Site-directed PCR Research Articles

Multiple highly expressed phosphoenolpyruvate carboxylase genes have divergent enzyme kinetic properties in two C4 grasses.

Phosphoenolpyruvate (PEP) carboxylase (PEPC) catalyses the irreversible carboxylation of PEP with bicarbonate to produce oxaloacetate. This reaction powers the carbon-concentrating mechanism (CCM) in plants that perform C4 photosynthesis. This CCM is generally driven by a single PEPC gene product that is highly expressed in the cytosol of mesophyll cells. We found two C4 grasses, Panicum miliaceum and Echinochloa colona, that each have two highly expressed PEPC genes. We characterized the kinetic properties of the two most abundant PEPCs in E. colona and P. miliaceum to better understand how the enzyme's amino acid structure influences its function. Coding sequences of the two most abundant PEPC proteins in E. colona and P. miliaceum were synthesized by GenScript and were inserted into bacteria expression plasmids. Point mutations resulting in substitutions at conserved amino acid residues (e.g. N-terminal serine and residue 890) were created via site-directed PCR mutagenesis. The kinetic properties of semi-purified plant PEPCs from Escherichia coli were analysed using membrane-inlet mass spectrometry and a spectrophotometric enzyme-coupled reaction. The two most abundant P. miliaceum PEPCs (PmPPC1 and PmPPC2) have similar sequence identities (>95 %), and as a result had similar kinetic properties. The two most abundant E. colona PEPCs (EcPPC1 and EcPPC2) had identities of ~78 % and had significantly different kinetic properties. The PmPPCs and EcPPCs had different responses to allosteric inhibitors and activators, and substitutions at the conserved N-terminal serine and residue 890 resulted in significantly altered responses to allosteric regulators. The two, significantly expressed C4Ppc genes in P. miliaceum were probably the result of genomes combining from two closely related C4Panicum species. We found natural variation in PEPC's sensitivity to allosteric inhibition that seems to bypass the conserved 890 residue, suggesting alternative evolutionary pathways for increased malate tolerance and other kinetic properties.

Mutations in SARS-CoV-2 nucleocapsid in variants of concern impair the sensitivity of SARS-CoV-2 detection by rapid antigen tests

Rapid antigen tests (RATs) are used as convenient SARS-CoV-2 tests to minimize infection risks in the private and public domain (e.g., access to shops, concerts, sports, and other social events). RATs are: however, less sensitive than quantitative reverse transcription Polymerase chain reaction (RT-qPCR) assays; hence, samples with low viral loads may be misdiagnosed. Reports on the ability of RATs to detect SARS-CoV-2 variants of concern (VOCs) Delta and Omicron are often only qualitative. We, therefore, examined the analytical sensitivities of four different RATs for the detection of both full virus and recombinant proteins of relevant VOCs. Since most RATs are based on the detection of the SARS-CoV-2 nucleocapsid protein (N-protein), we constructed multiple N-protein mutants (mirroring specific amino acid exchanges of VOC N-proteins) using prokaryotic expression plasmids and site-directed PCR mutagenesis. Testing of recombinant proteins by four RATs revealed amino acid substitutions R203K and R203M, are critical for the sensitivity of some RATs. Interestingly, R203M mutation completely abrogated antigen detection even at high protein concentrations in the Delta variant. As a proof-of-concept study, we show that one or two specific amino acid changes in the N-protein can negatively impact the analytical sensitivity of RATs. Hence, antibodies used in such lateral flow assays should be optimized and target preferentially more conserved regions of N-protein.

Enhancement of solubility of recombinant alcohol dehydrogenase from Rhodococcus ruber using predictive tool.

