RESUMO
Recombination, or chimera formation, is known to occur between related template sequences present in a single PCR amplification. To characterize the conditions under which such recombinant amplification products form we monitored the exchange of sequence between two homologous templates carrying different restriction sites separated by 282 bp. Using a typical cycling program the rates of recombination between the two restriction sites were 1 and 7% using Taq and Vent polymerases respectively over 12 doublings. However, by using long elongation times and cycling only to the mid-point of the amplification recombination could be suppressed below visual detection with both polymerases. Conversely, cycling programs designed to promote incomplete primer elongation and subsequent template strand exchange stimulated recombination to >20%.
Assuntos
Reação em Cadeia da Polimerase/métodos , Recombinação Genética , Sequência de Bases , Primers do DNA , DNA Polimerase Dirigida por DNA , Modelos Genéticos , Mapeamento por Restrição , Taq Polimerase , Moldes GenéticosRESUMO
Novel RNA enzymes, or ribozymes, are sought in large pools of random RNA sequences. Because of the large number of random positions in an individual pool molecule, only a vanishingly small fraction of the possible sequences are actually present. Even so, increasing the length of the individual pool molecules significantly increases the probability of finding a particular complex ribozyme. Because ribozymes are typically composed of conserved sequences interleaved with regions that can vary in sequence and length, a longer molecule allows a greater number of possible arrangements of a given ribozyme motif, increasing the likelihood that it will be present in the pool. Once a ribozyme motif has been found, rational and irrational optimization techniques can be used to identify related ribozyme sequences with greater activity.
Assuntos
RNA Catalítico/química , RNA Catalítico/genética , Sequência de Bases , Biotecnologia , Dados de Sequência Molecular , Estrutura Molecular , Conformação de Ácido NucleicoRESUMO
To activate transcription of the glnA gene, the dimeric NTRC protein (nitrogen regulatory protein C) of enteric bacteria binds to an enhancer located approximately 100 bp upstream of the promoter. The enhancer is composed of two binding sites for NTRC that are three turns of the DNA helix apart. One role of the enhancer is to tether NTRC in high local concentration near the promoter to allow for its frequent interaction with sigma 54 holoenzyme by DNA looping. We have found that a second role of the enhancer is to ensure oligomerization of NTRC into a complex of at least two dimers that is required for transcriptional activation. Formation of this complex is greatly facilitated by a protein-protein interaction between NTRC dimers that is increased when the protein is phosphorylated.
