RESUMO
Variation in chromatin composition and organization often reflects differences in genome function. Histone variants, for example, replace canonical histones to contribute to regulation of numerous nuclear processes including transcription, DNA repair, and chromosome segregation. Here we focus on H2A.Bbd, a rapidly evolving variant found in mammals but not in invertebrates. We report that in human cells, nucleosomes bearing H2A.Bbd form unconventional chromatin structures enriched within actively transcribed genes and characterized by shorter DNA protection and nucleosome spacing. Analysis of transcriptional profiles from cells depleted for H2A.Bbd demonstrated widespread changes in gene expression with a net downregulation of transcription and disruption of normal mRNA splicing patterns. In particular, we observed changes in exon inclusion rates and increased presence of intronic sequences in mRNA products upon H2A.Bbd depletion. Taken together, our results indicate that H2A.Bbd is involved in formation of a specific chromatin structure that facilitates both transcription and initial mRNA processing.
Assuntos
Histonas/genética , Processamento Pós-Transcricional do RNA , RNA Mensageiro/genética , Transcrição Gênica , Linhagem Celular Tumoral , Cromatina/genética , Regulação para Baixo , Éxons , Expressão Gênica , Variação Genética , Células HeLa , Humanos , Íntrons , Nucleossomos/genética , Proteômica/métodos , Splicing de RNARESUMO
Analysis of large-scale interphase genome positioning with reference to a nuclear landmark has recently been studied using sequencing-based single cell approaches. However, these approaches are dependent upon technically challenging, time consuming and costly high throughput sequencing technologies, requiring specialized bioinformatics tools and expertise. Here, we propose a novel, affordable and robust microscopy-based single cell approach, termed Topokaryotyping, to analyze and reconstruct the interphase positioning of genomic loci relative to a given nuclear landmark, detectable as banding pattern on mitotic chromosomes. This is accomplished by proximity-dependent histone labeling, where biotin ligase BirA fused to nuclear envelope marker Emerin was coexpressed together with Biotin Acceptor Peptide (BAP)-histone fusion followed by (i) biotin labeling, (ii) generation of mitotic spreads, (iii) detection of the biotin label on mitotic chromosomes and (iv) their identification by karyotyping. Using Topokaryotyping, we identified both cooperativity and stochasticity in the positioning of emerin-associated chromatin domains in individual cells. Furthermore, the chromosome-banding pattern showed dynamic changes in emerin-associated domains upon physical and radiological stress. In summary, Topokaryotyping is a sensitive and reliable technique to quantitatively analyze spatial positioning of genomic regions interacting with a given nuclear landmark at the single cell level in various experimental conditions.
Assuntos
Cariotipagem/métodos , Mitose , Membrana Nuclear/metabolismo , Análise de Célula Única/métodos , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromatina/genética , Cromatina/metabolismo , Células HEK293 , Células HeLa , Histonas/metabolismo , Humanos , Hibridização in Situ Fluorescente , Interfase , Proteínas de Membrana/metabolismo , Microscopia Confocal , Membrana Nuclear/genética , Proteínas Nucleares/metabolismo , Reprodutibilidade dos TestesRESUMO
Facioscapulohumeral dystrophy (FSHD) is an epi/genetic satellite disease associated with at least two satellite sequences in 4q35: (i) D4Z4 macrosatellite and (ii) ß-satellite repeats (BSR), a prevalent part of the 4qA allele. Most of the recent FSHD studies have been focused on a DUX4 transcript inside D4Z4 and its tandem contraction in FSHD patients. However, the D4Z4-contraction alone is not pathological, which would also require the 4qA allele. Since little is known about BSR, we investigated the 4qA BSR functional role in the transcriptional control of the FSHD region 4q35. We have shown that an individual BSR possesses enhancer activity leading to activation of the Adenine Nucleotide Translocator 1 gene (ANT1), a major FSHD candidate gene. We have identified ZNF555, a previously uncharacterized protein, as a putative transcriptional factor highly expressed in human primary myoblasts that interacts with the BSR enhancer site and impacts the ANT1 promoter activity in FSHD myoblasts. The discovery of the functional role of the 4qA allele and ZNF555 in the transcriptional control of ANT1 advances our understanding of FSHD pathogenesis and provides potential therapeutic targets.
