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PubMed2001

Expression plasmid with a very tight two-step control: Int/att-mediated gene inversion with respect to the stationary promoter.

A very tightly controlled expression vector was constructed, which was originally designed as to be able to use any promoter, constitutive or regulated. Moreover, in vector pNH46T1, the repressible P(tac)/P(lac) promoters were used to transcribe genes cloned in the proximal multiple cloning site (MCS), which was flanked by convergent attB and attP sites. The gene of interest was cloned into MCS in the OFF orientation, i.e. facing the promoter(s). In such OFF orientation, the cloned gene could not be expressed, and only its anti-sense mRNA could be produced. Four strong rrnBT1 terminators, in a tandem arrangement and proximal to the N-terminal end of the cloned non-inverted gene, were protecting it from any inadvertent transcription originating in the vector. Moreover, the P(tac)/P(lac) promoters/operators are controlled by the LacI(q)ts and LacI(+) repressor(s) that further reduce the basal gene expression in the uninduced state. When induced, the total vector population is converted to the ON orientation by expression of the Int function that inverts the attB and attP-flanked MCS including the cloned gene. This places the gene under direct control of the P(tac)/P(lac) promoters, and thus results in very high expression. An additional feature is the anti-termination system that consists of the promoter-proximal nutL site and the inducible gene N, whose role in the ON state is to overcome the rrnBT1 terminators and any other adventitiously cloned terminators.

Attachment Sites, Microbiological↗
PubMed2002

The dawn of gene isolation.

Ever since it became clear through the work of Watson and Crick that the gene is a stretch of double stranded helical DNA and is understandable in chemical terms, biochemists have striven to get their hands on isolated genes. The isolation of the ribosomal genes of Xenopus laevis in 1966 provided a first instance where a purified DNA of known function could be investigated, long before the advent of gene cloning technologies. The second instance was the purification of the Lac operon from Escherichia coli. Later, but still before the gene cloning days the 5S RNA genes of X. laevis and the histone genes of the sea urchin Psammechinus miliaris were isolated by physico-chemical methods, but their isolation marked the end of an era. By 1975, gene cloning technology was well established and the isolation of genes quickly became an everyday occurrence.

Animals↗
PubMed1997

Inducible high-level expression vector for mammalian cells, pEF-LAC carrying human elongation factor 1alpha promoter and lac operator.

We have constructed an inducible high-level expression vector, pEF-LAC. pEF-LAC has a modified human polypeptide chain elongation factor 1alpha (EF-1alpha) promoter containing three lactose operator sequences. Using the cat reporter gene, we characterized the transcriptional activity of pEF-LAC. In the transient transfection of NIH3T3 and BaF3 cells, the transcriptional activity of pEF-LAC was higher than that of the original human elongation factor 1alpha promoter, simian virus 40 (SV40) promoter, and Rous sarcoma virus (RSV) long terminal repeat (LTR). Cotransfection of the lactose repressor expression plasmid effectively suppressed the promoter activity of pEF-LAC, and the activity was fully recovered by addition of isopropyl beta-D-thiogalactopyranoside (IPTG). Even in the stable transfection of Rat-1 cells, the promoter activity of the integrated pEF-LAC was much higher than that of the RSV-LTR and regulated in an IPTG-dependent manner. These results suggest that pEF-LAC is a useful vector for the inducible high-level expression of the cloned gene in a variety of mammalian cells.

3T3 Cells↗
PubMed1998

Cold-sensitive E-lysis systems.

