Separable Sites Research Articles

G-C3N4-modulated bifunctional SnO2@g-C3N4@SnS2 hollow nanospheres for efficient electrochemical overall water splitting

An efficient bifunctional electrocatalyst is developed by bifunctional group construction method for electrochemical overall water splitting. Herein, a self-assembly method using silicon dioxide (SiO2) as template is proposed, in which tin dioxide (SnO2) and tin disulfide (SnS2) are constructed on the inner and outer walls of graphitic carbon nitride (g-C3N4) hollow nanospheres respectively, realizing the dual function of different interfaces controlled by g-C3N4 as the main body. Benefit from this design, the inner wall SnO2@g-C3N4 play a major contribution to oxygen evolution reaction (OER), while the outer wall g-C3N4@SnS2 contribute more to hydrogen evolution reaction (HER). Specifically, the adjustment of C element enhances the adsorption of *H at the edge S site, and the adjustment of N element optimizes the adsorption of OER reactant and the intermediate, thereby improving the bifunctional electrocatalytic activity. The test results show that the electrocatalytic material SnO2@g-C3N4@SnS2 exhibits an excellent electrocatalytic performance of OER and HER in alkaline medium. In addition, low voltage and 48 h stability of SnO2@g-C3N4@SnS2 are also demonstrated in the overall water splitting test. This work paves an avenue for design and control of bifunctional electrocatalysts with separable active sites.

Three classes of ligands each bind to distinct sites on the orphan G protein-coupled receptor GPR84

Medium chain fatty acids can activate the pro-inflammatory receptor GPR84 but so also can molecules related to 3,3′-diindolylmethane. 3,3′-Diindolylmethane and decanoic acid acted as strong positive allosteric modulators of the function of each other and analysis showed the affinity of 3,3′-diindolylmethane to be at least 100 fold higher. Methyl decanoate was not an agonist at GPR84. This implies a key role in binding for the carboxylic acid of the fatty acid. Via homology modelling we predicted and confirmed an integral role of arginine172, located in the 2nd extracellular loop, in the action of decanoic acid but not of 3,3′-diindolylmethane. Exemplars from a patented series of GPR84 antagonists were able to block agonist actions of both decanoic acid and 3,3′-diindolylmethane at GPR84. However, although a radiolabelled form of a related antagonist, [3H]G9543, was able to bind with high affinity to GPR84, this was not competed for by increasing concentrations of either decanoic acid or 3,3′-diindolylmethane and was not affected adversely by mutation of arginine172. These studies identify three separable ligand binding sites within GPR84 and suggest that if medium chain fatty acids are true endogenous regulators then co-binding with a positive allosteric modulator would greatly enhance their function in physiological settings.

Positive Allosteric Modulation of Kv Channels by Sevoflurane: Insights into the Structural Basis of Inhaled Anesthetic Action.

Inhalational general anesthesia results from the poorly understood interactions of haloethers with multiple protein targets, which prominently includes ion channels in the nervous system. Previously, we reported that the commonly used inhaled anesthetic sevoflurane potentiates the activity of voltage-gated K+ (Kv) channels, specifically, several mammalian Kv1 channels and the Drosophila K-Shaw2 channel. Also, previous work suggested that the S4-S5 linker of K-Shaw2 plays a role in the inhibition of this Kv channel by n-alcohols and inhaled anesthetics. Here, we hypothesized that the S4-S5 linker is also a determinant of the potentiation of Kv1.2 and K-Shaw2 by sevoflurane. Following functional expression of these Kv channels in Xenopus oocytes, we found that converse mutations in Kv1.2 (G329T) and K-Shaw2 (T330G) dramatically enhance and inhibit the potentiation of the corresponding conductances by sevoflurane, respectively. Additionally, Kv1.2-G329T impairs voltage-dependent gating, which suggests that Kv1.2 modulation by sevoflurane is tied to gating in a state-dependent manner. Toward creating a minimal Kv1.2 structural model displaying the putative sevoflurane binding sites, we also found that the positive modulations of Kv1.2 and Kv1.2-G329T by sevoflurane and other general anesthetics are T1-independent. In contrast, the positive sevoflurane modulation of K-Shaw2 is T1-dependent. In silico docking and molecular dynamics-based free-energy calculations suggest that sevoflurane occupies distinct sites near the S4-S5 linker, the pore domain and around the external selectivity filter. We conclude that the positive allosteric modulation of the Kv channels by sevoflurane involves separable processes and multiple sites within regions intimately involved in channel gating.