Assuntos
Translocador 1 do Nucleotídeo Adenina/genética , Cromossomos Humanos Par 4 , Distrofia Muscular Facioescapuloumeral/genética , Fatores de Transcrição/metabolismo , Ativação Transcricional , Translocador 1 do Nucleotídeo Adenina/biossíntese , Alelos , Sítios de Ligação , Células Cultivadas , DNA Satélite , Elementos Facilitadores Genéticos , Loci Gênicos , Humanos , Proteínas dos Microfilamentos , Mioblastos/metabolismo , Proteínas Nucleares/biossíntese , Proteínas Nucleares/genética , Proteínas de Ligação a RNA , Fatores de Transcrição/antagonistas & inibidoresRESUMO
We have developed an approach termed PUB-NChIP (proximity utilizing biotinylation with native ChIP) to purify and study the protein composition of chromatin in proximity to a nuclear protein of interest. It is based on coexpression of (1) a protein of interest, fused with the bacterial biotin ligase BirA, together with (2) a histone fused to a biotin acceptor peptide (BAP), which is specifically biotinylated by BirA-fusion in the proximity of the protein of interest. Using the RAD18 protein as a model, we demonstrate that the RAD18-proximal chromatin is enriched in some H4 acetylated species. Moreover, the RAD18-proximal chromatin containing a replacement histone H2AZ has a different pattern of H4 acetylation. Finally, biotin pulse-chase experiments show that the H4 acetylation pattern starts to resemble the acetylation pattern of total H4 after the proximity of chromatin to RAD18 has been lost.
Assuntos
Imunoprecipitação da Cromatina/métodos , Cromatina/metabolismo , Proteínas Nucleares/metabolismo , Acetilação , Biotinilação/métodos , Linhagem Celular , Histonas , Humanos , Processamento de Proteína Pós-TraducionalRESUMO
pAp (3'-5' phosphoadenosine phosphate) is a by-product of sulfur and lipid metabolism and has been shown to have strong inhibitory properties on RNA catabolism. In the present paper we report a new target of pAp, PARP-1 [poly(ADP-ribose) polymerase 1], a key enzyme in the detection of DNA single-strand breaks. We show that pAp can interact with PARP-1 and inhibit its poly(ADP-ribosyl)ation activity. In vitro, inhibition of PARP-1 was detectable at micromolar concentrations of pAp and altered both PARP-1 automodification and heteromodification of histones. Analysis of the kinetic parameters revealed that pAp acted as a mixed inhibitor that modulated both the Km and the Vmax of PARP-1. In addition, we showed that upon treatment with lithium, a very potent inhibitor of the enzyme responsible for pAp recycling, HeLa cells exhibited a reduced level of poly(ADP-ribosyl)ation in response to oxidative stress. From these results, we propose that pAp might be a physiological regulator of PARP-1 activity.
Assuntos
Difosfato de Adenosina/farmacologia , Lítio/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases , Células HeLa , Histonas/genética , Histonas/metabolismo , Humanos , Cinética , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Especificidade por SubstratoRESUMO
Starvation in bacteria is a complex adaptive response to deprivation of nutrients that has been shown to implicate a number of stress networks that modulate pathogenicity and antibiotic resistance. Starvation in nature is qualitatively different from in-culture late stationary phase energy source depletion. To look into proteome-level alterations elicited by complete elimination of carbon source, we used Escherichia coli HT115-derived SLE1 strain cells and a combination of label-free and metabolic isotope labeling approaches. We isolated pathways differentially affected by carbon starvation and observed robust upregulation of proteins implicated in networks belonging to Gene Ontology terms 'Biological adhesion' and 'Methylglyoxal metabolic process'.