The release of recombinant bacteria into the environment is undesirable because of possible risks associated with the genetically modified organisms. The aim of this study was to establish a cold-sensitive killing system with a lethal gene, activated when bacteria encounter lower environmental temperatures. To obtain cold-sensitive lysis vectors, the lambdacI857 repressor/pR promoter expression system was combined with either the lacI/lacZpo or the phage 434 cI/pR system that control the expression of the lysis gene E of bacteriophage phiX174. Escherichia coli strains harbouring such suicide vectors are able to grow at 37 degrees C, but cell lysis takes place at temperatures below 30 degrees C. By replacing gene E with a beta-galactosidase reporter gene we also showed that the onset of beta-galactosidase activity corresponds with the onset of lysis at 28 degrees C. Results indicate that these newly combined promoter/repressor systems can also be used to confer cold-sensitive expression to any gene of interest.

Bacterial Proteins↗
PubMed2000

Enhanced Tn10 and mini-Tn10 precise excision in DNA replication mutants of Escherichia coli K12.

The precise excision of transposon Tn10 and a mini-Tn10 derivative, inserted in the gal or lac operons, was studied in dnaB252 and dnaE486 temperature-sensitive mutants of Escherichia coli. dnaB codes for a DNA replication helicase and dnaE for the alpha subunit of DNA polymerase III. Mutations in these genes were found to enhance, at the permissive temperature, the precise excision of both genetic elements. The increase factor was much more pronounced for the dnaB252 mutant with the transposons inserted in gal. The stimulated excision was only partially affected by a recA null mutation but was significantly reduced by introduction of recF null or ruvA mutations. A model involving template switching of the polymerase between the direct repeats flanking the transposons, on the same strand or between sister strands, could account for the observed results.

Bacterial Proteins↗
PubMed1997

Influence of nucleotide excision repair of Escherichia coli on radiation-induced mutagenesis of double-stranded M13 DNA.

To investigate a possible role of nucleotide excision repair (NER) of E. coli in the removal of gamma-radiation-induced DNA lesions, double-stranded M13mp10 DNA, which contains a part of the lac operon, including the promoter/operator region, the lacZ alpha gene and a 144 basepair (bp) inframe insert in the lacZ alpha gene, as mutational target was gamma-irradiated in a phosphate buffer under N2. Subsequently, the radiation-exposed DNA was transfected to wild-type or NER-deficient (uvrA-) E. coli, mutants in the mutational target selected, followed by characterization of the mutants by sequence analysis. Both the mutations obtained from wild-type and uvrA- E. coli appeared to consist mainly of bp substitutions. However, in contrast to wild-type cells, a relatively large proportion of the mutations obtained from the NER-deficient cells (about 25%) is represented by -1 bp deletions, indicating that NER may be responsible for the removal of lesions which cause this particular type of frameshift. Comparison of the bp substitutions between both E. coli strains showed considerable differences. Thirty per cent of all bp substitutions in the NER-deficient host are T/A-->C/G transitions which are virtually absent in wild-type E. coli. This indicates that NER is involved in the elimination of lesions responsible for these transitions. This may also be true for a part of the lesions which cause C/G-->T/A transitions, which make up 52% of the bp substitutions in uvrA- cells versus 17% in wild-type cells. Strikingly, C/G-->G/C transversions appeared to be only formed in wild-type, where they make up 22% of all bp substitutions, and not in the NER-deficient E. coli. This result suggests, that due to the action of NER, a particular type of mutation may be introduced. A similar indication holds for C/G-->A/T transversions, which are predominant in wild-type (58%) and in the minority in uvrA- cells (15%).

Base Sequence↗
PubMed2001

Vectors to express foreign genes and techniques to monitor gene expression in Pseudomonads.

Improved tools for Pseudomonas research include small, broad-host-range vectors that allow regulated expression from the lac operon and T7 promoters whose biology is well understood and adaptable to many bacteria. To facilitate studies on gene regulation, tracking and monitoring of bacteria in diverse environments, and the construction of biosensors, various reporter genes with versatile assay formats have been developed that can be delivered on plasmid, transposon and integration-proficient vectors.

Bacterial Proteins↗
PubMed1996

Lac repressor at last.