Combined Deficiency of Coagulation Factors V and VIII: An Update

Combined deficiency of factor V (FV) and FVIII (F5F8D) is an autosomal recessive bleeding disorder characterized by simultaneous decreases of both coagulation factors. This review summarizes recent reports on the clinical presentations, treatments, and molecular mechanism of F5F8D. Genetic studies identified LMAN1 and MCFD2 as causative genes for this disorder, revealing a previously unknown intracellular transport pathway shared by the two important blood coagulation factors. LMAN1 and MCFD2 form a Ca2+-dependent cargo receptor complex that functions in the transport of FV/FVIII from the endoplasmic reticulum (ER) to the Golgi. Disrupting the LMAN1-MCFD2 receptor, complex formation is the primary molecular defect of missense mutations leading to F5F8D. The EF-hand domains of MCFD2 are necessary and sufficient for the interactions with both LMAN1 and FV/FVIII. Similarly, the carbohydrate recognition domain of LMAN1 contains distinct and separable binding sites for both MCFD2 and FV/FVIII. Therefore, FV and FVIII likely carry duel sorting signals that are separately recognized by LMAN1 and MCFD2 and necessary for the efficient ER-to-Golgi transport. FV and FVIII likely bind LMAN1 through the high-mannose N-linked glycans under the higher Ca2+ conditions in the ER and dissociate in the lower Ca2+ environment of the ER-Golgi intermediate compartment.

Molecular basis of LMAN1 in coordinating LMAN1-MCFD2 cargo receptor formation and ER-to-Golgi transport of FV/FVIII

AbstractThe LMAN1-MCFD2 (lectin, mannose binding 1/multiple coagulation factor deficiency protein 2) cargo receptor complex transports coagulation factors V (FV) and VIII (FVIII) from the endoplasmic reticulum (ER) to the ER-Golgi intermediate compartment (ERGIC). LMAN1 (ERGIC-53) is a hexameric transmembrane protein with a carbohydrate recognition domain (CRD) on the ER luminal side. Here, we show that mutations in the first beta sheet of the CRD abolish MCFD2 binding without affecting the mannose binding, suggesting that LMAN1 interacts with MCFD2 through its N-terminal beta sheet, consistent with recently reported crystal structures of the CRD-MCFD2 complex. Mutations in the Ca2+- and sugar-binding sites of the CRD disrupt FV and FVIII interactions, without affecting MCFD2 binding. This interaction is independent of MCFD2, as LMAN1 mutants defective in MCFD2 binding can still interact with FVIII. Thus, the CRD of LMAN1 contains distinct, separable binding sites for both its partner protein (MCFD2) and the cargo proteins (FV/FVIII). Monomeric LMAN1 mutants are defective in ER exit and unable to interact with MCFD2, suggesting that the oligomerization of LMAN1 is necessary for its cargo receptor function. These results point to a central role of LMAN1 in regulating the binding in the ER and the subsequent release in the ERGIC of FV and FVIII.

Fatigue-induced spontaneous termination point – Nonequilibrium phase transitions and critical behavior in quasi-isometric exertion

The aim of the present study was to unravel the integration among component processes that jointly lead to a fatigue-induced spontaneous termination point (FISTP), and to pinpoint possible mechanisms underlying this phenomenon. On 5 days during 2 weeks, six participants, who were familiar with the task, performed a quasi-isometric arm-curl exercise holding an Olympic bar with an initial elbow flexion of 90° to the point of spontaneous termination of the exercise due to exhaustion. The repeated measurements ANOVA of windowed variance measures based on the time series of the elbow angles revealed a highly significant effect of the exertion time on the intra- and inter-trial enhancement of the elbow-angle variability when approaching a FISTP. Spectral analysis showed that the variability was generated predominantly in time spans of 1 to about 10 s suggesting that slower and hence higher order control loops are destabilized close to termination points. There was a significant, positive correlation between termination angle and angle variability. The discontinuous change of the elbow angle at the moment of exercise termination was preceded by an enhancement of intra- and inter-trial fluctuations of the elbow angle. This may hint at a nonlinear coupling between the participating neuromuscular components or, more generally, a nonlinear dynamical process underlying the FISTP phenomenon. This dynamical characteristic may indeed explain why other accounts based on separable sites of local, physiological limitations fail to elucidate the occurrence of FISTP.

