RESUMO
Identification of nucleotides used for RNA chain initiation or for contacting DNA binding proteins is basic to our understanding of gene regulation. Normally, a radioactive primer is used to copy RNA or DNA. The polymerase extension stops at free ends of mRNA (as in promoter mapping) or at the position of template cleavage or modification (as in footprinting). The locations of these positions are then analyzed by polyacrylamide gel electrophoresis. These analyses have been improved using fluorescently labeled primers and commonly available DNA sequencing machines. The protocol, which we call fluorescently labeled oligonucleotide extension (FLOE), eliminates the need for handling radioactivity and polyacrylamide. The DNA sequencer delivers data as a "trace" that is ready for quantification, which eliminates the need to trace gels separately. The data analysis is further improved by new software, Scanalyze, which we present here. We demonstrate that by using promoter mapping and footprinting, FLOE shortens experimental time, extends the stretch of analyzable sequence, and simplifies quantification compared to radioactive methods and is as sensitive in terms of detecting templates.
Assuntos
Algoritmos , Primers do DNA , Hibridização in Situ Fluorescente/métodos , Sondas de Oligonucleotídeos , Análise de Sequência de DNA/métodos , Espectrometria de Fluorescência/métodos , Coloração e Rotulagem/métodos , Escherichia coli/genética , Escherichia coli/metabolismo , Corantes Fluorescentes , Perfilação da Expressão Gênica , Software , Transcrição Gênica/genéticaRESUMO
Analyzing gene expression profiles from cells en masse provides an average profile for the population which may obscure differences in individual cells. Using an optimized workflow for qRT-PCR, gene expression profiles of undifferentiated pluripotent stem cells reveal distinct gene expression profiles for individual cells, and a large expression level range of almost every gene. Importantly, this technique allows for the identification and characterization of small subpopulations.
Assuntos
Análise de Célula Única , Transcriptoma , Animais , Células Cultivadas , Expressão Gênica , Humanos , Células-Tronco Pluripotentes/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
TaqMan Array Cards are high-throughput, accurate, sensitive, and simple-to-use tools for quantitative analysis of mRNA or miRNA transcripts using a real-time PCR protocol. They utilize a microfluidic card with 384 reaction chambers and eight sample loading ports. For studies of coding transcripts, the reaction chambers are preloaded with user selected or predefined panels of Applied Biosystems TaqMan Gene Expression Assays. These assays enable real-time monitoring of a PCR reaction via hydrolysis of an oligonucleotide probe which has been dual labeled with fluorescent dye and quencher. Applications of TaqMan Array Cards include verification and follow on testing of microarray results, as well as hypothesis driven testing of panels of genes selected for their biological functions and relationships. This chapter describes a protocol for assaying transcription in cultured cells using methods optimized to minimize hands-on time and pipetting steps by skipping RNA isolation and generating cDNA directly in Ambion Cells-to-C(T) lysis solution.
Assuntos
Indústria Farmacêutica/métodos , Análise de Sequência com Séries de Oligonucleotídeos/instrumentação , Taq Polimerase/metabolismo , Animais , Extratos Celulares/química , Células Cultivadas , Indústria Farmacêutica/instrumentação , Humanos , Camundongos , Técnicas Analíticas Microfluídicas , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Reação em Cadeia da Polimerase , Ratos , Transcrição Reversa , Fatores de TempoRESUMO
Genome duplication and segregation normally are completed before cell division in all organisms. The temporal relation of duplication and segregation, however, can vary in bacteria. Chromosomal regions can segregate towards opposite poles as they are replicated or can stay cohered for a considerable period before segregation. The bacterium Vibrio cholerae has two differently sized circular chromosomes, chromosome I (chrI) and chrII, of about 3 and 1 Mbp, respectively. The two chromosomes initiate replication synchronously, and the shorter chrII is expected to complete replication earlier than the longer chrI. A question arises as to whether the segregation of chrII also is completed before that of chrI. We fluorescently labeled the terminus regions of chrI and chrII and followed their movements during the bacterial cell cycle. The chrI terminus behaved similarly to that of the Escherichia coli chromosome in that it segregated at the very end of the cell division cycle: cells showed a single fluorescent focus even when the division septum was nearly complete. In contrast, the single focus representing the chrII terminus could divide at the midcell position well before cell septation was conspicuous. There were also cells where the single focus for chrII lingered at midcell until the end of a division cycle, like the terminus of chrI. The single focus in these cells overlapped with the terminus focus for chrI in all cases. It appears that there could be coordination between the two chromosomes through the replication and/or segregation of the terminus region to ensure their segregation to daughter cells.