Solubility is one of key factors influencing the heterologous production of recombinant proteins in biotechnology. Among many aggregation-prone proteins, alcohol dehydrogenase (ADH-A) from Rhodococcus ruber (in this work abbreviated RrADH) shows a great potential in processes involved in the biotransformation of natural compounds. As ADH-A is a potentially high value asset in industrial biotransformation processes, improvement of its solubility would be of major commercial benefit. Predictive tools and in silico analysis provide a fast means for improving protein properties, for selecting appropriate changes, and ultimately for saving costs. We have therefore focused on enhancement of the solubility of RrADH using an online accesible predictive tool Aggrescan 3D 2.0. Selected mutations were introduced into the protein amino acid sequence by using site-directed PCR. This led to a 17% increase in the protein solubility of RrADHmut1 and a 98% increase for RrADHmut2. Moreover, the basic kinetics of the enzyme reaction were positively affected, further optimizing the overall performance of the production process.

Successful Application of Site-directed Mutagenesis Polymerase Chain Reaction to Mutate TMS 11 of the Staphylococcal Multidrug Efflux Protein QacA

Staphylococcus aureus is a major problem in both the clinical setting and within the community. S. aureus can quickly develop resistance to a wide range of antibiotics through a number of different mechanisms, of which, using transporters located in the cell membrane to pump antibiotics out of the cell is the most serious concern. In staphylococcal species, QacA, one such important transporter, is encoded by qacA. QacA is 55kD in size and has 14 transmembrane segments (TMS) (TMS 1-TMS 14). This research describes the mutation process of the amino acid residues in TMS 11 of QacA using site-direct PCR. In this research, 15 primers were successfully designed for site-directed mutagenesis PCR. The site-mutagenesis PCR was successfully conducted to create 15 qacA mutants. These mutants will be used in further functional research of QacA.

Mitochondrial cytopathies: Their causes and correction pathways

Mitochondrial cytopathies are a heterogeneous group of systemic disorders caused by mutations in mitochondrial or nuclear genome. The review presents some data on pathogenic mutations in mitochondrial DNA leading to the imbalance in the oxidation phosphorylation processes and energy metabolism in the cells and eventually to the development of mitochondrial cytopathy. The pathways of medicated correction are examined, which are aimed at obtaining optimal energy efficiency of mitochondria with impaired functions, increase of the efficiency of energy metabolism in the tissues, as well as prevention of mitochondrial membrane damage by free radicals using antioxidants and membrane protectors. A conclusion is drawn on the inefficiency of currently used therapeutic strategies and the necessity of new approaches, which can be gene therapy of mitochondrial diseases. Some modern methods for gene defects correction, capable of restoring or removing the damaged gene, expressing full gene product, or blocking the mutant or strange genes work are analyzed. It is shown that the described approaches to the gene therapy of human mitochondrial diseases demand the introduction of foreign sequences into nuclear or mitochondrial genome of a living person, which completely excludes their practical application because of the uncertainty of the outcome. A perspective approach in solving this problem may be a creation of a system allowing the correction of defect genes without introducing synthetic nucleotides into the human genome. Phenotypic selection combined with a capacity of homologous recombination, artificially imparted to mitochondria of yeast Yarrowia lipolytica, allows for replication of intact human mitochondrial DNA in yeast mitochondria, supporting a full-size native human mitochondrial DNA in the yeast cells and eliminating pathogenic mutations by means of standard sitedirected PCR mutagenesis. After the correction in the Y. lipolytica cells, copies of mitochondrial DNA of an individual patient may be returned to him using the transfection of mesenchymal stromal cells followed by selection of transfectants grown in minimal culture media, in which the cells with higher respiratory mitochondrial activity will gain the advantage.

