Lac Repressor Research Articles

Nuclear domain 10-associated proteins recognize and segregate intranuclear DNA/protein complexes to negate gene expression

BackgroundDNA viruses, such as herpes simplex virus type 1 (HSV-1), Simian virus 40 (SV40), and Cytomegaloviruses (CMV), start their replicative processes and transcription at specific nuclear domains known as ND10 (nuclear domain 10, also called PML bodies). It has been previously determined that for HSV-1 and SV40, a short DNA sequence and its binding protein are required and sufficient for cell localization of viral DNA replication and gene transcription.ResultsOur recent observations provide evidence that a foreign (not endogenous) DNA/protein complex in the nucleus recruits ND10 proteins. First, the complexes formed from the bacterial lac operator DNA and its binding protein (lac repressor), or from HPV11 (human papillomavirus 11) origin DNA and its binding protein (E2), co-localized with different ND10 proteins. Second, the HSV-1 amplicon without inserted lac operator DNA repeats distributed in the nucleus randomly, whereas the amplicon with lac operator DNA repeats associated with ND10, suggesting that DNA-binding proteins are required to localize at ND10. The cellular intrinsic DNA/protein complex (as detected for U2 DNA) showed no association with ND10. Furthermore, our examination of PML−/−, Daxx−/−, and Sp100-negative cells led to our discovering that DNA/protein complexes recruit ND10 protein independently. Using the GFP-LacI/Operator system, we were able to direct the transfected DNA to ND10 and found that gene expression was significantly repressed when the transfected DNA was directed to ND10.ConclusionTaken together, the results suggest that cells recognize DNA/protein complexes through a mechanism that involves interaction with the ND10-associated proteins.

Lactose Repressor Experimental Folding Landscape: Fundamental Functional Unit and Tetramer Folding Mechanisms

The fundamental principles that govern monomer folding are believed to be congruent with those of protein oligomers. However, the effects of protein assembly during the folding reaction can result in a series of complex transitions that are considerably more challenging to deconvolute. Here we developed the experimental protein folding mechanism for the lactose repressor (LacI), for both the dimeric and the tetrameric states, using equilibrium unfolding and kinetic experiments, and by leveraging the previously reported monomer folding landscape. Reaction details for LacI oligomers were observed by way of circular dichroism, intrinsic fluorescence, and Förster resonance energy transfer (FRET) and as a function of protein concentration. In general, the dimer and tetramer are four-phase folding reactions in which the first three transitions are tantamount to the folding of constituent monomers. The final reaction phase of the LacI dimer can be attributed to protein assembly, based on the concentration dependence of the observed folding rates and intermolecular FRET measurements. Unlike the dimer, the latter reaction phase of the LacI tetramer is not dependent on protein concentration, likely because of a strong tethering of the monomers, which simplifies the folding reaction by eliminating an explicit protein assembly phase. Finally, folding of the LacI dimer and tetramer was assessed in the presence of polyethylene glycol to rule out inert molecular crowding as the driving force for the protein folding reaction; in addition, these data provide insight into the folding mechanism in vivo.

Protein-induced changes in DNA structure and dynamics observed with noncovalent site-directed spin labeling and PELDOR

Site-directed spin labeling and pulsed electron–electron double resonance (PELDOR or DEER) have previously been applied successfully to study the structure and dynamics of nucleic acids. Spin labeling nucleic acids at specific sites requires the covalent attachment of spin labels, which involves rather complicated and laborious chemical synthesis. Here, we use a noncovalent label strategy that bypasses the covalent labeling chemistry and show that the binding specificity and efficiency are large enough to enable PELDOR or DEER measurements in DNA duplexes and a DNA duplex bound to the Lac repressor protein. In addition, the rigidity of the label not only allows resolution of the structure and dynamics of oligonucleotides but also the determination of label orientation and protein-induced conformational changes. The results prove that this labeling strategy in combination with PELDOR has a great potential for studying both structure and dynamics of oligonucleotides and their complexes with various ligands.