Assuntos
Segregação de Cromossomos , Cromossomos Bacterianos/metabolismo , Replicação do DNA/genética , Vibrio cholerae/genética , Cromossomos Bacterianos/genética , DNA Bacteriano/biossíntese , Corantes Fluorescentes , Origem de Replicação , Coloração e RotulagemRESUMO
In bacteria, many DNA-protein interactions that initiate transcription, replication and recombination require the mediation of DNA architectural proteins such as IHF and HU. For replication initiation, plasmid P1 requires three origin binding proteins: the architectural protein HU, a plasmid-specific initiator, RepA, and the Escherichia coli chromosomal initiator, DnaA. The two initiators bind in the origin of replication to multiple sites, called iterons and DnaA boxes respectively. We show here that all five known DnaA boxes can be deleted from the plasmid origin provided the origin is extended by about 120 bp. The additional DNA provides an IHF site and most likely a weak DnaA binding site, because replacing the putative site with an authentic DnaA box enhanced plasmid replication in an IHF-dependent manner. IHF most likely brings about interactions between distally bound DnaA and RepA by bending the intervening DNA. The role of IHF in activating P1 origin by allowing DnaA binding to a weak site is reminiscent of the role the protein plays in initiating the host chromosomal replication.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Fatores Hospedeiros de Integração/metabolismo , Plasmídeos/genética , Origem de Replicação/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Replicação do DNA/genética , Proteínas de Ligação a DNA/genética , Ensaio de Desvio de Mobilidade Eletroforética , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Fatores Hospedeiros de Integração/genética , Ligação Proteica , Transativadores/genética , Transativadores/metabolismo , Ativação TranscricionalRESUMO
With current concerns of antibiotic-resistant bacteria and biodefense, it has become important to rapidly identify infectious bacteria. Traditional technologies involving isolation and amplification of the pathogenic bacteria are time-consuming. We report a rapid and simple method that combines in vivo biotinylation of engineered host-specific bacteriophage and conjugation of the phage to streptavidin-coated quantum dots. The method provides specific detection of as few as 10 bacterial cells per milliliter in experimental samples, with an approximately 100-fold amplification of the signal over background in 1 h. We believe that the method can be applied to any bacteria susceptible to specific phages and would be particularly useful for detection of bacterial strains that are slow growing, e.g., Mycobacterium, or are highly infectious, e.g., Bacillus anthracis. The potential for simultaneous detection of different bacterial species in a single sample and applications in the study of phage biology are discussed.
Assuntos
Bacteriófagos/química , Biotina/análise , Bactérias Gram-Negativas/isolamento & purificação , Bactérias Gram-Positivas/isolamento & purificação , Nanotecnologia/métodos , Pontos Quânticos , Biotina/química , Citometria de Fluxo , Microscopia de Fluorescência , Sensibilidade e Especificidade , Fatores de TempoRESUMO
Eukaryotic chromosomes contain a locus, the centromere, at which force is applied to separate replicated chromosomes. A centromere analogue is also found in some bacterial plasmids and chromosomes, although not yet identified in the well-studied Escherichia coli chromosome. We aimed to identify centromere-like sequences in E. coli with the premise that such sequences would be the first to migrate towards the cell poles, away from the cell centre where DNA replication is believed to occur. We have labelled different loci on the chromosome by integrating arrays of binding sites for LacI-EYFP and phage lambdacI-ECFP and supplying these fusion proteins in trans. Comparison of such pairs of loci suggests the presence of a centromere-like site close to the origin of replication. Polar migration of the site was dependent on migS, a locus recently implicated in chromosome migration, thus providing strong support for migS being the E. coli centromere.
Assuntos
Segregação de Cromossomos , Cromossomos Bacterianos , DNA Bacteriano/metabolismo , Escherichia coli/genética , Sequência de Bases , Centrômero , Segregação de Cromossomos/fisiologia , Cromossomos Bacterianos/fisiologia , Escherichia coli/fisiologia , Genes Reporter , Proteínas Luminescentes/genética , Microscopia de Fluorescência , Recombinação Genética , Origem de ReplicaçãoRESUMO
Many organisms control initiation of DNA replication by limiting supply or activity of initiator proteins. In plasmids, such as P1, initiators are limited primarily by transcription and dimerization. However, the relevance of initiator limitation to plasmid copy number control has appeared doubtful, because initiator oversupply increases the copy number only marginally. Copy number control instead has been attributed to initiator-mediated plasmid pairing ("handcuffing"), because initiator mutations to handcuffing deficiency elevates the copy number significantly. Here, we present genetic evidence of a role for initiator limitation in plasmid copy number control by showing that autorepression-defective initiator mutants also can elevate the plasmid copy number. We further show, by quantitative modeling, that initiator dimerization is a homeostatic mechanism that dampens active monomer increase when the protein is oversupplied. This finding implies that oversupplied initiator proteins are largely dimeric, partly accounting for their limited ability to increase copy number. A combination of autorepression, dimerization, and handcuffing appears to account fully for control of P1 plasmid copy number.