Assuntos
Escherichia coli , Proteômica , Escherichia coli/metabolismo , Carbono/metabolismo , Aldeído Pirúvico/metabolismo , Regulação para CimaRESUMO
Protein tags are peptide sequences genetically embedded into a recombinant protein for various purposes, such as affinity purification, Western blotting, and immunofluorescence. Another recent application of peptide tags is in vivo labeling and analysis of protein-protein interactions (PPI) by proteomics methods. One of the common workflows involves site-specific in vivo biotinylation of an AviTag-fused protein in the presence of the biotin ligase BirA. However, due to the rapid kinetics of labeling, this tag is not ideal for analysis of PPI. Here we describe the rationale, design, and protocol for the new biotin acceptor peptides BAP1070 and BAP1108 using modular assembling of biotin acceptor fragments, DNA sequencing, transient expression of proteins in cells, and Western blotting methods. These tags were used in the Proximity Utilizing Biotinylation (PUB) method, which is based on coexpression of BAP-X and BirA-Y in mammalian cells, where X or Y are candidate interacting proteins of interest. By changing the sequence of these peptides, a low level of background biotinylation is achieved, which occurs due to random collisions of proteins in cells. Over 100 plasmid constructs, containing genes of transcription factors, histones, gene repressors, and other nuclear proteins were obtained during implementation of projects related to this method.
RESUMO
Protein footprinting is a new methodology that is based on probing, typically with the use of MS, of reactivity of different amino acid residues to a modifying reagent. Data thus obtained allow one to make inferences about protein conformations and their intermolecular interactions. Most of the protein footprinting studies so far have been performed on individual proteins in vitro. We explore whether a similar approach is possible with the proteins inside of living cells, employing dimethylsulfate (DMS), a reagent widely used for the in vivo footprinting of nucleic acids. DMS can induce methylation of the lysine, histidine and glutamate residues on proteins. Using models of the histone H2B/H2AZ heterodimer assembled in vitro and from chromatin treated in vivo, we show that the methylation by deuterated DMS allows one to distinguish the accessibility of a particular residue in and out of the protein's environmental/structural context. The detection of changes in protein conformations or their interactions in vivo can provide a new approach to the identification of proteins involved in various intracellular pathways and help in the search for perspective drug targets and biomarkers of diseases.
Assuntos
Histonas/metabolismo , Proteoma/metabolismo , Proteômica/métodos , Ésteres do Ácido Sulfúrico/metabolismo , Sequência de Aminoácidos , Animais , Cromatina/metabolismo , Ácido Glutâmico/metabolismo , Células HeLa , Histidina/metabolismo , Humanos , Lisina/metabolismo , Metilação , Dados de Sequência Molecular , Dobramento de Proteína , Multimerização Proteica , XenopusRESUMO
The common techniques to study protein-protein proximity in vivo are not well adapted to the capabilities and the expertise of a standard proteomics laboratory, typically based on the use of mass spectrometry. With the aim of closing this gap, we have developed PUB-MS (for proximity utilizing biotinylation and mass spectrometry), an approach to monitor protein-protein proximity, based on biotinylation of a protein fused to a biotin-acceptor peptide (BAP) by a biotin-ligase, BirA, fused to its interaction partner. The biotinylation status of the BAP can be further detected by either Western analysis or mass spectrometry. The BAP sequence was redesigned for easy monitoring of the biotinylation status by LC-MS/MS. In several experimental models, we demonstrate that the biotinylation in vivo is specifically enhanced when the BAP- and BirA-fused proteins are in proximity to each other. The advantage of mass spectrometry is demonstrated by using BAPs with different sequences in a single experiment (allowing multiplex analysis) and by the use of stable isotopes. Finally, we show that our methodology can be also used to study a specific subfraction of a protein of interest that was in proximity with another protein at a predefined time before the analysis.
