In vitro gene fusions that join an enzymatically active beta-galactosidase segment to amino-terminal fragments of exogenous proteins: Escherichia coli plasmid vectors for the detection and cloning of translational initiation signals

Abstract

We report the construction and use of a series of plasmid vectors suitable for the detection and cloning of translational control signals and 5' coding sequences of exogenously derived genes. In these plasmids, the first eight codons of the amino-terminal end of the lactose operon beta-galactosidase gene, lacZ, were removed, and unique BamHI, EcoRI, and SmaI (XmaI) endonuclease cleavage sites were incorporated adjacent to the eighth codon of lacZ. Introduction of deoxyribonucleic acid fragments containing appropriate regulatory signals and 5' coding sequences into such lac fusion plasmids led to the production of hybrid proteins consisting of the carboxyl-terminal segment of a beta-galactosidase remnant plus a peptide fragment that contained the amino-terminal amino acids encoded by the exogenous deoxyribonucleic acid sequence. These hybrid peptides retained beta-galactosidase enzymatic activity and yielded a Lac+ phenotype. Such hybrid proteins are useful for purifying peptide sequences encoded by exogenous deoxyribonucleic acid fragments and for studies relating the structure and function of specific peptide segments.

Ice nucleation activity of Pseudomonas fluorescens: mutagenesis, complementation analysis and identification of a gene product.

“…Allele number 315 was derived from allele 115, as follows. A 7.1-kb lacZYcontaining fragment was excised from plasmid pMC871 (Casadaban et al, 1980) by digestion with BamHI+Sall. The plasmid containing allele 115 was partially digested by Sail and ligated together with the fragment from pMC871.…”

Photosynthesis-independent regulation of bacteriochlorophyll synthesis by light intensity in Rhodobacter capsulatus

“…Two types of fusions were evaluated. Plasmid pA-ZIV contains the entire 1ucZ gene and translational control sequences preceded by a small segment of the E. coli trp operon (the luc 903 fragment [15]), which in turn is preceded by the pufB' gene truncated at codon 25, and the put operon major promoter region [7,10,12]. Thus, the level of P-galactosidase expressed from pAZIV should reflect put promoter activity, but would not indicate possible translational control of put gene expression.…”

“…Thus, the level of P-galactosidase expressed from pAZIV should reflect put promoter activity, but would not indicate possible translational control of put gene expression. Plasmid pAZV contains the same put operon region, but with pucB' fused (at codon 25) translationally in-frame to the 8th codon of the 1ac'Z 931 fragment [7,10,15]. Therefore, the amount of P-galactosidase activity obtained with pAZV should reflect possible regulation of translation initiation of the pucB message as well as the activity of the put promoter.…”

Analysis of the regulatory region of the lysC gene of Escherichia coli

“…Plasmid pTK275 (Ap r , lacPZYA) was used for the in vitro construction of transcriptional fusions to lacPZ. It is derived from pMC1403 [8] and carries a Shine Dalgarno (SD) sequence and the translation start codon in front of the 8th amino acid codon of lacPZ. Plasmid pUC18 was used for DNA cloning and construction.…”

“…Minimal medium M63 [9] supplemented with 1% (w/v) glucose and 0.002% (w/v) L-leucine was used for E. coli MC1061 [8]. Ampicillin (100 mg l 3I ) and 5-bromo-4-chloro-3-indolyl-L-galatoside (X-gal) were added as required.…”