Analysis of gene control signals by DNA fusion and cloning in Escherichia coli

Transposition and fusion of the lac genes to selected promoters in Escherichia coli using bacteriophage lambda and Mu.

Escherichia coli is the organism of choice for the production of recombinant proteins. Its use as a cell factory is well-established and it has become the most popular expression platform. For this reason, there are many molecular tools and protocols at hand for the high-level production of recombinant proteins, such as a vast catalog of expression plasmids, a great number of engineered strains and many cultivation strategies. We review the different approaches for the synthesis of recombinant proteins in E. coli and discuss recent progress in this ever-growing field.

/pdf/recombinant-protein-expression-in-escherichia-coli-advances-13zp3322p0.pdf

Recombinant protein expression in Escherichia coli: advances and challenges.

The use of gel electrophoresis for quantitative studies of DNA-protein interactions is described. This rapid and simple technique involves separation of free DNA from DNA-protein complexes based on differences in their electrophoretic mobilities in polyacrylamide gels. Under favorable conditions both unbound DNA and DNA associated with protein can be quantified. This gel method is applied to the study of the E. coli lactose operon regulatory system. At ionic strengths in the physiological range, the catabolite activator protein (CAP) is shown to form a long-lived complex with the wild type lac promotor, but not with a CAP-insensitive mutant. Formation of a stable "open" or "melted-in" complex of RNA polymerase with the wild type promoter requires the participation of CAP and cyclic AMP. Further, it is demonstrated that even when pre-formed in the presence of CAP-cAMP, the polymerase-promoter open complex becomes unstable if CAP is then selectively removed.

A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions: application to components of the Escherichia coli lactose operon regulatory system

The “initiator” as a transcription control element

The maximum rate of expression of the lac operon is severely reduced in lac promoter mutants. Revertants of these mutations which produce higher levels of enzyme were isolated. Some of these revertants had lost sensitivity to catabolite repression and transient repression. The mutations responsible for these losses took place at sites very close to the original promoter mutations. From these results we conclude that the promoter itself is the target site for both catabolite and transient repression of the lac operon.

https://www.pnas.org/content/pnas/66/3/773.full.pdf

Catabolite-Insensitive Revertants of Lac Promoter Mutants

Terminal deoxynucleotidyl transferase is an enzyme which has the unique property of polymerizing polydeoxynucleotides onto a primer in the absence of a template (1,2). This enzyme is found both in the thymus and the bone marrow of birds, rodents, and humans (3-7). Whether the marrow cells that contain terminal transferase are related to thymocytes, or are on a separate pathway of differentiation, is not yet known (7,8). To determine the lineage of the murine bone marrow cells that have terminal transferase, we have investigated whether these cells have the antigen Thy-1 induced on the cells by treatment with thymopoietin (9). Thymopoietin is known to induce a set of characteristic T-cell markers including the Thy-1 alloantigen on the surface of a subpopulation of bone marrow cells committed to T-cell differentiation (prothymocytes) (10). Destruction of Thy- 1-positive cells after exposure to thymopoietin allows elimination of a substantial fraction of those bone marrow cells that can repopulate an irradiated thymus (11). We find that such an elimination after induction with the thymic polypeptide removes a substantial amount of terminal transferase from the bone marrow cell population, suggesting that at least one-half of the marrow cells bearing this enzyme are related to those found in the thymus.

https://rupress.org/jem/article-pdf/144/2/543/1087958/543.pdf

Terminal deoxynucleotidyl transferase is found in prothymocytes

A partial deletion in the lac p region makes the lac operon insensitive to catabolite repression.

Catabolite sensitive site of the lac operon.

A cDNA library was prepared from a terminal deoxynucleotidyltransferase-containing thymoma in the lambda phage vector lambda gt11. By screening plaques with anti-terminal transferase antibody, positive clones were identified of which some had beta-galactosidase-cDNA fusion proteins identifiable after electrophoretic fractionation by immunoblotting with anti-terminal transferase antibody. The predominant class of cross-hybridizing clones was determined to represent cDNA for terminal transferase by showing that one representative clone hybridized to a 2200-nucleotide mRNA in close-matched enzyme-positive but not to enzyme-negative cells and that the cDNA selected a mRNA that translated to give a protein of the size and antigenic characteristics of terminal transferase. Only a small amount of genomic DNA hybridized to the longest available clone, indicating that the sequence is virtually unique in the mouse genome.

/pdf/cloning-of-terminal-transferase-cdna-by-antibody-screening-58qcrrt5hc.pdf

Cloning of terminal transferase cDNA by antibody screening.

Different levels of β-galactosidase are found in various trp-lac fusion strains. These levels of β-galactosidase fall within a 60-fold range. The amount of thiogalactoside transacetylase activity detected in these same strains only varies 10-fold and is found in amounts greater than those predicted from the β-galactosidase levels. The observation that the β-galactosidase and thiogalactoside transacetylase levels are not directly proportional, that the lacZ messenger ribonucleic acid (mRNA) levels are not proportional to the β-galactosidase activity, that, at least for the one fusion strain tested, the SuA polarity suppressor does not affect the β-galactosidase level, and that, in all but one strain, the β-galactosidase activity appears to reside in normal β-galactosidase molecules suggests that the disproportionately low production of β-galactosidase is due to a decrease in the frequency of translation initiation of lacZ mRNA in these strains. Several mechanisms are proposed to explain this decrease. Some possible bases for the disproportional production of β-galactosidase and thiogalactoside transacetylase are also described. The preferred explanation for these disproportional enzyme levels is that only a fraction of the full complement of ribosomes need initiate translation at lacZ for the functional synthesis of lac mRNA to occur and that once the lac ribonucleic acid is made a full complement of ribosomes can bind at internal translation initiation sites at Y and A.

Allen E. Silverstone

Papers

Catabolite-Insensitive Revertants of Lac Promoter Mutants

Terminal deoxynucleotidyl transferase is found in prothymocytes

Catabolite sensitive site of the lac operon.

Cloning of terminal transferase cDNA by antibody screening.

Inhibition of lacZ Gene Translation Initiation in trp-lac Fusion Strains