Surface display of Acetobacter pasteurianus AdhA on Bacillus subtilis spores to enhance ethanol tolerance for liquor industrial potential

Bacillus subtilis have always been regarded as one of the most important functional bacteria for flavor formation in liquor industry. During the flavor formation process, B. subtilis facilitate the conversion of ethanol into aroma compounds by esterification and Maillard reactions mainly due to their vigorous enzymatic secretion and metabolic systems. However, high concentrations of ethanol could be a significant stressor to inhibit Bacillus spp. propagation and thus impair their effective flavor-forming ability. Therefore, in order to obtain improved B. subtilis strains with enhanced ethanol tolerance for liquor industrial potential, we adopted spore surface display strategy in the present study. Acetobacter pasteurianus alcohol dehydrogenase A (adhA) gene was employed as foreign gene, and recombinant integrative plasmid pJS700-adhA was constructed by using B. subtilis spore coat protein CotC as a fusion partner. A combination of amylase activity assay, site-directed PCR identification, Western blot analysis, and immunofluorescence microscopy sequentially demonstrated that AdhA was successfully expressed and displayed on recombinant B. subtilis mutant spore surface. In addition, ethanol tolerance assay showed that the recombinant mutant exhibited enhanced ethanol resistance than its wild-type form when confronted with high concentrations of ethanol, indicating both of its validity in methodology and application potential for liquor industry.

N-Glycosylation at the SynCAM (Synaptic Cell Adhesion Molecule) Immunoglobulin Interface Modulates Synaptic Adhesion

Select adhesion molecules connect pre- and postsynaptic membranes and organize developing synapses. The regulation of these trans-synaptic interactions is an important neurobiological question. We have previously shown that the synaptic cell adhesion molecules (SynCAMs) 1 and 2 engage in homo- and heterophilic interactions and bridge the synaptic cleft to induce presynaptic terminals. Here, we demonstrate that site-specific N-glycosylation impacts the structure and function of adhesive SynCAM interactions. Through crystallographic analysis of SynCAM 2, we identified within the adhesive interface of its Ig1 domain an N-glycan on residue Asn(60). Structural modeling of the corresponding SynCAM 1 Ig1 domain indicates that its glycosylation sites Asn(70)/Asn(104) flank the binding interface of this domain. Mass spectrometric and mutational studies confirm and characterize the modification of these three sites. These site-specific N-glycans affect SynCAM adhesion yet act in a differential manner. Although glycosylation of SynCAM 2 at Asn(60) reduces adhesion, N-glycans at Asn(70)/Asn(104) of SynCAM 1 increase its interactions. The modification of SynCAM 1 with sialic acids contributes to the glycan-dependent strengthening of its binding. Functionally, N-glycosylation promotes the trans-synaptic interactions of SynCAM 1 and is required for synapse induction. These results demonstrate that N-glycosylation of SynCAM proteins differentially affects their binding interface and implicate post-translational modification as a mechanism to regulate trans-synaptic adhesion.

Thyroid Hormone Receptor β (TRβ) and Liver X Receptor (LXR) Regulate Carbohydrate-response Element-binding Protein (ChREBP) Expression in a Tissue-selective Manner

Thyroid hormone (TR) and liver X (LXR) receptors are transcription factors involved in lipogenesis. Both receptors recognize the same consensus DNA-response element in vitro. It was previously shown that their signaling pathways interact in the control of cholesterol elimination in the liver. In the present study, carbohydrate-response element-binding protein (ChREBP), a major transcription factor controlling the activation of glucose-induced lipogenesis in liver, is characterized as a direct target of thyroid hormones (TH) in liver and white adipose tissue (WAT), the two main lipogenic tissues in mice. Using genetic and molecular approaches, ChREBP is shown to be specifically regulated by TRbeta but not by TRalpha in vivo, even in WAT where both TR isoforms are expressed. However, this isotype specificity is not found in vitro. This TRbeta specific regulation correlates with the loss of TH-induced lipogenesis in TRbeta(-/-) mice. Fasting/refeeding experiments show that TRbeta is not required for the activation of ChREBP expression particularly marked in WAT following refeeding. However, TH can stimulate ChREBP expression in WAT even under fasting conditions, suggesting completely independent pathways. Because ChREBP has been described as an LXR target, the interaction of LXR and TRbeta in ChREBP regulation was assayed both in vitro and in vivo. Each receptor recognizes a different response element on the ChREBP promoter, located only 8 bp apart. There is a cross-talk between LXR and TRbeta signaling on the ChREBP promoter in liver but not in WAT where LXR does not regulate ChREBP expression. The molecular basis for this cross-talk has been determined in in vitro systems.