Crystal and cocrystal structures of the LacI and PurR repressors reveal a novel use of hinge alpha helices which bind in the minor groove of the operator and mediate transmission of the allosteric signals that modulate DNA-binding activity.

Bacterial Proteins↗
PubMed1988

Expression of active rat DNA polymerase beta in Escherichia coli.

A recombinant plasmid for expression of rat DNA polymerase beta was constructed in a plasmid/phage chimeric vector, pUC118, by an oligonucleotide-directed mutagenesis technique. The insert contained a 1005 bp coding sequence for the whole rat DNA polymerase beta. The recombinant plasmid was designed to use the regulatory sequence of Escherichia coli lac operon and the initiation ATG codon for beta-galactosidase as those for DNA polymerase beta. The recombinant clone, JMp beta 5, obtained by transfection of E. coli JM109 with the plasmid, produced high levels of DNA polymerase activity and a 40-kDa polypeptide that were not detected in JM109 cell extract. Inducing this recombinant E. coli with isopropyl beta-thiogalactopyranoside (IPTG) yielded amounts of 40-kDa polypeptide as high as 19.3% of total protein. Another recombinant clone, JMp beta 2-1, which was constructed by an oligonucleotide-directed mutagenesis to use the second ATG codon for the initiation codon, thus deleting the first 17 amino acid residues from the amino terminus, produced neither high DNA polymerase activity nor the 40-kDa polypeptide. The evidence suggests that this amino-terminal structure is important for stability of this enzyme in E. coli. The DNA polymerase was purified to homogeneity from the IPTG-induced JMp beta 5 cells by fewer steps than the procedure for purification of DNA polymerase beta from animal cells. The properties of this enzyme in activity, chromatographic behavior, size, antigenicity, and also lack of associated nuclease activity were indistinguishable from those of DNA polymerase beta purified from rat cells, indicating the identity of the overproduced DNA polymerase in the JMp beta 5 and the rat DNA polymerase beta.

Amino Acid Sequence↗
PubMed1980

Production of biologically active N alpha-desacetylthymosin alpha 1 in Escherichia coli through expression of a chemically synthesized gene.

Thymosin alpha 1, an immune restorative polypeptide hormone, was synthesized in Escherichia coli by using recombinant DNA cloning techniques. Based on the known amino acid sequence, a gene coding for the thymosin alpha 1 polypeptide chain was designed and enzymatically assembled from chemically synthesized oligodeoxyribonucleotide fragments. The gene was ligated into plasmid pBR322 and placed under lac operon control, and N alpha-desacetylthymosin alpha 1 was expressed as part of a beta-galactosidase chimeric protein. Cyanogen bromide cleavage of this protein gave a mixture of polypeptides, among which thymosin alpha 1 activity was detected by radioimmunoassay (RIA). The E. coli product is identical with native thymosin alpha 1 isolated from calf thymus in the amino acid sequence but lacks the N-terminal acetyl group. Results of a guinea pig migration inhibition factor (MIF) assay, a terminal deoxyribonucleotidyl transferase (TdT) assay, and radioimmunoassay indicate that the N alpha-desacetylthymosin alpha 1 produced by deoxyribonucleic acid (DNA) cloning techniques has biological activity equivalent to that of the native hormone.

Amino Acid Sequence↗
PubMed1979

Lactose carrier protein of Escherichia coli. Transport and binding of 2'-(N-dansyl)aminoethyl beta-D-thiogalactopyranoside and p-nitrophenyl alpha-d-galactopyranoside.