Assuntos
Espectrometria de Massas/métodos , Proteômica/métodos , Sequência de Aminoácidos , Biotina/química , Biotinilação , Fibroblastos/citologia , Transferência Ressonante de Energia de Fluorescência/métodos , Células HEK293 , Humanos , Isótopos/química , Dados de Sequência Molecular , Mapeamento de Interação de Proteínas/métodos , Proteínas Recombinantes/químicaRESUMO
MyoD regulates skeletal myogenesis. Since proteins associated with MyoD exert regulatory functions, their identification is expected to contribute important insights into the mechanisms governing gene expression in skeletal muscle. We have found that the RNA helicases p68/p72 are MyoD-associated proteins and that the noncoding RNA SRA also immunoprecipitates with MyoD. In vitro and in vivo experiments indicated that both p68/p72 and SRA are coactivators of MyoD. RNA interference toward either p68/p72 or SRA prevented proper activation of muscle gene expression and cell differentiation. Unexpectedly, reducing the levels of p68/p72 proteins impaired recruitment of the TATA binding protein TBP; RNA polymerase II; and the catalytic subunit of the ATPase SWI/SNF complex, Brg-1, and hindered chromatin remodeling. These findings reveal that p68/p72 play a critical role in promoting the assembly of proteins required for the formation of the transcription initiation complex and chromatin remodeling.
Assuntos
Diferenciação Celular/fisiologia , Músculo Esquelético/fisiologia , Proteína MyoD/metabolismo , RNA Helicases/metabolismo , RNA não Traduzido/metabolismo , Sequência de Aminoácidos , Células HeLa , Humanos , Dados de Sequência Molecular , Músculo Esquelético/citologia , Proteína MyoD/genética , RNA Helicases/química , Homologia de Sequência de AminoácidosRESUMO
Escherichia coli possesses only one essential oligoribonuclease (Orn), an enzyme that can degrade oligoribonucleotides of five residues and shorter in length (nanoRNA). Firmicutes including Bacillus subtilis do not have an Orn homolog. We had previously identified YtqI (NrnA) as functional analog of Orn in B. subtilis. Screening a genomic library from B. subtilis for genes that can complement a conditional orn mutant, we identify here YngD (NrnB) as a second nanoRNase in B. subtilis. Like NrnA, NrnB is a member of the DHH/DHHA1 protein family of phosphoesterases. NrnB degrades nanoRNA 5-mers in vitro similarily to Orn. Low expression levels of NrnB are sufficient for orn complementation. YhaM, a known RNase present in B. subtilis, degrades nanoRNA efficiently in vitro but requires high levels of expression for only partial complementation of the orn(-) strain. A triple mutant (nrnA(-), nrnB(-), yhaM(-)) in B. subtilis is viable and shows almost no impairment in growth. Lastly, RNase J1 seems also to have some 5'-to-3' exoribonuclease activity on nanoRNA and thus can potentially finish degradation of RNA. We conclude that, unlike in E. coli, degradation of nanoRNA is performed in a redundant fashion in B. subtilis.
Assuntos
Bacillus subtilis/enzimologia , Oligorribonucleotídeos/metabolismo , Ribonucleases/metabolismo , Bacillus subtilis/genética , DNA/metabolismo , Teste de Complementação Genética , Mutação , Oligorribonucleotídeos/química , Filogenia , RNA/química , RNA/metabolismo , Ribonucleases/classificação , Ribonucleases/genéticaRESUMO
Tumor-derived mutant forms of p53 compromise its DNA binding, transcriptional, and growth regulatory activity in a manner that is dependent upon the cell-type and the type of mutation. Given the high frequency of p53 mutations in human tumors, reactivation of the p53 pathway has been widely proposed as beneficial for cancer therapy. In support of this possibility p53 mutants possess a certain degree of conformational flexibility that allows for re-induction of function by a number of structurally different artificial compounds or by short peptides. This raises the question of whether physiological pathways for p53 mutant reactivation also exist and can be exploited therapeutically. The activity of wild-type p53 is modulated by various acetyl-transferases and deacetylases, but whether acetylation influences signaling by p53 mutant is still unknown. Here, we show that the PCAF acetyl-transferase is down-regulated in tumors harboring p53 mutants, where its re-expression leads to p53 acetylation and to cell death. Furthermore, acetylation restores the DNA-binding ability of p53 mutants in vitro and expression of PCAF, or treatment with deacetylase inhibitors, promotes their binding to p53-regulated promoters and transcriptional activity in vivo. These data suggest that PCAF-mediated acetylation rescues activity of at least a set of p53 mutations. Therefore, we propose that dis-regulation of PCAF activity is a pre-requisite for p53 mutant loss of function and for the oncogenic potential acquired by neoplastic cells expressing these proteins. Our findings offer a new rationale for therapeutic targeting of PCAF activity in tumors harboring oncogenic versions of p53.