Binding of PDZ-RhoGEF to ATP-binding Cassette Transporter A1 (ABCA1) Induces Cholesterol Efflux through RhoA Activation and Prevention of Transporter Degradation

ATP-binding cassette transporter A1 (ABCA1)-mediated lipid efflux to apolipoprotein A1 (apoA-I) initiates the biogenesis of high density lipoprotein. Here we show that the Rho guanine nucleotide exchange factors PDZ-RhoGEF and LARG bind to the C terminus of ABCA1 by a PDZ-PDZ interaction and prevent ABCA1 protein degradation by activating RhoA. ABCA1 is a protein with a short half-life, and apoA-I stabilizes ABCA1 protein; however, depletion of PDZ-RhoGEF/LARG by RNA interference suppressed the apoA-I stabilization of ABCA1 protein in human primary fibroblasts. Exogenous PDZ-RhoGEF expression activated RhoA and increased ABCA1 protein levels and cholesterol efflux activity. Likewise, forced expression of a constitutively active RhoA mutant significantly increased ABCA1 protein levels, whereas a dominant negative RhoA mutant decreased them. The constitutively active RhoA retarded ABCA1 degradation, thus accounting for its ability to increase ABCA1 protein. Moreover, stimulation with apoA-I transiently activated RhoA, and the pharmacological inhibition of RhoA or the dominant negative RhoA blocked the ability of apoA-I to stabilize ABCA1. Finally, depletion of RhoA or RhoGEFs/RhoA reduces the cholesterol efflux when transcriptional regulation via PPARgamma is eliminated. Taken together, our results have identified a novel physical and functional interaction between ABCA1 and PDZ-RhoGEF/LARG, which activates RhoA, resulting in ABCA1 stabilization and cholesterol efflux activity.

Segments Crucial for Membrane Translocation and Pore-forming Activity of Bordetella Adenylate Cyclase Toxin

Bordetella adenylate cyclase toxin-hemolysin (CyaA, AC-Hly, or ACT) permeabilizes cell membranes by forming small cation-selective (hemolytic) pores and subverts cellular signaling by delivering into host cells an adenylate cyclase (AC) enzyme that converts ATP to cAMP. Both AC delivery and pore formation were previously shown to involve a predicted amphipathic alpha-helix(502-522) containing a pair of negatively charged Glu(509) and Glu(516) residues. Another predicted transmembrane alpha-helix(565-591) comprises a Glu(570) and Glu(581) pair. We examined the roles of these glutamates in the activity of CyaA. Substitutions of Glu(516) increased specific hemolytic activity of CyaA by two different molecular mechanisms. Replacement of Glu(516) by positively charged lysine residue (E516K) increased the propensity of CyaA to form pores, whereas proline (E516P) or glutamine (E516Q) substitutions extended the lifetime of open single pore units. All three substitutions also caused a drop of pore selectivity for cations. Substitutions of Glu(570) and Glu(581) by helix-breaking proline or positively charged lysine residue reduced (E570K, E581P) or ablated (E570P, E581K) AC membrane translocation. Moreover, E570P, E570K, and E581P substitutions down-modulated also the specific hemolytic activity of CyaA. In contrast, the E581K substitution enhanced the hemolytic activity of CyaA 4 times, increasing both the frequency of formation and lifetime of toxin pores. Negative charge at position 570, but not at position 581, was found to be essential for cation selectivity of the pore, suggesting a role of Glu(570) in ion filtering inside or close to pore mouth. The pairs of glutamate residues in the predicted transmembrane segments of CyaA thus appear to play a key functional role in membrane translocation and pore-forming activities of CyaA.