HIV-1 Nef Targets MHC-I and CD4 for Degradation Via a Final Common β-COP–Dependent Pathway in T Cells

To facilitate viral infection and spread, HIV-1 Nef disrupts the surface expression of the viral receptor (CD4) and molecules capable of presenting HIV antigens to the immune system (MHC-I). To accomplish this, Nef binds to the cytoplasmic tails of both molecules and then, by mechanisms that are not well understood, disrupts the trafficking of each molecule in different ways. Specifically, Nef promotes CD4 internalization after it has been transported to the cell surface, whereas Nef uses the clathrin adaptor, AP-1, to disrupt normal transport of MHC-I from the TGN to the cell surface. Despite these differences in initial intracellular trafficking, we demonstrate that MHC-I and CD4 are ultimately found in the same Rab7+ vesicles and are both targeted for degradation via the activity of the Nef-interacting protein, β-COP. Moreover, we demonstrate that Nef contains two separable β-COP binding sites. One site, an arginine (RXR) motif in the N-terminal α helical domain of Nef, is necessary for maximal MHC-I degradation. The second site, composed of a di-acidic motif located in the C-terminal loop domain of Nef, is needed for efficient CD4 degradation. The requirement for redundant motifs with distinct roles supports a model in which Nef exists in multiple conformational states that allow access to different motifs, depending upon which cellular target is bound by Nef.

Cripto Is a Noncompetitive Activin Antagonist That Forms Analogous Signaling Complexes with Activin and Nodal

Cripto plays critical roles during embryogenesis and has been implicated in promoting the growth and spread of tumors. Cripto is required for signaling by certain transforming growth factor-beta superfamily members, such as Nodal, but also antagonizes others, such as activin. The opposing effects of Cripto on Nodal and activin signaling seem contradictory, however, because these closely related ligands utilize the same type I (ALK4) and type II (ActRII/IIB) receptors. Here, we have addressed this apparent paradox by demonstrating that Cripto forms analogous receptor complexes with Nodal and activin and functions as a noncompetitive activin antagonist. Our results show that activin-A and Nodal elicit similar maximal signaling responses in the presence of Cripto that are substantially lower than that of activin-A in the absence of Cripto. In addition, we provide biochemical evidence for complexes containing activin-A, Cripto, and both receptor types and show that the assembly of such complexes is competitively inhibited by Nodal. We further demonstrate that Nodal and activin-A share the same binding site on ActRII and that ALK4 has distinct and separable binding sites for activin-A and Cripto. Finally, we show that ALK4 mutants with disrupted activin-A binding retain Cripto binding and prevent the effects of Cripto on both activin-A and Nodal signaling. Together, our data indicate that Cripto facilitates Nodal signaling and inhibits activin signaling by forming receptor complexes with these ligands that are structurally and functionally similar.

Thrombospondin-1 differentially induces chemotaxis and DNA synthesis of human venous smooth muscle cells at the receptor-binding level

Thrombospondin-1 is a large matricellular protein that acts as a pleiotropic growth factor for human vascular smooth muscle cells, and may play a role in the progression of vascular disease. Although we have previously demonstrated the dependence of both thrombospondin-1-stimulated cell chemotaxis and proliferation on tyrosine kinases, the receptor mechanisms involved remain obscure. This investigation aims to determine the nature of the receptor(s) involved in the cellular responses to thrombospondin-1. Cellular signals were identified by western blotting following cell stimulation, while cellular responses were assessed by measuring DNA synthesis and chemotaxis. These data demonstrate that thrombospondin-1-induced cell chemotaxis can be inhibited by a peptide containing the Arg-Gly-Asp motif, a function-blocking alpha(v)beta(3) antibody, a function-blocking integrin-associated protein (IAP) antibody and pertussis toxin, while thrombospondin-1-stimulated DNA synthesis is inhibited by a function-blocking alpha(3)beta(1) antibody. Similarly the Arg-Gly-Asp-containing peptide inhibits tyrosine phosphorylation of focal adhesion kinase and the p85 regulatory subunit of phosphatidylinositol 3-kinase, but does not significantly affect tyrosine phosphorylation, or activation, of extracellular-regulated kinase. These data suggest that soluble thrombospondin-1 interacts with human vascular smooth muscle cells via two independent and separable receptor-binding sites, to differentially stimulate cell chemotaxis and DNA synthesis.