Assuntos
DNA/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Fatores de Transcrição de p300-CBP/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Cromatina/metabolismo , Neoplasias Colorretais/metabolismo , Humanos , Camundongos , Mutação , Ligação Proteica , Proteína Supressora de Tumor p53/genética , Fatores de Transcrição de p300-CBP/genéticaRESUMO
Protein-protein interactions of core pluripotency transcription factors play an important role during cell reprogramming. Cell identity is controlled by a trio of transcription factors: Sox2, Oct4, and Nanog. Thus, methods that help to quantify protein-protein interactions may be useful for understanding the mechanisms of pluripotency at the molecular level. Here, a detailed protocol for the detection and quantitative analysis of in vivo protein-protein proximity of Sox2 and Oct4 using the proximity-utilizing biotinylation (PUB) method is described. The method is based on the coexpression of two proteins of interest fused to a biotin acceptor peptide (BAP)in one case and a biotin ligase enzyme (BirA) in the other. The proximity between the two proteins leads to more efficient biotinylation of the BAP, which can be either detected by Western blotting or quantified using proteomics approaches, such as a multiple reaction monitoring (MRM) analysis. Coexpression of the fusion proteins BAP-X and BirA-Y revealed strong biotinylation of the target proteins when X and Y were, alternatively, the pluripotency transcription factors Sox2 and Oct4, compared with the negative control where X or Y was green fluorescent protein (GFP), which strongly suggests that Sox2 and Oct4 come in close proximity to each other and interact.
Assuntos
DNA/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Mapeamento de Interação de Proteínas/métodos , Fatores de Transcrição SOXB1/metabolismo , Biotinilação , Carbono-Nitrogênio Ligases/metabolismo , Proteínas de Escherichia coli/metabolismo , Células HEK293 , Humanos , Mapas de Interação de Proteínas , Proteínas Repressoras/metabolismoRESUMO
Rapalogs have become standard-of-care in patients with metastatic breast, kidney, and neuroendocrine cancers. Nevertheless, tumor escape occurs after several months in most patients, highlighting the need to understand mechanisms of resistance. Using a panel of cancer cell lines, we show that rapalogs downregulate the putative protein kinase TRIB3 (tribbles pseudokinase 3). Blood samples of a small cohort of patients with cancer treated with rapalogs confirmed downregulation of TRIB3. Downregulation of TRIB3 was mediated by LRRFIP1 independently of mTOR and disrupted its interaction with the spliceosome, where it participated in rapalog-induced deregulation of RNA splicing. Conversely, overexpression of TRIB3 in a panel of cancer cell lines abolished the cytotoxic effects of rapalogs. These findings identify TRIB3 as a key component of the spliceosome, whose repression contributes significantly to the mechanism of resistance to rapalog therapy. SIGNIFICANCE: Independent of mTOR signaling, rapalogs induce cytoxicity by dysregulating spliceosome function via repression of TRIB3, the loss of which may, in the long term, contribute to therapeutic resistance.