Inducible genetic system for the axolotl

Transgenesis promises a powerful means for assessing gene function during amphibian limb regeneration. This approach is complicated, however, by the need for embryonic appendage development to proceed unimpeded despite the genetic alterations one wishes to test later in the context of regeneration. Achieving conditional gene regulation in this amphibian has not proved to be as straightforward as in many other systems. In this report we describe a unique method for obtaining temporal control over exogenous gene expression in the axolotl. Based on technology derived from the Escherichia coli Lac operon, uninduced transgenes are kept in a repressed state by the binding of constitutively expressed Lac repressor protein (LacI) to operator sequences within the expression construct. Addition of a lactose analog, IPTG, to the swimming water of the axolotl is sufficient for the sugar to be taken up by cells, where it binds the LacI protein, thereby inducing expression of the repressed gene. We use this system to demonstrate an in vivo role for thrombospondin-4 in limb regeneration. This inducible system will allow for systematic analysis of phenotypes at defined developmental or regenerative time points. The tight regulation and robustness of gene induction combined with the simplicity of this strategy will prove invaluable for studying many aspects of axolotl biology.

Regulatory consequences of gene translocation in bacteria

Gene translocations play an important role in the plasticity and evolution of bacterial genomes. In this study, we investigated the impact on gene regulation of three genome organizational features that can be altered by translocations: (i) chromosome position; (ii) gene orientation; and (iii) the distance between a target gene and its transcription factor gene (‘target-TF distance’). Specifically, we quantified the effect of these features on constitutive expression, transcription factor binding and/or gene expression noise using a synthetic network in Escherichia coli composed of a transcription factor (LacI repressor) and its target gene (yfp). Here we show that gene regulation is generally robust to changes in chromosome position, gene orientation and target-TF distance. The only demonstrable effect was that chromosome position alters constitutive expression, due to changes in gene copy number and local sequence effects, and that this determines maximum and minimum expression levels. The results were incorporated into a mathematical model which was used to quantitatively predict the responses of a simple gene network to gene translocations; the predictions were confirmed experimentally. In summary, gene translocation can modulate constitutive gene expression levels due to changes in chromosome position but it has minimal impact on other facets of gene regulation.

Transcription factors slide to find binding sites in vivo

In vitro studies have previously shown that transcription factors find their binding sites through a mechanism of 'facilitated' diffusion that combines three-dimensional diffusion with sliding along DNA. Using a single-molecule approach, Hammar et al. have now shown that this mechanism also occurs in living bacterial cells. They used a fluoroescently labelled lac repressor protein (LacI) and monitored its binding to the lac operator. They show that LacI slides on average 45 ± 10 bp, and in most cases it slides over the operator sequence at least once before binding to it. During evolution, transcription factors may need to maintain the ability to slide over non-specific sequences as well as to bind specifically.

Exploring the sequence–function relationship in transcriptional regulation by the lac O1 operator

Understanding how binding of a transcription factor to an operator is influenced by the operator sequence is an ongoing quest. It facilitates discovery of alternative binding sites as well as tuning of transcriptional regulation. We investigated the behavior of the Escherichia coli Lac repressor (LacI) protein with a large set of lac O(1) operator variants. The 114 variants examined contained a mean of 2.9 (range 0-4) mutations at positions -4, -2, +2 and +4 in the minimally required 17 bp operator. The relative affinity of LacI for the operators was examined by quantifying expression of a GFP reporter gene and Rosetta structural modeling. The combinations of mutations in the operator sequence created a wide range of regulatory behaviors. We observed variations in the GFP fluorescent signal among the operator variants of more than an order of magnitude under both uninduced and induced conditions. We found that a single nucleotide change may result in changes of up to six- and 12-fold in uninduced and induced GFP signals, respectively. Among the four positions mutated, we found that nucleotide G at position -4 is strongly correlated with strong repression. By Rosetta modeling, we found a significant correlation between the calculated binding energy and the experimentally observed transcriptional repression strength for many operators. However, exceptions were also observed, underscoring the necessity for further improvement in biophysical models of protein-DNA interactions.

Constructing protein nano‐fiber and estimation of the electronic state around metal ions

AbstractWe aim to construct a nano‐fiber using proteins by mixing two kinds of proteins. The binding sites are expected to be formed between two α‐helices of one protein and two α‐helices of another protein. As model proteins, we use Lac repressor four helix protein (LARFH), sulerythrin, and 3‐isopropylmalate dehydrogenase. With these proteins, we performed molecular dynamics (MD) simulations with a coarse‐grained (CG) model. In MD simulations with the CG model for LARFH, we found that the molecules approached faster in the system where the mutant protein with positively charged amino acids was placed with the mutant protein with negatively charged amino acids than in the system containing two wild‐type protein molecules. In the system with the variants, the number of contact interfaces with the positive variant‐negative variant combination after 150 ns simulation is larger than that with the positive–positive variants combination or the negative–negative variants combination. Sulerythrin has two pairs of Fe2+ and Zn2+. Electronic structure calculation was performed around metal ions in sulerythrin. By electronic structure calculation for sulerythrin around metal ions, the electric charge and spin density were estimated. © 2012 Wiley Periodicals, Inc.

