sequência de Bases , DNA/análise , Dados de Sequência Molecular , Alinhamento de SequênciaRESUMO
After sexual reproduction in ciliated protozoa a new macronucleus differentiates from a micronuclear derivative. In the course of macronuclear development dramatic DNA- and chromatin reorganisation processes occur, which include splicing of DNA sequences such as IES (internal eliminated sequences) and transposon-like elements during formation of polytene chromosomes, degradation of the polytene chromosomes and specific elimination of micronuclear-specific DNA, de novo addition of telomeres and specific amplification of DNA sequences. In order to understand the molecular basis of this nuclear differentiation process, analysis of developmentally regulated genes seems to be a necessary prerequisite. We performed a microarray analysis to identify genes differentially expressed during macronuclear differentiation. 467 sequences from cDNA libraries were identified as possible candidates from which 384 sequences were further characterised by sequence analysis. These sequences were identified, if possible, by DNA and protein BLAST analysis. Expression of one of these sequences was silenced by RNAi and a preliminary functional analysis performed. Results presented in this study provide the basis for a functional characterisation of genes differentially expressed during this nuclear differentiation process.
Assuntos
Núcleo Celular/fisiologia , Cilióforos/genética , Regulação da Expressão Gênica no Desenvolvimento , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Animais , Cilióforos/crescimento & desenvolvimento , DNA Complementar/química , DNA Complementar/genética , DNA de Protozoário/análise , DNA de Protozoário/genética , Perfilação da Expressão Gênica , Genes de Protozoários/genética , Dados de Sequência Molecular , Proteínas de Protozoários/genética , Interferência de RNA , RNA de Protozoário/análise , RNA de Protozoário/genética , Análise de Sequência de DNARESUMO
Telomere replication of eukaryotic chromosomes is achieved by a specialized enzyme, the telomerase. Although the biochemistry of end-replication is well understood, little is known about the organization of the end-replication machinery, its regulation throughout the cell cycle or the biological function of the telomerase-associated proteins. Here we investigate the function of the telomerase-associated protein p43 within the macronucleus of the ciliated protozoa Euplotes. It has been shown that p43 binds in vitro to the RNA subunit of telomerase and shares homology with the La autoantigen family. It therefore has been suggested that it is involved in the assembly and/or nuclear retention of telomerase. We show that the p43-telomerase complex is bound to a subnuclear structure in vivo and is resistant to electroelution. Upon inhibition of p43 or telomerase expression by RNAi, which in this study was used for the first time in spirotrichs, this complex is no longer retained in the nucleus. Further analysis revealed that the p43-telomerase complex is bound to the nuclear matrix in vivo and that after inhibition of p43 expression, telomerase is released from this structure, strongly suggesting that p43 is involved in anchoring of telomerase in the nucleus. This is the first in vivo demonstration of the biological function of this telomerase-associated component involved in telomere replication and allows us to propose a model for the organization of the end-replication machinery in the eukaryotic cell.
Assuntos
Euplotes/metabolismo , Proteínas de Protozoários/metabolismo , Telomerase/metabolismo , Animais , Sequência de Bases , Núcleo Celular/metabolismo , DNA de Protozoário/genética , Euplotes/genética , Substâncias Macromoleculares , Modelos Biológicos , Ligação Proteica , Subunidades Proteicas , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Interferência de RNA , Telomerase/química , Telomerase/genéticaRESUMO
Macronuclear DNA of stichotrichous ciliates occurs in small 'gene-sized' molecules with sizes of about 0.5 to 40 kb. Each of these molecules is terminated by telomeric sequences of defined length. A single macronucleus contains up to 10(8) DNA molecules; due to the high concentration of telomeric sequences in this nucleus it is an attractive model to study telomere behaviour. We recently provided evidence that macronuclear telomeres are attached to the nuclear matrix and that this interaction is mediated by the telomere binding protein (TeBP). Using various experimental approaches, we now demonstrate that telomeres as well as both subunits of the telomere binding protein are associated with the nuclear matrix. However, there is no direct binding of telomeric DNA to the matrix but telomere matrix interaction is exclusively mediated by the TeBP. In addition, we show that telomeric sequences adopt in vivo the antiparallel G-quartet structure when bound to the nuclear matrix. These data not only allow us to propose a model for macronuclear architecture but may also be relevant for further analysis of telomere-matrix interactions in higher eukaryotes.