Assuntos
Proteínas de Ciclo Celular/genética , Neoplasias/genética , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Repressoras/genética , Sirolimo/farmacologia , Antineoplásicos/farmacologia , Proteínas de Ciclo Celular/biossíntese , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Everolimo/farmacologia , Expressão Gênica/efeitos dos fármacos , Humanos , Células MCF-7 , Neoplasias/sangue , Neoplasias/metabolismo , Regiões Promotoras Genéticas/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/biossíntese , Proteínas Serina-Treonina Quinases/genética , Splicing de RNA/efeitos dos fármacos , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Repressoras/biossíntese , Sirolimo/análogos & derivados , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismoRESUMO
We describe a modification of the tandem affinity purification method for purification and analysis of multiprotein complexes, termed here DEF-TAP (for differential elution fractionation after tandem affinity purification). Its essential new feature is the use for last purification step of 6xHis-Ni(++) interaction, which is resistant to a variety of harsh washing conditions, including high ionic strength and the presence of organic solvents. This allows us to use various fractionation schemes before the protease digestion, which is expected to improve the coverage of the analyzed protein mixture and also to provide an additional insight into the structure of the purified macromolecular complex and the nature of protein-protein interactions involved. We illustrate our new approach by analysis of soluble nuclear complexes containing histone H4 purified from HeLa cells. In particular, we observed different fractionation patterns of HAT1 and RbAp46 proteins as compared with RbAp48 protein, all identified as interaction partners of H4 histone. In addition, we report all components of the licensing MCM2-7 complex and the apoptosis-related DAXX protein among the interaction partners of the soluble H4. Finally, we show that HAT1 requires N-terminal tail of H4 for its stable association with this histone.
Assuntos
Histonas/análise , Proteômica/métodos , Acetilação , Sequência de Aminoácidos , Sequência de Bases , Vetores Genéticos , Histona Acetiltransferases/análise , Histona Acetiltransferases/metabolismo , Histonas/química , Histonas/genética , Histonas/metabolismo , Humanos , Dados de Sequência Molecular , Ligação Proteica , Proteína 4 de Ligação ao Retinoblastoma/análise , Proteína 4 de Ligação ao Retinoblastoma/metabolismo , Proteína 7 de Ligação ao Retinoblastoma/análise , Proteína 7 de Ligação ao Retinoblastoma/metabolismoRESUMO
Chromatin is considered to be a principal carrier of epigenetic information due to the ability of alternative chromatin states to persist through generations of cell divisions and to spread on DNA. Replacement histone variants are novel candidates for epigenetic marking of chromatin. We developed a novel approach to analyze the chromatin environment of nucleosomes containing a particular replacement histone. We applied it to human H2AZ, one of the most studied alternative histones. We find that neither H2AZ itself nor other features of the H2AZ-containing nucleosome spread to the neighboring nucleosomes in vivo, arguing against a role for H2AZ as a self-perpetuating epigenetic mark.
Assuntos
Epigênese Genética , Histonas/genética , Histonas/metabolismo , Sequência de Aminoácidos , Animais , Cromatina/genética , Cromatina/metabolismo , Inativação Gênica , Variação Genética , Células HeLa , Histonas/química , Humanos , Camundongos , Modelos Biológicos , Dados de Sequência Molecular , Células NIH 3T3 , Nucleossomos/metabolismo , Processamento de Proteína Pós-Traducional , Estrutura Quaternária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Oligoribonuclease is the only RNase in Escherichia coli that is able to degrade RNA oligonucleotides five residues and shorter in length. Firmicutes including Bacillus subtilis do not have an Oligoribonuclease (Orn) homologous protein and it is not yet understood which proteins accomplish the equivalent function in these organisms. We had previously identified oligoribonucleases Orn from E. coli and its human homolog Sfn in a screen for proteins that are regulated by 3'-phosphoadenosine 5'-phosphate (pAp). Here, we identify YtqI as a potential functional analog of Orn through its interaction with pAp. YtqI degrades RNA oligonucleotides in vitro with preference for 3-mers. In addition, YtqI has pAp-phosphatase activity in vitro. In agreement with these data, YtqI is able to complement both orn and cysQ mutants in E. coli. An ytqI mutant in B. subtilis shows impairment of growth in the absence of cysteine, a phenotype resembling that of a cysQ mutant in E. coli. Phylogenetic distribution of YtqI, Orn and CysQ supports bifunctionality of YtqI.
