RESUMO
Driven by the needs of functional genomics, DNA engineering by homologous recombination in Escherichia coli has emerged as a major addition to existing technologies. Two alternative approaches, RecA-dependent engineering and ET recombination, allow a wide variety of DNA modifications, including some which are virtually impossible by conventional methods. These approaches do not rely on the presence of suitable restriction sites and can be used to insert, delete or substitute DNA sequences at any desired position on a target molecule. Furthermore, ET recombination can be used for direct subcloning and cloning of DNA sequences from complex mixtures, including bacterial artificial chromosomes and genomic DNA preparations. The strategies reviewed in this article are applicable to modification of DNA molecules of any size, including very large ones, and present powerful new avenues for DNA manipulation in general.
Assuntos
Clonagem Molecular/métodos , DNA/metabolismo , Engenharia Genética/métodos , Escherichia coli/genética , Escherichia coli/metabolismo , Modelos Genéticos , Recombinases Rec A/metabolismo , Proteínas Recombinantes/metabolismo , Recombinação Genética , Saccharomyces cerevisiae/metabolismoRESUMO
The cloning of foreign DNA in Escherichia coli episomes is a cornerstone of molecular biology. The pioneering work in the early 1970s, using DNA ligases to paste DNA into episomal vectors, is still the most widely used approach. Here we describe a different principle, using ET recombination, for directed cloning and subcloning, which offers a variety of advantages. Most prominently, a chosen DNA region can be cloned from a complex mixture without prior isolation. Hence cloning by ET recombination resembles PCR in that both involve the amplification of a DNA region between two chosen points. We apply the strategy to subclone chosen DNA regions from several target molecules resident in E. coli hosts, and to clone chosen DNA regions from genomic DNA preparations. Here we analyze basic aspects of the approach and present several examples that illustrate its simplicity, flexibility, and remarkable efficiency.
Assuntos
Clonagem Molecular/métodos , DNA Bacteriano/genética , Escherichia coli/genética , Recombinação Genética , Sequência de Bases , DNA Bacteriano/metabolismo , Exodesoxirribonucleases/metabolismo , Dados de Sequência Molecular , Plasmídeos/genéticaRESUMO
The initial steps of double-stranded break (DSB) repair by homologous recombination mediated by the 5'-3' exonuclease/annealing protein pairs, RecE/RecT and Redalpha/Redbeta, were analyzed. Recombination was RecA-independent and required the expression of both components of an orthologous pair, even when the need for exonuclease activity was removed by use of preresected substrates. The required orthologous function correlated with a specific protein-protein interaction, and recombination was favored by overexpression of the annealing protein with respect to the exonuclease. The need for both components of an orthologous pair was observed regardless of whether recombination proceeded via a single-strand annealing or a putative strand invasion mechanism. The DSB repair reactions studied here are reminiscent of the RecBCD/RecA reaction and suggest a general mechanism that is likely to be relevant to other systems, including RAD52 mediated recombination.
Assuntos
Proteínas de Bactérias/metabolismo , Reparo do DNA/fisiologia , Proteínas de Ligação a DNA , Proteínas de Escherichia coli , Exodesoxirribonucleases/metabolismo , Recombinação Genética , Proteínas de Bactérias/genética , Sequência de Bases , DNA/genética , DNA/metabolismo , Exodesoxirribonucleases/genética , Dados de Sequência Molecular , Recombinases Rec A/genética , Recombinases Rec A/metabolismoRESUMO
Bacterial artificial chromosomes (BACs) offer many advantages for functional studies of large eukaryotic genes. To utilize the potential applications of BACs optimally, new approaches that allow rapid and precise engineering of these large molecules are required. Here, we describe a simple and flexible two-step approach based on ET recombination, which permits point mutations to be introduced into BACs without leaving any other residual change in the recombinant product. Introduction of other modifications, such as small insertions or deletions, is equally feasible. The use of ET recombination to achieve site-directed mutagenesis opens access to a powerful use of BACs and is extensible to DNA molecules of any size in Escherichia coli, including the E. coli chromosome.