The Crystal Structure and Mutational Binding Analysis of the Extracellular Domain of the Platelet-activating Receptor CLEC-2

The human C-type lectin-like molecule CLEC-2 is expressed on the surface of platelets and signaling through CLEC-2 causes platelet activation and aggregation. CLEC-2 is a receptor for the platelet-aggregating snake venom protein rhodocytin. It is also a newly identified co-receptor for human immunodeficiency virus type 1 (HIV-1). An endogenous ligand has not yet been identified. We have solved the crystal structure of the extracellular domain of CLEC-2 to 1.6-A resolution, and identified the key structural features involved in ligand binding. A semi-helical loop region and flanking residues dominate the surface that is available for ligand binding. The precise distribution of hydrophobic and electrostatic features in this loop will determine the nature of any endogenous ligand with which it can interact. Major ligand-induced conformational change in CLEC-2 is unlikely as its overall fold is compact and robust. However, ligand binding could induce a tilt of a 3-10 helical portion of the long loop region. Mutational analysis and surface plasmon resonance binding studies support these observations. This study provides a framework for understanding the effects of rhodocytin venom binding on CLEC-2 and for understanding the nature of likely endogenous ligands and will provide a basis for rational design of drugs to block ligand binding.

Versatility and Differential Roles of Cysteine Residues in Human Prostacyclin Receptor Structure and Function

Prostacyclin plays important roles in vascular homeostasis, promoting vasodilatation and inhibiting platelet thrombus formation. Previous studies have shown that three of six cytoplasmic cysteines, particularly those within the C-terminal tail, serve as important lipidation sites and are differentially conjugated to palmitoyl and isoprenyl groups (Miggin, S. M., Lawler, O. A., and Kinsella, B. T. (2003) J. Biol. Chem. 278, 6947-6958). Here we report distinctive roles for extracellular- and transmembrane-located cysteine residues in human prostacyclin receptor structure-function. Within the extracellular domain, all cysteines (4 of 4) appear to be involved in disulfide bonding interactions (i.e. a highly conserved Cys-92-Cys-170 bond and a putative non-conserved Cys-5-Cys-165 bond), and within the transmembrane (TM) region there are several cysteines (3 of 8) that maintain critical hydrogen bonding interactions (Cys-118 (TMIII), Cys-251 (TMVI), and Cys-202 (TMV)). This study highlights the necessity of sulfhydryl (SH) groups in maintaining the structural integrity of the human prostacyclin receptor, as 7 of 12 extracellular and transmembrane cysteines studied were found to be differentially indispensable for receptor binding, activation, and/or trafficking. Moreover, these results also demonstrate the versatility and reactivity of these cysteine residues within different receptor environments, that is, extracellular (disulfide bonds), transmembrane (H-bonds), and cytoplasmic (lipid conjugation).

Functional Divergence of a Unique C-terminal Domain of Leucyl-tRNA Synthetase to Accommodate Its Splicing and Aminoacylation Roles

Leucyl-tRNA synthetase (LeuRS) performs dual essential roles in group I intron RNA splicing as well as protein synthesis within the yeast mitochondria. Deletions of the C terminus differentially impact the two functions of the enzyme in splicing and aminoacylation in vivo. Herein, we determined that a fiveamino acid C-terminal deletion of LeuRS, which does not complement a null strain, can form a ternary complex with the bI4 intron and its maturase splicing partner. However, the complex fails to stimulate splicing activity. The x-ray co-crystal structure of LeuRS showed that a C-terminal extension of about 60 amino acids forms a discrete domain, which is unique among the LeuRSs and interacts with the corner of the L-shaped tRNALeu. Interestingly, deletion of the entire yeast mitochondrial LeuRS C-terminal domain enhanced its aminoacylation and amino acid editing activities. In striking contrast, deletion of the corresponding C-terminal domain of Escherichia coli LeuRS abolished aminoacylation of tRNALeu and also amino acid editing of mischarged tRNA molecules. These results suggest that the role of the leucine-specific C-terminal domain in tRNA recognition for aminoacylation and amino acid editing has adapted differentially and with surprisingly opposite effects. We propose that the secondary role of yeast mitochondrial LeuRS in RNA splicing has impacted the functional evolution of this critical C-terminal domain.

