Construction of a multiframe vector to express coding sequences in Escherichia coli.
Cloning of foreign DNA fragments for coding sequence analysis in Escherichia coli usually involves sets of three vectors. To simplify this, we constructed an expression vector named pMFV7 containing three ATG codons in different frames downstream of a Shine-Dalgarno sequence, assuming that the ribosome can use any of the three start codons in an alternative manner. Translation beginning at either of the start codons would drive the expression of any coding fragment cloned downstream. To test the feasibility of this proposal, we cloned DNA fragments of the lacZ gene in each of the possible reading frames downstream from pMFV7 start codons. Sequence analysis of the N-terminus regions around the fusion sites indicates that ribosomes indeed initiate translation at each of the three initiation codons. In one case, levels of beta-galactosidase activity depended largely on the N-terminus of the translation products. We conclude that pMFV7 may be useful for expressing coding sequences regardless of their reading frame.
Domínguez-Martínez
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V
Facultad de Ciencias Biológicas e Instituto, Salud Pública, Universidad Veracruzana, Xalapa, Ver., México.
Guarneros-Peña
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G
Segura-Nieto
M
M
Curiel-Quesada
E
E
D003001:Cloning, Molecular / Q000379:methods*; D018387:Codon, Initiator; D004274:DNA, Recombinant; D004926:Escherichia coli / Q000235:genetics* / Q000502:physiology*; D015870:Gene Expression; D005798:Genes, Bacterial; D017930:Genes, Reporter; D005822:Genetic Vectors; D007763:Lac Operon; D010442:Peptide Chain Initiation, Translational; D010957:Plasmids; D016364:Reading Frames; D012270:Ribosomes / Q000502:physiology; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D018387:Codon, Initiator; D004274:DNA, Recombinant; D001616:beta-Galactosidase