The elevated level of lactose carrier protein present in cytoplasmic membranes derived from Escherichia coli strain T31RT, which carries the Y gene of the lac operon on a plasmid vector (Teather, R. M., et al. (1978) Mol. Gen. Genet. 159, 239--248), has allowed the detection of a complex between the carrier and the fluorescent substrate 2'-(N-dansyl)-aminoethyl beta-D-thiogalactopyranoside (Dns2-S-Gal). Binding is accompanied by a 50-nm blue shift in the emission maximum of the dansyl residue. The complex (dissociation constant, KD = 30 micron) rapidly dissociates upon addition of competing substrates such as beta-D-galactopyranosyl 1-thio-beta-D-galactopyranoside or upon reaction with the thiol reagent p-chloromercuribenzenesulfonate. Binding of both Dns2-S-Gal and p-nitrophenyl alpha-D-galactopyranoside (alpha-NPG) occurs spontaneously in the absence of an electrochemical potential gradient across the membrane. Comparison of equilibrium binding experiments using Dns2-S-Gal or alpha-NPG and differential labeling of the carrier with radioactive amino acids shows that the carrier binds 1 mol of substrate per mol of polypeptide (molecular weight 30 000). In addition to specific binding to the lactose carrier, Dns2-S-gal binds unspecifically to lipid vesicles or membranes, as described by a partition coefficient, K = 60, resulting in a 25-nm blue shift in the emission maximum of the dansyl group. Both Dns2-S-Gal and alpha-NPG are not only bound by the lactose carrier but also transported across the membrane by this transport protein in cells and membrane vesicles. The fluorescence changes observed with dansylated galactosides in membrane vesicles in the presence of an electrochemical gradient (Schuldiner et al. (1975) J. Biol. Chem. 250, 1361--1370)) are interpreted as an increase in unspecific binding after translocation.

Biological Transport, Active↗
PubMed1977

Interpretation of monovalent and divalent cation effects on the lac repressor-operator interaction.

We have investigated the effects of mixed Na+: Mg2+ ionic solutions on the stability of the nonspecific lac repressor-DNA complex. The effects of Mg2+ are simply interpreted in terms of its role as a competitor (with repressor) for DNA sites. From these studies, the binding constant of the Mg-DNA complex can be determined as a function of the concentration of Na+. We have used this information to interpret the data of Riggs and collaborators (Riggs, A.D., et al. (1970), J. Mol. Biol. 48, 67-83; 53, 401-417) on the ion dependence of the repressor-operator interaction. We find that there are approximately 70% as many ionic interactions in the repressor-operator complex as in the nonspecific complex. Our best estimate is that 8 +/- 1 ion pairs are formed. We calculate that the release of counterions in the formation of the specific complex contributes approximately 40% of the favorable free energy change in the association reaction under in vivo ionic conditions. Implications of these findings for the control of the lac operon and for the molecular relationship between the specific and nonspecific complexes are considered.

Bacterial Proteins↗
PubMed2003

Structural basis for the altered activity of Gly794 variants of Escherichia coli beta-galactosidase.

The open-closed conformational switch in the active site of Escherichia coli beta-galactosidase was studied by X-ray crystallography and enzyme kinetics. Replacement of Gly794 by alanine causes the apoenzyme to adopt the closed rather than the open conformation. Binding of the competitive inhibitor isopropyl thio-beta-D-galactoside (IPTG) requires the mutant enzyme to adopt its less favored open conformation, weakening affinity relative to wild type. In contrast, transition-state inhibitors bind to the enzyme in the closed conformation, which is favored for the mutant, and display increased affinity relative to wild type. Changes in affinity suggest that the free energy difference between the closed and open forms is 1-2 kcal/mol. By favoring the closed conformation, the substitution moves the resting state of the enzyme along the reaction coordinate relative to the native enzyme and destabilizes the ground state relative to the first transition state. The result is that the rate constant for galactosylation is increased but degalactosylation is slower. The covalent intermediate may be better stabilized than the second transition state. The substitution also results in better binding of glucose to both the free and the galactosylated enzyme. However, transgalactosylation with glucose to produce allolactose (the inducer of the lac operon) is slower with the mutant than with the native enzyme. This suggests either that the glucose is misaligned for the reaction or that the galactosylated enzyme with glucose bound is stabilized relative to the transition state for transgalactosylation.