High-throughput screening of cellulase F mutants from multiplexed plasmid sets using an automated plate assay on a functional proteomic robotic workcell

BackgroundThe field of plasmid-based functional proteomics requires the rapid assay of proteins expressed from plasmid libraries. Automation is essential since large sets of mutant open reading frames are being cloned for evaluation. To date no integrated automated platform is available to carry out the entire process including production of plasmid libraries, expression of cloned genes, and functional testing of expressed proteins.ResultsWe used a functional proteomic assay in a multiplexed setting on an integrated plasmid-based robotic workcell for high-throughput screening of mutants of cellulase F, an endoglucanase from the anaerobic fungus Orpinomyces PC-2. This allowed us to identify plasmids containing optimized clones expressing mutants with improved activity at lower pH. A plasmid library of mutagenized clones of the celF gene with targeted variations in the last four codons was constructed by site-directed PCR mutagenesis and transformed into Escherichia coli. A robotic picker integrated into the workcell was used to inoculate medium in a 96-well deep well plate, combining the transformants into a multiplexed set in each well, and the plate was incubated on the workcell. Plasmids were prepared from the multiplexed culture on the liquid handler component of the workcell and used for in vitro transcription/translation. The multiplexed expressed recombinant proteins were screened for improved activity and stability in an azo-carboxymethylcellulose plate assay. The multiplexed wells containing mutants with improved activity were identified and linked back to the corresponding multiplexed cultures stored in glycerol. Spread plates were prepared from the glycerol stocks and the workcell was used to pick single colonies from the spread plates, prepare plasmid, produce recombinant protein, and assay for activity. The screening assay and subsequent deconvolution of the multiplexed wells resulted in identification of improved CelF mutants and corresponding optimized clones in expression-ready plasmids.ConclusionThe multiplex method using an integrated automated platform for high-throughput screening in a functional proteomic assay allows rapid identification of plasmids containing optimized clones ready for use in subsequent applications including transformations to produce improved strains or cell lines.

FpvIR Control of fpvA Ferric Pyoverdine Receptor Gene Expression in Pseudomonas aeruginosa : Demonstration of an Interaction between FpvI and FpvR and Identification of Mutations in Each Compromising This Interaction

FpvR is a presumed cytoplasmic membrane-associated anti-sigma factor that controls the activities of extracytoplasmic function sigma factors PvdS and FpvI responsible for transcription of pyoverdine biosynthetic genes and the ferric pyoverdine receptor gene, fpvA, respectively. Using deletion analysis and an in vivo bacterial two-hybrid system, FpvR interaction with these sigma factors was confirmed and shown to involve the cytoplasmic N-terminal 67 amino acid resides of FpvR. FpvR bound specifically to a C-terminal region of FpvI corresponding to region 4 of the sigma(70) family of sigma factors. FpvR and FpvI mutant proteins compromised for this interaction were generated by random and site-directed PCR mutagenesis and invariably contained secondary structure-altering proline substitution in predicted alpha-helices within the FpvR N terminus or FpvI region 4. PvdS was shown to bind to the same N-terminal region of FpvR, and FpvR mutations compromising FpvI binding also compromised PvdS binding, although some mutations had a markedly greater impact on PvdS binding. Apparently, these two sigma factors bind to FpvR in a substantially similar but not identical fashion. Intriguingly, defects in FpvR binding correlated with a substantial drop in yields of the FpvI and to a lesser extent PvdS sigma factors, suggesting that FpvR-bound FpvI and PvdS are stable while free and active sigma factor is prone to turnover.