Structure and Function

Many ligand-gated ion channels that are activated upon binding to neurotransmitter molecules become inactivated, or desensitized, shortly after the transmembrane pore of the channel opens. This phenomenon is particularly important in fast chemical synapses of the nervous system where the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic (AMPA) and kainate-type glutamate receptors desensitize in milliseconds. However, it has not been clear how desensitization occurs when agonists are still tightly associated with the receptor. Su et al. provide some structural insight into the coupling of activation and desensitization of the GluR2-type AMPA receptor. The receptor consists of four transmembrane subunits, and glutamate-binding pockets are formed at dimer interfaces. Isolated glutamate-binding regions of the AMPA receptor showed less of a tendency to dimerize than did a form harboring a mutation known to block desensitization. A mutation that accelerates desensitization reduced dimer affinity. The authors explain this inverse correlation by proposing that a conformation change in the ligand-binding pocket may induce a conformational change within the transmembrane domains that forces the ion channel to open. However, the tension generated stimulates a subsequent conformational change in the ligand-binding region to relieve this tension. This uncouples the ligand-binding region from the channel region and permits the ligand-associated desensitized state. Hence, the interface between dimer subunits appears to control receptor activation and desensitization.Although structural and functionally similar to AMPA receptor, it has not been clear whether neuronal kainate receptors exhibit conductance properties that reflect increasing amounts of individual subunit activation, as seen with AMPA receptors. Through site-directed mutagenesis and use of a high-affinity agonist, Swanson et al. demonstrate that kainate receptors comprising GluR5 and KA-2 subunits do behave like AMPA receptors in that there are indeed subunit-associated conductances. The authors identify high- and low-affinity agonist-binding sites on the GluR5 and KA-2 subunits, respectively, Occupation of two pharmacologically separable ligand-binding sites may underlie the complex channel sensitivities and activation and desensitization kinetics displayed by these heteromeric glutamate channels.Y. Sun, R. Olsom, M. Horning, N. Armstrong, M. Mayer, E. Gouaux, Mechanism of glutamate receptor desensitization. Nature 417, 245-253 (2002). [Online Journal]G.T. Swanson, T. Green, R. Sakai, A. Contractor, W. Che, H. Kamiya, S.F. Heinemann, Differential activation of individual subunits in heteromeric kainate receptors. Neuron 34, 589-598 (2002). [Online Journal]

Structure and Function

Many ligand-gated ion channels that are activated upon binding to neurotransmitter molecules become inactivated, or desensitized, shortly after the transmembrane pore of the channel opens. This phenomenon is particularly important in fast chemical synapses of the nervous system where the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic (AMPA) and kainate-type glutamate receptors desensitize in milliseconds. However, it has not been clear how desensitization occurs when agonists are still tightly associated with the receptor. Su et al. provide some structural insight into the coupling of activation and desensitization of the GluR2-type AMPA receptor. The receptor consists of four transmembrane subunits, and glutamate-binding pockets are formed at dimer interfaces. Isolated glutamate-binding regions of the AMPA receptor showed less of a tendency to dimerize than did a form harboring a mutation known to block desensitization. A mutation that accelerates desensitization reduced dimer affinity. The authors explain this inverse correlation by proposing that a conformation change in the ligand-binding pocket may induce a conformational change within the transmembrane domains that forces the ion channel to open. However, the tension generated stimulates a subsequent conformational change in the ligand-binding region to relieve this tension. This uncouples the ligand-binding region from the channel region and permits the ligand-associated desensitized state. Hence, the interface between dimer subunits appears to control receptor activation and desensitization. Although structural and functionally similar to AMPA receptor, it has not been clear whether neuronal kainate receptors exhibit conductance properties that reflect increasing amounts of individual subunit activation, as seen with AMPA receptors. Through site-directed mutagenesis and use of a high-affinity agonist, Swanson et al. demonstrate that kainate receptors comprising GluR5 and KA-2 subunits do behave like AMPA receptors in that there are indeed subunit-associated conductances. The authors identify high- and low-affinity agonist-binding sites on the GluR5 and KA-2 subunits, respectively, Occupation of two pharmacologically separable ligand-binding sites may underlie the complex channel sensitivities and activation and desensitization kinetics displayed by these heteromeric glutamate channels. Y. Sun, R. Olsom, M. Horning, N. Armstrong, M. Mayer, E. Gouaux, Mechanism of glutamate receptor desensitization. Nature 417 , 245-253 (2002). [Online Journal] G.T. Swanson, T. Green, R. Sakai, A. Contractor, W. Che, H. Kamiya, S.F. Heinemann, Differential activation of individual subunits in heteromeric kainate receptors. Neuron 34 , 589-598 (2002). [Online Journal]