Alanine Screening Mutagenesis Establishes the Critical Inactivating Damage of IrradiatedE. coliLactose Repressor

The function of the E. coli lactose operon requires the binding of lactose repressor to operator DNA. We have previously shown that γ rradiation destabilizes the repressor-operator complex because the repressor loses its DNA-binding ability. It was suggested that the observed oxidation of the four tyrosines (Y7, Y12, Y17, Y47) and the concomitant structural changes of the irradiated DNA-binding domains (headpieces) could be responsible for the inactivation. To pinpoint the tyrosine whose oxidation has the strongest effect, four headpieces containing the product of tyrosine oxidation, 3,4-dihydroxyphenylalanine (DOPA), were simulated by molecular dynamics. We have observed that replacing Y47 by DOPA triggers the largest change of structure and stability of the headpiece and have concluded that Y47 oxidation is the greatest contributor to the decrease of repressor binding to DNA. To experimentally verify this conclusion, we applied the alanine screening mutagenesis approach. Tetrameric mutated repressors bearing an alanine instead of each one of the tyrosines were prepared and their binding to operator DNA was checked. Their binding ability is quite similar to that of the wild-type repressor, except for the Y47A mutant whose binding is strongly reduced. Circular dichroism determinations revealed small reductions of the proportion of α helices and of the melting temperature for Y7A, Y12A and Y17A headpieces, but much larger ones were revealed for Y47A headpiece. These results established the critical role of Y47 oxidation in modifying the structure and stability of the headpiece, and in reduction of the binding ability of the whole lactose repressor.

Generalized Facilitated Diffusion Model for DNA-Binding Proteins with Search and Recognition States

Transcription factors (TFs) such as the lac repressor find their target sequence on DNA at remarkably high rates. In the established Berg-von Hippel model for this search process, the TF alternates between three-dimensional diffusion in the bulk solution and one-dimensional sliding along the DNA chain. To overcome the so-called speed-stability paradox, in similar models the TF was considered as being present in two conformations (search state and recognition state) between which it switches stochastically. Combining both the facilitated diffusion model and alternating states, we obtain a generalized model. We explicitly treat bulk excursions for rodlike chains arranged in parallel and consider a simplified model for coiled DNA. Compared to previously considered facilitated diffusion models, corresponding to limiting cases of our generalized model, we surprisingly find a reduced target search rate. Moreover, at optimal conditions there is no longer an equipartition between the time spent by the protein on and off the DNA chain.

An arrayed human genomic library constructed in the PAC shuttle vector pJCPAC-Mam2 for genome-wide association studies

Genomic libraries constructed in the original Bacterial Artificial Chromosome (BAC) and P1 Artificial Chromosome (PAC) cloning systems were very useful for completion of the Human Genome Project. Libraries constructed in these vector systems were used to generate physical maps of all twenty three-chromosome pairs and served as the templates for DNA sequencing. The PAC vector pJCPAC-Mam2 is a 23 kilobase (kb) shuttle vector that is highly versatile and useful for functional studies in human tissue culture cell lines. pJCPAC-Mam2 contains the P1 single-copy replicon for low copy expression and a multi-copy lytic replicon under the control of the lac repressor for high copy expression in Escherichia coli, wild type and mutant loxP sites for generation of bidirectional nested deletions in any clone of interest and the Epstein Barr Virus (EBV) latent replication origin oriP, the Epstein Barr Nuclear Antigen 1 (EBNA1) gene, and a puromycin-resistance gene for propagation in mammalian cells. We constructed an arrayed 115,000-member human genomic library in pJCPAC-Mam2 that is housed in twelve hundred 96 well dishes. Subsequent pooling using a column and rows strategy reduced the library to 19 final pools for Polymerase Chain Reaction (PCR) screening. We estimate that the average insert size is approximately 57 kb based on NotI digestion and Field Inversion Gel Electrophoresis (FIGE) analysis of 473 random PAC clones. The library appears to represent a two- to three-fold coverage of the human genome based on screening of the library with PCR primers representing nine genes. This library should serve as a valuable resource to validate potential disease genes identified by genome-wide association studies (GWAS) in human tissue culture cell lines and in animal models. Also, individual library members could potentially be used for gene therapy trials for a variety of recessive disorders.