Phosphorylation of GRK1 and GRK7 by cAMP-dependent Protein Kinase Attenuates Their Enzymatic Activities

Phosphorylation of G protein-coupled receptors is a critical step in the rapid termination of G protein signaling. In rod cells of the vertebrate retina, phosphorylation of rhodopsin is mediated by GRK1. In cone cells, either GRK1, GRK7, or both, depending on the species, are speculated to initiate signal termination by phosphorylating the cone opsins. To compare the biochemical properties of GRK1 and GRK7, we measured the K(m) and V(max) of these kinases for ATP and rhodopsin, a model substrate. The results demonstrated that these kinases share similar kinetic properties. We also determined that cAMP-dependent protein kinase (PKA) phosphorylates GRK1 at Ser(21) and GRK7 at Ser(23) and Ser(36) in vitro. These sites are also phosphorylated when FLAG-tagged GRK1 and GRK7 are expressed in HEK-293 cells treated with forskolin to stimulate the endogenous production of cAMP and activation of PKA. Rod outer segments isolated from bovine retina phosphorylated the FLAG-tagged GRKs in the presence of dibutyryl-cAMP, suggesting that GRK1 and GRK7 are physiologically relevant substrates. Although both GRKs also contain putative phosphorylation sites for PKC and Ca(2+)/calmodulin-dependent protein kinase II, neither kinase phosphorylated GRK1 or GRK7. Phosphorylation of GRK1 and GRK7 by PKA reduces the ability of GRK1 and GRK7 to phosphorylate rhodopsin in vitro. Since exposure to light causes a decrease in cAMP levels in rod cells, we propose that phosphorylation of GRK1 and GRK7 by PKA occurs in the dark, when cAMP levels in photoreceptor cells are elevated, and represents a novel mechanism for regulating the activities of these kinases.

A Structural Basis for 14-3-3σ Functional Specificity*♦

The 14-3-3 family of proteins includes seven isotypes in mammalian cells that play numerous diverse roles in intracellular signaling. Most 14-3-3 proteins form homodimers and mixed heterodimers between different isotypes, with overlapping roles in ligand binding. In contrast, one mammalian isoform, 14-3-3sigma, expressed primarily in epithelial cells, appears to play a unique role in the cellular response to DNA damage and in human oncogenesis. The biological and structural basis for these 14-3-3sigma-specific functions is unknown. We demonstrate that endogenous 14-3-3sigma preferentially forms homodimers in cells. We have solved the x-ray crystal structure of 14-3-3sigma bound to an optimal phosphopeptide ligand at 2.4 angstroms resolution. The structure reveals the presence of stabilizing ring-ring and salt bridge interactions unique to the 14-3-3sigma homodimer structure and potentially destabilizing electrostatic interactions between subunits in 14-3-3sigma-containing heterodimers, rationalizing preferential homodimerization of 14-3-3sigma in vivo. The interaction of the phosphopeptide with 14-3-3 reveals a conserved mechanism for phospho-dependent ligand binding, implying that the phosphopeptide binding cleft is not the critical determinant of the unique biological properties of 14-3-3sigma. Instead, the structure suggests a second ligand binding site involved in 14-3-3sigma-specific ligand discrimination. We have confirmed this by site-directed mutagenesis of three sigma-specific residues that uniquely define this site. Mutation of these residues to the alternative sequence that is absolutely conserved in all other 14-3-3 isotypes confers upon 14-3-3sigma the ability to bind to Cdc25C, a ligand that is known to bind to other 14-3-3 proteins but not to sigma.