Identification of an involucrin promoter transcriptional response element with activity restricted to keratinocytes

The involucrin proximal promoter was examined for response elements that confer cell-type specificity. Using a segment spanning positions -157 to +41, three possible response elements were identified by their protein-binding activity using DNase I footprinting. From distal to proximal, they were: an activator protein-1 (AP-1) site (previously identified) overlapping an Ets-like site; a second Ets-like site located 13 bp more proximally; and an extended region designated footprinted site A (FPA). Mutation of the distal Ets-like site had essentially no effect on the transcriptional activity in transfections, while mutation of the proximal site reduced the activity by half. FPA was shown by electrophoretic mobility-shift assay (EMSA) to be comprised of two separable binding sites, FPA1 (distal) and FPA2 (proximal). While mutation of FPA2 had only a modest effect on transcriptional activity in transient transfections, mutation of FPA1 reduced transcriptional activity to approx. 20% of that obtained with the intact promoter. Additional mutations of FPA1 indicated that the active region comprises positions -85 to -73 (GTGGTGAAACCTGT). The molecular masses of the major proteins binding to this site were shown by UV cross-linking to be approx. 40 and 50 kDa, while minor bands were observed at 80 and 110 kDa. Since the involucrin promoter exhibits much higher transcriptional activity in keratinocytes than in other cell types in transfection assays (indicating that cell type specificity of expression is retained), the comparative influence of FPA1 was examined. While mutation of the AP-1 site affected transcriptional activity similarly in all cell lines tested, mutation of FPA1 decreased activity substantially in keratinocytes, but not in NIH-3T3 and HeLa cells, evidence for a contribution to cell-type specificity of expression. Furthermore, a correlation between the sensitivity to FPA1 mutation and amount of involucrin expression in different keratinocyte cell lines was evident. EMSA showed that NIH-3T3 and HeLa cells lacked the same FPA1 DNA-protein complex as keratinocytes. However, the amount of complex formed with nuclear extracts from several keratinocyte lines did not correlate well with the level of involucrin expression. Other factors, such as differences in post-translational modification or co-activators, must account for varied transcriptional response mediated by this site among keratinocyte lines.

Identification of an involucrin promoter transcriptional response element with activity restricted to keratinocytes

The involucrin proximal promoter was examined for response elements that confer cell-type specificity. Using a segment spanning positions -157 to +41, three possible response elements were identified by their protein-binding activity using DNase I footprinting. From distal to proximal, they were: an activator protein-1 (AP-1) site (previously identified) overlapping an Ets-like site; a second Ets-like site located 13 bp more proximally; and an extended region designated footprinted site A (FPA). Mutation of the distal Ets-like site had essentially no effect on the transcriptional activity in transfections, while mutation of the proximal site reduced the activity by half. FPA was shown by electrophoretic mobility-shift assay (EMSA) to be comprised of two separable binding sites, FPA1 (distal) and FPA2 (proximal). While mutation of FPA2 had only a modest effect on transcriptional activity in transient transfections, mutation of FPA1 reduced transcriptional activity to approx. 20% of that obtained with the intact promoter. Additional mutations of FPA1 indicated that the active region comprises positions -85 to -73 (GTGGTGAAACCTGT). The molecular masses of the major proteins binding to this site were shown by UV cross-linking to be approx. 40 and 50 kDa, while minor bands were observed at 80 and 110 kDa. Since the involucrin promoter exhibits much higher transcriptional activity in keratinocytes than in other cell types in transfection assays (indicating that cell type specificity of expression is retained), the comparative influence of FPA1 was examined. While mutation of the AP-1 site affected transcriptional activity similarly in all cell lines tested, mutation of FPA1 decreased activity substantially in keratinocytes, but not in NIH-3T3 and HeLa cells, evidence for a contribution to cell-type specificity of expression. Furthermore, a correlation between the sensitivity to FPA1 mutation and amount of involucrin expression in different keratinocyte cell lines was evident. EMSA showed that NIH-3T3 and HeLa cells lacked the same FPA1 DNA-protein complex as keratinocytes. However, the amount of complex formed with nuclear extracts from several keratinocyte lines did not correlate well with the level of involucrin expression. Other factors, such as differences in post-translational modification or co-activators, must account for varied transcriptional response mediated by this site among keratinocyte lines.