Development of an IPTG inducible expression vector adapted for Bacteroides fragilis

The genus Bacteroides are gram-negative, obligate anaerobes indigenous to the gastrointestinal tract of humans and animals. The Bacteroides and other members of the Bacteroidetes phylum have diverged from the Proteobacteria. These organisms evolved a unique promoter structure for the initiation of transcription, hence common genetic tools are of limited use in the Bacteroides. An expression vector that can control gene expression in the Bacteroides was constructed by engineering the lacO1,3 repressor binding sites into the promoter of the cfxA β-lactamase gene. The gene for the LacI repressor was placed under control of the Bacteroides tetQ gene promoter for constitutive expression and inserted into the vector. Studies utilizing the xylosidase reporter gene, Xa, showed that the gene was induced by Isopropyl β-d-1-thiogalactopyransoide (IPTG) in a time and concentration dependent manner from 10 to 250μM over a 10–240min time frame. The utility of the vector was demonstrated by insertion of the Bacteroides fragilis trxA gene into the plasmid. TrxA synthesis was monitored by Western hybridization and the results indicated that it was regulated by the presence of IPTG in the media. This is the first transcriptional regulatory system developed for the Bacteroides that has incorporated components from the Proteobacteria and demonstrates the feasibility of modifying existing genetic tools for use in these organisms.

Biochemical Analysis of the Human Mismatch Repair Proteins hMutSα MSH2G674A-MSH6 and MSH2-MSH6T1219D

The heterodimeric human MSH2-MSH6 protein initiates DNA mismatch repair (MMR) by recognizing mismatched bases that result from replication errors. Msh2(G674A) or Msh6(T1217D) mice that have mutations in or near the ATP binding site of MSH2 or ATP hydrolysis catalytic site of MSH6 develop cancer and have a reduced lifespan due to loss of the MMR pathway (Lin, D. P., Wang, Y., Scherer, S. J., Clark, A. B., Yang, K., Avdievich, E., Jin, B., Werling, U., Parris, T., Kurihara, N., Umar, A., Kucherlapati, R., Lipkin, M., Kunkel, T. A., and Edelmann, W. (2004) Cancer Res. 64, 517-522; Yang, G., Scherer, S. J., Shell, S. S., Yang, K., Kim, M., Lipkin, M., Kucherlapati, R., Kolodner, R. D., and Edelmann, W. (2004) Cancer Cell 6, 139-150). Mouse embryonic fibroblasts from these mice retain an apoptotic response to DNA damage. Mutant human MutSα proteins MSH2(G674A)-MSH6(wt) and MSH2(wt)-MSH6(T1219D) are profiled in a variety of functional assays and as expected fail to support MMR in vitro, although they retain mismatch recognition activity. Kinetic analyses of DNA binding and ATPase activities and examination of the excision step of MMR reveal that the two mutants differ in their underlying molecular defects. MSH2(wt)-MSH6(T1219D) fails to couple nucleotide binding and mismatch recognition, whereas MSH2(G674A)-MSH6(wt) has a partial defect in nucleotide binding. Nevertheless, both mutant proteins remain bound to the mismatch and fail to promote efficient excision thereby inhibiting MMR in vitro in a dominant manner. Implications of these findings for MMR and DNA damage signaling by MMR proteins are discussed.

Strategies to overexpress enterotoxigenic Escherichia coli (ETEC) colonization factors for the construction of oral whole-cell inactivated ETEC vaccine candidates