Assuntos
Replicação do DNA , Homeostase , Plasmídeos , DNA/metabolismo , DNA Helicases/química , DNA Helicases/genética , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Dimerização , Dosagem de Genes , Fenótipo , Transativadores/química , Transativadores/genética , Transativadores/metabolismoRESUMO
Most HIV transmission occurs on the mucosal surfaces of the gastrointestinal and cervicovaginal tracts, both of which are normally coated by a biofilm of nonpathogenic commensal bacteria. We propose to genetically engineer such naturally occurring bacteria to protect against HIV infection by secreting antiviral peptides. Here we describe the development and characterization of Nissle 1917, a highly colonizing probiotic strain of Escherichia coli, secreting HIV-gp41-hemolysin A hybrid peptides that block HIV fusion and entry into target cells. By using an appropriate combination of cis- and transacting secretory and regulatory signals, micromolar secretion levels of the anti-HIV peptides were achieved. The genetically engineered Nissle 1917 were capable of colonizing mice for periods of weeks to months, predominantly in the colon and cecum, with lower concentrations of bacteria present in the rectum, vagina, and small intestine. Histological and immunocytochemical examination of the colon revealed bacterial growth and peptide secretion throughout the luminal mucosa and in association with epithelial surfaces. The use of genetically engineered live microbes as anti-HIV microbicides has important potential advantages in economy, efficacy, and durability.
Assuntos
Fármacos Anti-HIV/farmacologia , Escherichia coli/metabolismo , Engenharia Genética/métodos , Proteína gp41 do Envelope de HIV/metabolismo , Infecções por HIV/prevenção & controle , HIV/efeitos dos fármacos , Proteínas Hemolisinas/metabolismo , Peptídeos/farmacologia , Animais , Fármacos Anti-HIV/metabolismo , Ceco/microbiologia , Colo/microbiologia , Escherichia coli/genética , Feminino , HIV/metabolismo , Humanos , Mucosa Intestinal/microbiologia , Camundongos , Modelos Biológicos , Peptídeos/metabolismo , Vagina/microbiologiaRESUMO
TraM is a DNA binding protein required for conjugative transfer of the self-transmissible IncF group of plasmids, including F, R1, and R100. F TraM binds to three sites in F oriT: two high affinity binding sites, sbmA and sbmB, which are direct repeats of nearly identical sequence involved in the autoregulation of the traM gene; and a lower affinity site, sbmC, an inverted repeat important for transfer, which is situated nearest to the nic site where transfer originates. TraM bound cooperatively to its binding sites at oriT; the presence of sbmA and sbmB increased the affinity for sbmC 10-fold. Bending of oriT DNA by TraM was minimal, suggesting that TraM, a tetramer, was able to loop the DNA when bound to sbmA and sbmB simultaneously. Hydroxyl radical footprinting of DNA of sbmA and sbmC revealed that TraM contacted the DNA within a region previously delineated by DNase I footprinting. TraM protected the CT bases within the sequence CTAG, which occurred at 12-base intervals on the top and bottom strand of sbmA, most consistently with other protected bases. The footprint on sbmC revealed that the predicted inverted repeats were protected by TraM with a pattern that began at the center of the repeats and radiated outward at 11-12 base intervals toward the 5'-ends of either strand. At high protein concentrations, this pattern extended beyond the footprint defined by DNase I, suggesting that the DNA was wrapped around the protein forming a nucleosome-like structure, which could aid in preparing the DNA for transfer.
Assuntos
Proteínas de Bactérias/metabolismo , DNA/metabolismo , Origem de Replicação , Proteínas de Bactérias/química , Sequência de Bases , Sítios de Ligação , Ligação Competitiva , Clonagem Molecular , Pegada de DNA , Desoxirribonuclease I/metabolismo , Proteínas de Drosophila , Ensaio de Imunoadsorção Enzimática , Radical Hidroxila/metabolismo , Cinética , Dados de Sequência Molecular , Plasmídeos/metabolismo , Canais de Potássio , Ligação Proteica , Proteínas Recombinantes/metabolismoRESUMO
The general amino acid control (GAAC) enables yeast cells to overcome amino acid deprivation by activation of the alpha subunit of translation initiation factor 2 (eIF2alpha) kinase GCN2 and consequent induction of GCN4, a transcriptional activator of amino acid biosynthetic genes. Binding of GCN2 to GCN1 is required for stimulation of GCN2 kinase activity by uncharged tRNA in starved cells. Here we show that YIH1, when overexpressed, dampens the GAAC response (Gcn- phenotype) by suppressing eIF2alpha phosphorylation by GCN2. The overexpressed YIH1 binds GCN1 and reduces GCN1-GCN2 complex formation, and, consistent with this, the Gcn- phenotype produced by YIH1 overexpression is suppressed by GCN2 overexpression. YIH1 interacts with the same GCN1 fragment that binds GCN2, and this YIH1-GCN1 interaction requires Arg-2259 in GCN1 in vitro and in full-length GCN1 in vivo, as found for GCN2-GCN1 interaction. However, deletion of YIH1 does not increase eIF2alpha phosphorylation or derepress the GAAC, suggesting that YIH1 at native levels is not a general inhibitor of GCN2 activity. We discovered that YIH1 normally resides in a complex with monomeric actin, rather than GCN1, and that a genetic reduction in actin levels decreases the GAAC response. This Gcn- phenotype was partially suppressed by deletion of YIH1, consistent with YIH1-mediated inhibition of GCN2 in actin-deficient cells. We suggest that YIH1 resides in a YIH1-actin complex and may be released for inhibition of GCN2 and stimulation of protein synthesis under specialized conditions or in a restricted cellular compartment in which YIH1 is displaced from monomeric actin.