Separable Binding Sites for the Natural Agonist Endothelin-1 and the Non-Peptide Antagonist Bosentan on Human Endothelin-A Receptors

A three-dimensional model for the transmembrane domains of human endothelin-A receptor was built using structural information from bacteriorhodopsin and sequence alignment to other guanine-nucleotide-binding regulatory(G) protein-coupled receptors. Based on this model, 18 amino acids located at the inside of the receptor were mutated and analyzed for binding of the natural ligand endothelin-1 and bosentan, a recently described potent orally active endothelin antagonist [Clozel, M., Breu, V., Gray, G., Kalina, B., Löffler, B.-M., Burri, K., Cassal, J.-M., Hirth, G., Müller, M., Neidhart, W. & Ramuz, H. (1994) Pharmacological characterization of bosentan, a new potent orally active nonpeptide endothelin receptor antagonist, J. Pharmacol. Exp. Ther. 270, 228-235]. Mutation of Gly97, Lys140, Lys159, Gln165 and Phe315, located in transmembrane region 1, 2, 3, 3, and 6, respectively, caused reduced specific binding of 125I-labelled endothelin-1, despite an expression level similar to wild-type endothelin-A receptor. Mutation of Tyr263, Arg326 and Asp351 preserved endothelin-1 binding but caused reduced binding of bosentan. These amino acids, located on transmembrane regions 5, 6 and 7, respectively, are conserved among endothelin-A and endothelin-B receptors but not in other G-protein-coupled receptors. These observations demonstrate a dissociation of the binding site for the peptidic natural agonist endothelin-1 and the synthetic non-peptide antagonist bosentan. They provide the molecular basis for bosentan being a specific antagonist for both, endothelin-A as well as endothelin-B receptors and may in combination with studies on structure/activity relationship support the design of novel and more potent endothelin receptor antagonists.

Separable Binding Sites for the Natural Agonist Endothelin-1 and the Non-Peptide Antagonist Bosentan on Human Endothelin-A Receptors

Separable Binding Sites for the Natural Agonist Endothelin-1 and the Non-Peptide Antagonist Bosentan on Human Endothelin-A Receptors

A retinoic acid-triggered cascade of HOXB1 gene activation.

Retinoic acid (RA) has been proposed to be a direct regulator of HOX gene complexes. However, the molecular mechanism of the RA signaling pathway during normal development is unclear. We have identified an RA-responsive element in the promoter of HOXB1 gene composed of two functionally separable sites: (i) a DR-2 sequence, which is the direct target of the RA receptor retinoid X receptor heterodimer; and (ii) a motif for an RA-inducible and tissue-specific coactivator termed retinoid-inducible protein. Through neither enhancer alone is functional, this combined element strongly activates the HOXB1 promoter in a cell-specific and retinoid-dependent manner. Finally, this activation is potentiated by a proximal autoregulatory site for HOXB1 gene itself. These data define a tripartite cascade leading to the establishment of HOXB1 gene activation.

DNA Damage-inducible Replication of the Escherichia coli Chromosome is Initiated at Separable Sites within the Minimal oriC

When Escherichia coli cells are subjected to genetic stress by exposure to agents or conditions that transiently block DNA replication, the mode of DNA replication is profoundly altered. One of the alterations is the induction of inducible stable DNA replication (iSDR) that does not require the initiator protein, DnaA, and occurs despite the presence of rifampin and chloramphenicol, which inhibit the initiation of usual chromosome replication at oriC. It has been demonstrated that iSDR starts primarily from both the oriC and terC regions of the chromosome. To precisely map the iSDR origin (oriM1) located in the oriC region, various oriC fragments were inserted into a plasmid vector derived from pSC101, and the copy number of these plasmid constructs was measured in the presence of rifampin and chloramphenicol after cells were induced for the SOS response by thymine starvation. The results indicated that there are at least two origins for iSDR, within the minimal oriC; one (oriM1A) is located between the BamHI (coordinate +1) and the AvaII(155) sites, and the other (oriM1B) between the AvaII(155) and the Hind III(244) sites. Furthermore, a 263 bp fragment containing oriM1 , which was placed at the attλ site of the chromosome, was found to initiate chromosome replication in the presence of the drugs when cells were starved of thymine. Introduction of additional copies of oriM1 into a cell stimulated initiation of iSDR at oriM1 on the chromosome. The result supported the model that iSDR starts from D-loops created between oriM1 sequences and that the amount of D-loops determines the level of the iSDR activity.