Enterotoxigenic Escherichia coli (ETEC) is an important cause of diarrheal disease and deaths among children in developing countries and the major cause of traveler's diarrhea (TD). Since surface protein colonization factors (CFs) of ETEC are important for pathogenicity and immune protection is mainly mediated by locally produced IgA antibodies in the gut, much effort has focused on the development of an oral CF-based vaccine. The most extensively studied ETEC candidate vaccine is the rCTB-CF ETEC vaccine, containing recombinantly produced cholera B subunit and the most commonly encountered ETEC CFs on the surface of whole inactivated bacteria. Initial clinical trials with this vaccine showed significant immune responses against the key antigens in different age groups in Bangladesh and Egypt and protection against more severe TD in Western travelers. However, when tested in a phase-III trial in Egyptian infants, the protective efficacy of the vaccine was found to be low, indicating the need to improve the immunogenicity of the vaccine, e.g., by increasing the levels of the protective antigens. This review describes different strategies for the construction of recombinant nontoxigenic E. coli and Vibrio cholerae candidate vaccine strains over-expressing higher amounts of ETEC CFs than clinical ETEC isolates selected to produce high levels of the respective CF, e.g., those ETEC strains which have been used in the rCTB-CF ETEC vaccine. Several different expression vectors containing the genes responsible for the expression and assembly of the examined CFs, all downstream of the powerful tac promoter, which could be maintained either with or without antibiotic selection, were constructed. Expression from the tac promoter was under the control of the lacI(q) repressor present on the plasmids. Following induction with isopropyl-β-D-thiogalactopyranoside, candidate vaccine strains over-expressing single CFs, unnatural combinations of two CFs, and also hybrid forms of ETEC CFs were produced. Specific monoclonal antibodies against the major subunits of the examined CF were used to quantify the amount of the surface-expressed CF by a dot-blot assay and inhibition ELISA. Oral immunization with formalin- or phenol-inactivated recombinant bacteria over-expressing the CFs was found to induce significantly higher antibody responses compared to immunization with the previously used vaccine strains. We therefore conclude that our constructs may be useful as candidate strains in an oral whole-cell inactivated CF ETEC vaccine.

FRET studies of a landscape of Lac repressor-mediated DNA loops

DNA looping mediated by the Lac repressor is an archetypal test case for modeling protein and DNA flexibility. Understanding looping is fundamental to quantitative descriptions of gene expression. Systematic analysis of LacI•DNA looping was carried out using a landscape of DNA constructs with lac operators bracketing an A-tract bend, produced by varying helical phasings between operators and the bend. Fluorophores positioned on either side of both operators allowed direct Förster resonance energy transfer (FRET) detection of parallel (P1) and antiparallel (A1, A2) DNA looping topologies anchored by V-shaped LacI. Combining fluorophore position variant landscapes allows calculation of the P1, A1 and A2 populations from FRET efficiencies and also reveals extended low-FRET loops proposed to form via LacI opening. The addition of isopropyl-β-d-thio-galactoside (IPTG) destabilizes but does not eliminate the loops, and IPTG does not redistribute loops among high-FRET topologies. In some cases, subsequent addition of excess LacI does not reduce FRET further, suggesting that IPTG stabilizes extended or other low-FRET loops. The data align well with rod mechanics models for the energetics of DNA looping topologies. At the peaks of the predicted energy landscape for V-shaped loops, the proposed extended loops are more stable and are observed instead, showing that future models must consider protein flexibility.

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using fluorochromes like Green Fluorescent Protein (GFP) directly attached to the protein of interest has become increasingly popular 1. Using GFP and similar fluorochromes the subcellular localisations and movements of proteins can be detected in a fluorescent microscope. Moreover, also the subnuclear localisation of a certain region of a chromosome can be studied using this technique. GFP is fused to the Lac Repressor protein (LacR) and ectopically expressed in the cell where tandem repeats of the lacO sequence has been inserted into the region of interest on the chromosome2. The LacR-GFP will bind to the lacO repeats and that area of the genome will be visible as a green dot in the fluorescence microscope. Yeast is especially suited for this type of manipulation since homologous recombination is very efficient and thereby enables targeted integration of the lacO repeats and engineered fusion proteins with GFP 3. Here we describe a quantitative method for live cell analysis of fission yeast. Additional protocols for live cell analysis of fission yeast can be found, for example on how to make a movie of the meiotic chromosomal behaviour 4. In this particular experiment we focus on subnuclear organisation and how it is affected during gene induction. We have labelled a gene cluster, named Chr1, by the introduction of lacO binding sites in the vicinity of the genes. The gene cluster is enriched for genes that are induced early during nitrogen starvation of fission yeast 5. In the strain the nuclear membrane (NM) is labelled by the attachment of mCherry to the NM protein Cut11 giving rise to a red fluorescent signal. The Spindle Pole body (SPB) compound Sid4 is fused to Red Fluorescent Protein (Sid4-mRFP) 6. In vegetatively growing yeast cells the centromeres are always attached to the SPB that is embedded in the NM 7. The SPB is identified as a large round structure in the NM. By imaging before and 20 minutes after depletion of the nitrogen source we can determine the distance between the gene cluster (GFP) and the NM/SPB. The mean or median distances before and after nitrogen depletion are compared and we can thus quantify whether or not there is a shift in subcellular localisation of the gene cluster after nitrogen depletion.