Assuntos
Difosfato de Adenosina/metabolismo , Bacillus subtilis/enzimologia , Proteínas de Bactérias/metabolismo , Exorribonucleases/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Escherichia coli/genética , Exorribonucleases/genética , Teste de Complementação Genética , Monoéster Fosfórico Hidrolases/genética , Filogenia , RNA/metabolismo , Especificidade por SubstratoRESUMO
BACKGROUND: A fundamental requirement for genomic studies is the availability of genetic material of good quality and quantity. The desired quantity and quality are often hard to obtain when target DNA is composed of complex mixtures of relatively short DNA fragments. Here, we sought to develop a method to representatively amplify such complex mixtures by converting them to long linear and circular concatamers, from minute amounts of starting material, followed by phi29-based multiple displacement amplification. RESULTS: We report here proportional amplification of DNA fragments that were first converted into concatamers starting from DNA amounts as low as 1 pg. Religations at low concentration (< 1 ng/microL) preferentially lead to fragment self-circularization, which are then amplified independently, and result in non-uniform amplification. To circumvent this problem, an additional (stuffer) DNA was added during religation (religation concentration > 10 ng/microL), which helped in the formation of long concatamers and hence resulted in uniform amplification. To confirm its usefulness in research, DP1 bound chromatin was isolated through ChIP and presence of DHFR promoter was detected using q-PCR and compared with an irrelevant GAPDH promoter. The results clearly indicated that when ChIP material was religated in presence of stuffer DNA (improved MDA), it allowed to recover the original pattern, while standard MDA and MDA without stuffer DNA failed to do so. CONCLUSION: We believe that this method allows for generation of abundant amounts of good quality genetic material from a complex mixture of short DNA fragments, which can be further used in high throughput genetic analysis.
Assuntos
DNA/análise , Técnicas de Amplificação de Ácido Nucleico/métodos , Imunoprecipitação da Cromatina , DNA/metabolismo , Células HeLa , HumanosRESUMO
Tagging of proteins in vivo by covalent attachment of a biotin moiety has emerged as a new prospective tool for protein detection and purification. Previously, we established a strategy for expression of in vivo biotinylated proteins in mammalian cells. It is based on coexpression of the protein of interest fused to a short biotin acceptor peptide and biotin ligase BirA cloned in the same vector. We show here that the in vivo biotinylation can be used for immunogold postembedding labeling in immunoelectron microscopy experiments. We show that immunoelectron microscopy with biotinylated nuclear proteins is compatible with a wide range of postembedding methods, facilitating combination of morphological and localization studies in a single experiment. We also show that the method works in both transient transfection and stable cell line expression protocols and can be used for colocalization studies. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.
Assuntos
Biotina/metabolismo , Proteínas Nucleares/metabolismo , Biotinilação , Carbono-Nitrogênio Ligases/genética , Linhagem Celular , Cromatina/metabolismo , Proteínas de Escherichia coli/genética , Histonas/metabolismo , Humanos , Microscopia Imunoeletrônica , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Ligação Proteica , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/genética , Inclusão do Tecido , TransfecçãoRESUMO
We identified Oligoribonuclease (Orn), an essential Escherichia coli protein and the only exonuclease degrading small ribonucleotides (5mer to 2mer) and its human homologue, small fragment nuclease (Sfn), in a screen for proteins that are potentially regulated by 3'-phosphoadenosine 5'-phosphate (pAp). We show that both enzymes are sensitive to micromolar amounts of pAp in vitro. We also demonstrate that Orn can degrade short DNA oligos in addition to its activity on RNA oligos, similar to what was documented for Sfn. pAp was shown to accumulate as a result of inhibition of the pAp-degrading enzyme by lithium, widely used to treat bipolar disorder, thus its regulatory targets are of significant medical interest. CysQ, the E.coli pAp-phosphatase is strongly inhibited by lithium and calcium in vitro and is a main target of lithium toxicity in vivo. Our findings point to remarkable conservation of the connection between sulfur- and RNA metabolism between E.coli and humans.