Uses of lac fusions for the study of biological problems.

A new genetic selection identifies essential residues in SecG, a component of the Escherichia coli protein export machinery.

“…Both recessive conditional lethal sec mutations resulting in a generalized secretion defect and dominant prl mutations affecting protein localization have been isolated. Early selections of sec mutants were based on the properties of strains expressing a hybrid protein containing the signal sequence of an exported protein fused to P-galactosidase, a cytoplasmic enzyme (Silhavy and Beckwith, 1985). These strains are Lacdue to the insertion of at least part of the fusion protein in the membrane.…”

Behaviour of IncP-1 plasmids and a miniMu transposon in a marine Vibrio sp.: isolation of starvation inducible lac operon fusions

“…Fi-nally, it is possible to use the antibiotic marker on the inserted genetic element to clone the desired gene. Mu phage mutagenesis has successfully been used to obtain lac fusion mutants of E. coil and S. typhimurium and bacterial gene regulation studies have benefited greatly from analysis of such mutants ~7, [9][10][11]. Belas et al [12] previously reported the use of the PlclrlOOCM phage for mobilization of their newly constructed mini-Mu (Tet r) transposon into the genome of Fibrio parahaemolyticus.…”

Insertion of a 27 amino acid viral peptide in different zones of Escherichia coli β-galactosidase: Effects on the enzyme activity

“…The /3-galactosidase enzyme (EC 3 2 1 23) encoded by the Eschertchta coh lac Z gene, forms a tetramer of four non-covalently hnked subunlts, each one consisting of 1023 amino aods [1] The presence of a domam structure m the monomerlc enzyme has been mainly shown by mtraclstronlc complementatlon studies [2,3] fl-Galactosldase lS responsible for the conversion of lactose into glucose and galactose, allowing this sugar to be used as a carbon source [4] Because it also catalyzes the hydrolysis of other substrates that are converted tn colored compounds, this enzyme has been widely used to monitor a wide range of blologxcal processes such as regulation of gene expression [5] In addmon, it has been routinely employed as a tool to identify recombinant cell clones when using clonmg vectors hke lambda gtll, pUC or pEX series Recent studies have shown that /3-galactosldase can support small insertions without leading to complete enzymatic mactwatlon This observation has been of great interest m different fields of basic and applied research Breul et al [6] randomly introduced octamerlc ohgonucleotldes an the lacZ gene in order to corroborate the domain structure in the monomer, and Baum et al [7] inserted decapeptldes corresponding to HIV and polio protease cleavage sites to assay viral protease activities Because of the high potential of these techniques in both structural and functional studies, we have searched in this work for regions of fl-galactosldase, predicted to be exposed at the molecule surface, that could accept larger insertions maintaining at least some of the enzymatic actwity We have chosen the antigenic site A of foot-and-mouth disease virus (FMDV) as the insertion peptlde This region, located in the VP1 protein, is known to be an exposed, flexible and hypervarlable loop [8] able to accept a large number of mutations without affecting the structure of the neighboring capsld domains Using this peptlde, we have identified a tolerant region revolving amino acids 275 to 279, in which insertions maintain the enzymatic activity However, our results also indicate that the enzyme is not as permlsswe to sequence modifications as preliminary data had suggested [7]…”