The cytoskeletal protein talin contains at least two distinct vinculin binding domains.

We have mapped the vinculin-binding sites in the cytoskeletal protein talin as well as those sequences which target the talin molecule to focal contacts. Using a series of overlapping talin-fusion proteins expressed in E. coli and 125I-vinculin in both gel-overlay and microtitre well binding assays, we present evidence for three separable binding sites for vinculin. All three are in the tail segment of talin (residues 434-2541) and are recognized by the same fragment of vinculin (residues 1-258). Two sites are adjacent to each other and span residues 498-950, and the third site is more than 700 residues distant in the primary sequence. Scatchard analysis of 125I-vinculin binding to talin also indicates three sites, each with a similar affinity (Kd = 2-6 x 10(-7) M). We also detect a substoichiometric interaction of higher affinity (Kd = 3 x 10(-8) M) which remains unexplained. By expressing regions of the chicken talin molecule in heterologous cells, we have shown that the sequences required to target talin to focal contacts overlap those which bind vinculin.

Effect of four classes of herbicides on growth and acetolactate-synthase activity in several variants of Arabidopsis thaliana

We have isolated a triazolopyrimidine-resistant mutant csrl-2, of Arabidopsis thaliana (L.) Heynh. Here, we compare csrl-2 with the previously isolated mutants csrl and csr1-1, and with wild-type Arabidopsis for responses to members of four classes of herbicides, namely, sulfonylureas, triazolopyrimidines, imidazolinones, and pyrimidyl-oxy-benzoates. Two separable herbicide binding sites have been identified previously on the protein of acetolactate synthase (ALS). Here, the mutation giving rise to csrl, originating in a coding sequence towards the 5' end of the ALS gene, and that in csrl-2, affected the inhibitory action on growth and ALS activity of sulfonylurea and triazolopyrimidine herbicides but not that of the imidazolinones or pyrimidyl-oxybenzoates. The other mutation, in csrl-1, originating in a coding sequence towards the 3' end of the ALS gene, affected the inhibitory action of imidazolinones and pyrimidyl-oxy-benzoates but not that of the sulfonylureas or triazolopyrimidines. Additional, stimulatory effects of some of these herbicides on growth of seedlings was unrelated to their effect on their primary target, ALS. The conclusion from these observations is that one of the two previously identified herbicide-binding sites may bind sulfonylureas and triazolopyrimidines while the other may bind imidazolinones and pyrimidyl-oxy-benzoates within a herbicide-binding domain on the ALS enzyme. Such a comparative study using near-isogenic mutants from the same species allows not only the further definition of the domain of herbicide binding on ALS but also could aid investigation of the relationship between herbicide-, substrate-, and allosteric-binding sites on this enzyme.This research was supported by an Operating Grant from the Natural Sciences and Engineering Research Council of Canada to J.K.

Lack of Cross-Resistance of Imidazolinone-Resistant Cell Lines of Datura innoxia P. Mill. to Chlorsulfuron

Two cell lines of Datura innoxia resistant to two imidazolinone herbicides, imazapyr and imazaquin, were isolated from mutagenized, predominantly haploid cell suspension cultures. Both of the resistant variants were >1000-fold more resistant than the wild-type to the two imidazolinones. The variant resistant to imazapyr showed cross-resistance to imazaquin and vice versa, but no cross-resistance to a structurally different inhibitor, chlorsulfuron, a sulfonylurea herbicide, was observed. The target enzyme, acetolactate synthase, extracted from imidazolinone-resistant cell lines was not inhibited by imazapyr or imazaquin but was sensitive to chlorsulfuron indicating separable sites of action for these inhibitors. The variation in resistance and cross-resistance of chlorsulfuron-resistant (PK Saxena, J King [1988] Plant Physiol 86: 863-867) and imidazolinone-resistant cell lines of Datura innoxia demonstrates the possibility of separate mutations of acetolactate synthase gene resulting in specific phenotypes.