Analysis of and expression of connexin 26 mutations

Problem: Mutations in the gap junction beta-2 (GJB2) gene, which encodes the connexin 26 protein, account for 50% of all the patients with autosomal recessive nonsyndromic hearing loss. Connexins are proteins that oligomerize to form a gap junction channel. Gap junctions enable intercellular communication between cell membranes of 2 neighboring cells. Mutations in these connexins lead to absent or malformed gap junctions and congenital deafness. The expression of different connexin mutations’ molecules has not been fully evaluated. Methods: Based on their prevalence and inheritance, 4 specific mutations were chosen (35delG, R184P, R184Q, and C202F). Primers were designed based on specific mutations and site-directed PCR mutagenesis was performed using Stratagene’s Quikchange site-directed mutagenesis kit. Mutations were confirmed via DNA sequencing. The mutated GJB2 genes and wild-type GJB2 were inserted into the EcoRI site of the pNUT vector and transfected into baby-kidney-cells (BHK) using Lipofectamine. Expression of mutated genes was analyzed using immunofluorescence. Results: Mutations (35delG, R184P, R184Q, C202F) were successfully created using site-directed PCR mutagenesis. DNA sequencing confirmed desired mutations. Mutations and wild-type GJB2 genes were successfully expressed in BHK cells and analyzed using immunofluorescence. Specific results and difference will be presented. Conclusion: Specific GJB2 mutations (35delG, R184P, 184Q, C202F) were successfully created and expressed. Immunofluorescence results confirm specific difference between wild-type GJB2 and GJB2 mutations. This study was the first to successfully express and analyze mutations R184Q and C202F and to express mutations 35delG and R184P in a mouse in vitro system. Significance: As noted above, mutations in the gap junction beta-2 (GJB2) gene, which encodes the connexin 26 protein, account for 50% of all the patients with autosomal recessive nonsyndromic hearing loss. Expression and analysis of these mutations are crucial to our understanding of this disease and to our ability to provide potential therapy via targeted gene therapy in the future. Support: None reported.

Functional determinants of the Escherichia coli fis promoter: roles of -35, -10, and transcription initiation regions in the response to stringent control and growth phase-dependent regulation.

Escherichia coli Fis is a small DNA binding and bending protein that has been implicated in a variety of biological processes. A minimal promoter sequence consisting of 43 bp is sufficient to generate its characteristic growth phase-dependent expression pattern and is also subject to negative regulation by stringent control. However, information about the precise identification of nucleotides contributing to basal promoter activity and its regulation has been scant. In this work, 72 independent mutations were generated in the fis promoter (fis P) region from -108 to +78 using both random and site-directed PCR mutagenesis. beta-Galactosidase activities from mutant promoters fused to the (trp-lac)W200 fusion on a plasmid were used to conclusively identify the sequences TTTCAT and TAATAT as the -35 and -10 regions, respectively, which are optimally separated by 17 bp. We found that four consecutive substitutions within the GC-rich sequence just upstream of +1 and mutations in the -35 region, but not in the -10 region, significantly reduced the response to stringent control. Analysis of the effects of mutations on growth phase-dependent regulation showed that replacing the predominant transcription initiation nucleotide +1C with a preferred nucleotide (A or G) profoundly altered expression such that high levels of fis P mRNA were detected during late logarithmic and early stationary phases. A less dramatic effect was seen with improvements in the -10 and -35 consensus sequences. These results suggest that the acute growth phase-dependent regulation pattern observed with this promoter requires an inefficient transcription initiation process that is achieved with promoter sequences deviating from the -10 and -35 consensus sequences and, more importantly, a dependence upon the availability of the least favored transcription initiation nucleotide, CTP.

Mutational analysis of the transcriptional regulator GcvA: amino acids important for activation, repression, and DNA binding.

The GcvA protein is required for both glycine-mediated activation and purine-mediated repression of the gcvTHP operon. Random and site-directed PCR mutagenesis was used to create nucleotide changes in gcvA to identify residues of the protein involved in activation, repression, and DNA binding. Single amino acid substitutions at L30 and F31 cause a defect in activation of a gcvT-lacZ fusion but have no effect on repression or DNA binding. Single amino acid substitutions at V32 and S38 cause the loss of binding of GcvA to DNA. A deletion of the carboxy-terminal 14 amino acids of GcvA results in the loss of purine-mediated repression and, consequently, a constitutive activation of a gcvT-lacZ fusion. The results of this study partially define regions of GcvA involved in activation, repression, and DNA binding and demonstrate that these functions of GcvA are genetically separable.