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using fluorochromes like Green Fluorescent Protein (GFP) directly attached to the protein of interest has become increasingly popular 1. Using GFP and similar fluorochromes the subcellular localisations and movements of proteins can be detected in a fluorescent microscope. Moreover, also the subnuclear localisation of a certain region of a chromosome can be studied using this technique. GFP is fused to the Lac Repressor protein (LacR) and ectopically expressed in the cell where tandem repeats of the lacO sequence has been inserted into the region of interest on the chromosome2. The LacR-GFP will bind to the lacO repeats and that area of the genome will be visible as a green dot in the fluorescence microscope. Yeast is especially suited for this type of manipulation since homologous recombination is very efficient and thereby enables targeted integration of the lacO repeats and engineered fusion proteins with GFP 3. Here we describe a quantitative method for live cell analysis of fission yeast. Additional protocols for live cell analysis of fission yeast can be found, for example on how to make a movie of the meiotic chromosomal behaviour 4. In this particular experiment we focus on subnuclear organisation and how it is affected during gene induction. We have labelled a gene cluster, named Chr1, by the introduction of lacO binding sites in the vicinity of the genes. The gene cluster is enriched for genes that are induced early during nitrogen starvation of fission yeast 5. In the strain the nuclear membrane (NM) is labelled by the attachment of mCherry to the NM protein Cut11 giving rise to a red fluorescent signal. The Spindle Pole body (SPB) compound Sid4 is fused to Red Fluorescent Protein (Sid4-mRFP) 6. In vegetatively growing yeast cells the centromeres are always attached to the SPB that is embedded in the NM 7. The SPB is identified as a large round structure in the NM. By imaging before and 20 minutes after depletion of the nitrogen source we can determine the distance between the gene cluster (GFP) and the NM/SPB. The mean or median distances before and after nitrogen depletion are compared and we can thus quantify whether or not there is a shift in subcellular localisation of the gene cluster after nitrogen depletion.

A Strategy for Searching a Rare Event: Interpretation by Bayes' Theorem

There is the astonishing order in organisms of life which consist of an astronomical amount of molecules and seem to proceed their moving randomly. All known living things on earth are based on the complex webs of chemical reactions with biomolecules like proteins, DNA, RNA, and so on. Some of interactions among the complex interaction networks can be rare events stochastically because of involving only few interacting molecules in crowding of various kinds of molecules within cells. One of examples is the regulation process through binding of transcription factors to DNA in a living E. coli cell. The protein of transcript factor, called a lac repressor, regulates gene expression by site-specific binding to the site of chromosomal DNA, called a lac operon. The protein has to find a targeting site among 10-10 decoy sites on a long DNA molecule. The kinetics experiment of this process reported earlier specific binding at rate as much as 100 times faster than the diffusion limit of a bimolecular reaction by Smoluchowski relation. Since then, a few facilitated mechanisms for this process were proposed to explain the discrepancy. However there has not been a clear consensus yet, even though reducing the dimensionality of diffusion process has regarded as the most probable mechanism. Also many other cases for searching a rare event seem to be unclear for their mechanisms yet. So far, the mechanisms proposed for searching a rare event usually focused on a binding step of a searcher molecule to a target site among multi-steps, like a lock-and-key binding model. In this study, I propose a different view based on the probability theory called Bayes’ theorem. It gives an idea that an unbinding step can be more important than a binding step in searching a rear event. Bayes' theorem shows how to determine inverse probability in conditional probability. It is also called as ‘subjective probability’ because it sometimes means personal belief of probability to happen a certain event. Suppose event A has occurred, given the mutually exclusive and exhaustive events Bi (i = 1, ··, j, ··, n). The probability that each event occurs is P(A) and P(Bi). The conditional probability of event A given event Bj is denoted by P(A|Bj), and its reverse probability of event Bj given event A, P(Bj |A). Bayes’ theorem is as follows:

A two‐step fluorescence‐activated cell sorting approach to isolate genetically modified mammalian cells with isopropyl‐beta‐D‐thiogalactoside (IPTG)‐inducible gene expression

inducible gene expression system is a versatile tool tostudy protein function. It provides an alternative to transientsystems wherein constitutive over-expression of ectopic pro-teins can introduce deleterious effects such as cell cycle arrest(1) or apoptosis (2). The ability to switch off/on the expres-sion of a specific protein is particularly useful for comparativestudies using the same cell system. To date, a range of induci-ble systems have been described such as those responsive tometal (3), glucocorticoid (4), or isopropyl-beta-D-thiogalac-toside (IPTG) (5). Since metal ions and glucocorticoids areknown to affect the expression of many endogenous proteins,IPTG remains a more popular choice of inducer. Nevertheless,these systems share a common drawback in that generation ofinducible cell lines can be labor-intensive, time-consuming(up to several months), and inefficient (1–2 cell lines perstudy). Here we use the LacSwitch II Inducible MammalianExpression System (Agilent Technologies, Santa Clara, CA) asa typical model to describe an improved approach to generateinducible cell lines.The LacSwitch system is designed for inducible geneexpression in mammalian cells. This system employs two vec-tors, the pCMVLacI vector which confers hygromycin resist-ance and encodes Lac repressor, and the lac-operator-contain-ing pOPRSVI/MCS or pOPI3CAT vector which confers G418resistance and encodes the gene of interest. When both vectorsare transfected into a cultured cell line, the Lac protein isexpressed and binds to the lac-operators to repress expressionof the gene of interest. Such repression is constitutive until theinducer IPTG is added to the cell culture to inactivate the Lacprotein. There are different approaches to generate induciblecell lines using this system. While the transfection conditionsvary among different studies, the screening procedures areapparently consistent in that individual transfected clones aremanually isolated and expanded for examination of geneexpression by Western blot analysis. Since transfection effi-ciency and expression levels of ectopic proteins can be highlyvariable, it is often necessary to screen dozens to hundreds ofindividual clones to select the specific ones with desired induc-ible phenotypic characteristics. Although multiple methodssuch as cloning rings (6), trypsin-soaked filter circles (7), andserial dilution (8) are available to facilitate the isolation ofindividual clones, the screening process remains extremelylaborious and poses additional risks for tissue culture contam-ination. To address these concerns, we propose a two-stepapproach using fluorescent protein as a selection marker andfluorescence-activated cell sorting (FACS) to automate thescreening procedures.This FACS-based approach starts with co-transfection ofboth the pCMVLacI vector encoding Lac repressor and thepOPRSVI/MCS or pOPI3CAT vector encoding fluorescentprotein. Since transfection efficiency of individual vectors var-ies among cells, four types of clone variants are anticipatedwith integrated gene expressing (i) Lac only; (ii) fluorescentprotein only; (iii) both Lac and fluorescent protein; or (iv) noectopic protein, due to undesired genomic recombination.The IPTG-dependent expression profiles of individual clonesare summarized (Fig. 1A). Given the unique property of thedesired inducible type III clone, specific isolation is possibleby applying different combinations of induction and cell sort-ing criteria (Fig. 1B). The strategy is to first keep the mixed

Single-Molecule Imaging Reveals Mechanisms of Roadblock Clearance by DNA Motor Enzymes

In the cell, nucleic acid motor proteins act on substrates occupied by other proteins, yet little is known regarding the inevitable collisions that must occur. Using nanofabricated curtains of DNA and real-time, multi-color single-molecule microscopy we visualized collisions between two model translocases and DNA-bound obstacles. We show that both RecBCD, a helicase necessary for initiating homologous DNA recombination, and FtsK, a DNA pump involved in chromosome dimer resolution, actively disrupts nucleoprotein complexes, including RNA polymerase (RNAP) holoenzyme. RecBCD pushed and eventually displaces RNAP, Lac repressor, EcoRI(E111Q) and even nucleosomes. FtsK pushed RNAP but was able to either push or bypass EcoRI(E111Q). We conclude that RecBCD acts as a powerful stripase that overwhelms potential roadblocks. In contrast, FtsK is able to bypass some roadblocks, possibly by dissociation and reassembly ahead of the block.