Assuntos
Cromossomos Artificiais Bacterianos/genética , Escherichia coli/genética , Mutagênese Sítio-Dirigida/genética , Mutação Puntual/genética , Recombinação Genética/genética , Cromossomos Bacterianos/genética , DNA Bacteriano/genética , DNA Recombinante/genética , Engenharia Genética/métodos , Hexosiltransferases/genética , Plasmídeos/genéticaAssuntos
Clonagem Molecular/métodos , DNA Recombinante/genética , Proteínas de Ligação a DNA , Proteínas de Escherichia coli , Recombinação Genética , Animais , Proteínas de Bactérias/metabolismo , Cromossomos Artificiais Bacterianos/genética , Cromossomos Bacterianos/genética , Escherichia coli/genética , Exodesoxirribonucleases/metabolismo , Engenharia Genética , Humanos , CamundongosRESUMO
memA was isolated by subtractive hybridization in which the mRNA repertoire was compared in a panel of human melanoma cell lines with different metastasizing potential. Expression of memA mRNA is elevated in the highly metastasizing human melanoma cell lines and derived xenografts, as compared with the non-metastasizing ones. In a collection of human tumor cell lines and melanoma metastasis lesions, memA mRNA expression could be detected in the A-431 (epidermoid carcinoma), HT-1080 (fibrosarcoma), JEG-3 and JAR (choriocarcinomas) cell lines and in three out of 11 melanoma metastasis lesions. The distribution of memA mRNA in a collection of healthy human organs is also tissue restricted. Sequence analysis revealed that the MEMA protein is identical with a 160 kDa nuclear 'domain rich in serines' (DRS) protein occurring free in the nucleoplasm and in U2-ribonucleoprotein structures. MEMA is also homologous to pinin, a 140 kDa protein associated with the desmosome-intermediate filament complex, and to a 32 kDa porcine neutrophilic protein that was copurified with components of the NADPH-oxidase enzyme complex. The encoded amino acid sequence predicts that the MEMA protein has three coiled-coil domains, one glycine loop domain, is very hydrophilic and contains regions rich in glutamine/proline, glutamic acid and serine residues.
Assuntos
Moléculas de Adesão Celular , Proteínas Nucleares/genética , RNA Mensageiro/análise , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Humanos , Rim/metabolismo , Pulmão/metabolismo , Melanoma/genética , Dados de Sequência Molecular , Metástase Neoplásica , Proteínas Nucleares/análise , Proteínas Nucleares/biossíntese , RNA Mensageiro/biossíntese , Alinhamento de Sequência , Neoplasias Cutâneas/genética , Células Tumorais CultivadasRESUMO
We present a method to modify bacterial artificial chromosomes (BACs) resident in their host strain. The method is based on homologous recombination by ET-cloning. We have successfully modified BACs at two distinct loci by recombination with a PCR product containing homology arms of 50 nt. The procedure we describe here is rapid, was found to work with high efficiency and should be applicable to any BAC modification desired.
Assuntos
Proteínas de Bactérias/metabolismo , Cromossomos Bacterianos/genética , Clonagem Molecular/métodos , Proteínas de Ligação a DNA , Proteínas de Escherichia coli , Exodesoxirribonucleases/metabolismo , Recombinação Genética , Modelos GenéticosRESUMO
A straightforward way to engineer DNA in E. coli using homologous recombination is described. The homologous recombination reaction uses RecE and RecT and is transferable between E. coli strains. Several target molecules were manipulated, including high copy plasmids, a large episome and the E. coli chromosome. Sequential steps of homologous or site-specific recombination were used to demonstrate a new logic for engineering DNA, unlimited by the disposition of restriction endonuclease cleavage sites or the size of the target DNA.