Amino Acid Substitution↗
PubMed2004

Using networks to identify fine structural differences between functionally distinct protein states.

The vast increase in available data from the "-omics" revolution has enabled the fields of structural proteomics and structure prediction to make great progress in assigning realistic three-dimensional structures to each protein molecule. The challenge now lies in determining the fine structural details that endow unique functions to sequences that assume a common fold. Similar problems are encountered in understanding how distinct conformations contribute to different phases of a single protein's dynamic function. However, efforts are hampered by the complexity of these large, three-dimensional molecules. To overcome this limitation, structural data have been recast as two-dimensional networks. This analysis greatly reduces visual complexity but retains information about individual residues. Such diagrams are very useful for comparing multiple structures, including (1) homologous proteins, (2) time points throughout a dynamics simulation, and (3) functionally different conformations of a given protein. Enhanced structural examination results in new functional hypotheses to test experimentally. Here, network representations were key to discerning a difference between unliganded and inducer-bound lactose repressor protein (LacI), which were previously presumed to be identical structures. Further, the interface of unliganded LacI was surprisingly similar to that of the K84L variant and various structures generated by molecular dynamics simulations. Apo-LacI appears to be poised to adopt the conformation of either the DNA- or inducer-bound structures, and the K84L mutation appears to freeze the structure partway through the conformational transition. Additional examination of the effector binding pocket results in specific hypotheses about how inducer, anti-inducer, and neutral sugars exert their effects on repressor function.

Bacterial Proteins↗
PubMed2005

Integrated insights from simulation, experiment, and mutational analysis yield new details of LacI function.

Protein structural change underlies many signal transduction processes. Although end-state structures are known for various allosteric proteins, intermediates are difficult to observe. Recently, targeted molecular dynamics simulation (TMD) was used to examine the conformational transition and predict relevant intermediates for wild-type lactose repressor (LacI). A catalog of involved residues suggests that the transition of this homodimer is asymmetric and that K84 is a prominent participant in the dynamic N-subdomain interface. Previous experiments indicated that hydrophobic substitutions at position 84 engender slowed, biphasic inducer binding kinetics, which might reflect the same phenomena observed in TMD. Here, we report biochemical confirmation that DNA and inducer binding remain allosterically linked in K84A and K84L, albeit with a differential smaller than that found in wild-type LacI. Other features of these mutant proteins are consistent with an allosteric conformational shift that approximates that of the wild type. As a consequence, these repressors can be utilized to explore an unanswered question about LacI function: How many inducers (one or two per dimer) are required to diminish operator affinity? The biphasic natures of the K84L and K84A inducer association rates allow direct correlation between the two distinct inducer binding events and operator release. Indeed, the kinetics of operator release for the K84A and K84L closely parallel those for the second inducer binding event. Together with implications from previous equilibrium results for wild-type and mutant proteins, these kinetic data demonstrate that binding of two inducers per dimeric DNA binding unit is required to release the operator in these variant LacI proteins.

Amino Acid Substitution↗
PubMed2005

Use of urea and glycine betaine to quantify coupled folding and probe the burial of DNA phosphates in lac repressor-lac operator binding.

