A broad-host-range vector system for cloning and translational lacZ fusion analysis

A broad-host-range vector system for studying translational fusions was constructed. The region that retains the origin of replication, nic, mob, and rep genes of the broad-host-range plasmid RSF1010 was isolated as either an HincII or a PstI-PvuII restriction fragment. These restriction fragments were ligated to tetracycline, kanamycin, or streptomycin/spectinomycin resistance genes to generate plasmids pUI501, pUI511, pUI504, and pUI506. A functional lacZ gene lacking downstream lac operon sequences together with the lac promoter was constructed from plasmids pMC1871 and pUC18. This lacZ gene was inserted into pUI501 and pUI511 to generate plasmids pUI502, pUI503, pUI512, and pUI513. An oligodeoxynucleotide sequence that carries three unique blunt-end restriction sites was synthesized, annealed, and ligated in frame to the amino-terminal end of the lacZ gene in each of these plasmids. This multiple cloning sequence will allow translational fusions to the lacZ gene in all three reading frames. The stability of these plasmids and the expression of the lacZ gene in both Escherichia coli and Rhodobacter sphaeroides were studied.     

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.

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.     

Translational coupling varying in efficiency between different pairs of genes in the central region of the atp operon of Escherichia coli

A series of atp::lacZ fusions has been constructed for use in a study of translational coupling in the central region of the Escherichia coli atp operon. Five genes, atpE, atpF, atpH, atpA and atpG, were shown to be translationally coupled to various degrees of tightness. A new lac promoter vector, compatible with the atp::lacZ fusion vectors, was used to express individual atp genes in the same hosts as the fusion genes. The H(+)-ATPase subunits thus synthesized exercised no significant trans-regulation on the expression of the atp::lacZ fusions, indicating that the coupling is primarily cis. The mechanism of this coupling was investigated using in vitro mutagenesis. At least in the case of the pair atpHA, coupling seems to involve facilitated binding of fresh ribosomes to the atpA translational initiation regions.     

Clonal analysis of the lac operons from Klebsiella M5al and the Lac plasmid (pRE2) from Klebsiella V9A

The chromosomal lac region of the coliform bacterium Klebsiella M5al was cloned into the multicopy plasmid pBR322 to give pHE7 and pHE8. pHE8 contains 12.6 kb of M5al DNA, including its complete lac operon, and pHE7 contains 2.5 kb of M5al DNA and includes the complete lac Y gene and a small segment of lacZ. The M5al operon has the same gene order as the Escherichia coli lac operon. The lac genes of the Lac plasmid of Klebsiella V9A were cloned into pBR322 to give pHE1 and pHE2, of approximately 39 and 43 kb. Both plasmids were unstable in an E. coli RecA-strain, in contrast to the stability of pHE8. Polyacrylamide gel electrophoresis tests suggested that the M5al beta-galactosidase monomer is about 5% longer, i.e. has about 50 more amino acids, than that of the E. coli Z gene. Tests made on the enzymes coded by the lac operons of M5al, another Klebsiella strain (V9A) and its resident Lac plasmid, and several Lac+ Enterobacteria, led to the conclusion that only Escherichia coli among the Enterobacteria contains an active lacA gene.