TY - JOUR
T1 - Construction of a multiframe vector to express coding sequences in Escherichia coli
AU - Domínguez-Martínez, V.
AU - Guarneros-Peña, G.
AU - Segura-Nieto, M.
AU - Curiel-Quesada, E.
PY - 2001
Y1 - 2001
N2 - 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 β-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.
AB - 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 β-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.
KW - Expression vectors
KW - In-frame gene cloning
KW - Translation initiation
UR - http://www.scopus.com/inward/record.url?scp=0035135028&partnerID=8YFLogxK
U2 - 10.1139/cjm-47-1-72
DO - 10.1139/cjm-47-1-72
M3 - Artículo
SN - 0008-4166
VL - 47
SP - 72
EP - 76
JO - Canadian Journal of Microbiology
JF - Canadian Journal of Microbiology
IS - 1
ER -