Thermodynamic analysis of urea-biopolymer interactions and effects of urea on folding of proteins and alpha-helical peptides shows that urea interacts primarily with polar amide surface. Urea is therefore predicted to be a quantitative probe of coupled folding, remodeling, and other large-scale changes in the amount of water-accessible polar amide surface in protein processes. A parallel analysis indicates that glycine betaine [N,N,N-trimethylglycine (GB)] can be used to detect burial or exposure of anionic (carboxylate, phosphate) biopolymer surface. To test these predictions, we have investigated the effects of these solutes (0-3 m) on the formation of 1:1 complexes between lac repressor (LacI) and its symmetric operator site (SymL) at a constant KCl molality. Urea reduces the binding constant K(TO) [initial slope dlnK(TO)/dm(urea) = -1.7 +/- 0.2], and GB increases K(TO) [initial slope dlnK(TO)/dm(GB) = 2.1 +/- 0.2]. For both solutes, this derivative decreases with an increase in solute concentration. Analysis of these initial slopes predicts that (1.5 +/- 0.3) x 10(3) A2 of polar amide surface and (4.5 +/- 1.0) x 10(2) A2 of anionic surface are buried in the association process. Analysis of published structural data, together with modeling of unfolded regions of free LacI as extended chains, indicates that 1.5 x 10(3) A2 of polar amide surface and 6.3 x 10(2) A2 of anionic surface are buried in complexation. Quantitative agreement between structural and thermodynamic results is obtained for amide surface (urea); for anionic surface (GB), the experimental value is approximately 70% of the structural value. For LacI-SymL binding, two-thirds of the structurally predicted change in amide surface (1.0 x 10(3) A2) occurs outside the protein-DNA interface in protein-protein interfaces formed by folding of the hinge helices and interactions of the DNA binding domain (DBD) with the core of the repressor. Since urea interacts principally with amide surface, it is particularly well-suited to detect and quantify the extent of coupled folding and other large-scale remodeling events in the steps of protein-nucleic acid interactions and other protein associations.

Algorithms↗
PubMed2006

Extracting biochemical parameters for cellular modeling: A mean-field approach.

Recent developments in molecular biology have made it feasible to carry out experimental verification of mathematical models for biochemical processes, offering the eventual prospect of creating a detailed, validated picture of gene expression. A persistent difficulty with this long-term goal is the incompleteness of the kinetic information available in the literature: Many rate constants cannot or have not yet been measured. Here, we present a method of filling in missing parameters using an approach conceptually analogous to mean-field approaches in statistical mechanics: When studying a particular gene, we extract key parameters by considering the averaged effect of all other genes in the system, analogously to considering the averaged magnetic field in a physical spin model. This methodology has been applied to account for the effect of the presence of the Escherichia coli genome on the availability of key enzymes involved in gene expression (RNA polymerases and ribosomes), yielding the number of free enzymes as a function of cellular growth rate. These conclusions have been obtained by deriving genome-wide averages and matching them to bulk literature values of E. coli K-12 and B/r. Average rate constants have been found for RNA polymerases and ribosomes binding to promoter and ribosome-binding sites, respectively; these results suggest that cells vary not only their production rates of RNA polymerase and ribosomes under different growth-rate conditions but also change their global level of transcriptional/translational activation and repression, thus altering the average binding rate constants for these enzymes. To test the mean-field method, the results from the genome-wide averages have been applied to the induced lac operon, where our derived on-rate for binding of RNA polymerase to the promoter is in good agreement with previous experimental results.

Computer Simulation↗
PubMed1981

cis-Interacting genes in the S region of the murine major histocompatibility complex.

Insight into the control of gene expression may be gained by analysing genetic systems marked by both regulatory and structural variants. In such systems one can determine whether a regulatory element controls structural genes on both chromosomes or only on the chromosome to which it is linked. The latter may be detected in individuals heterozygous at both the regulatory and structural loci, in which case the effect of each regulatory allele is seen to be exerted only on the cis-located structural allele. In prokaryotic organisms, the identification of cis interaction of this sort has allowed elucidation of many features of genetic regulation, first for the lac operon and subsequently for a variety of other systems. In higher organisms, however, there have been few opportunities to observe cis-interacting genes. The most thoroughly characterized mammalian system in this regard is the murine beta-glucuronidase locus described by Paigen and his colleagues, in which cis interaction has been shown to occur between two closely linked genetic elements-the beta-glucuronidase structural gene itself and an androgen-activated regulatory gene which controls the quantity of beta-glucuronidase expressed. We report here that cis-interacting genetic elements are also found in the S region of the mouse major histocompatibility complex H-2.

Animals↗