ncd and kinesin motor domains interact with both alpha- and beta-tubulin.

Motor domains of the Drosophila minus-end-directed microtubule (MT) motor protein ncd, were found to saturate microtubule binding sites at a stoichiometry of approximately one motor domain per tubulin dimer. To determine the tubulin subunit(s) involved in binding to ncd, mixtures of ncd motor domain and MTs were treated with the zero-length cross-linker 1-ethyl-3-(3-dimethylaminopropyl-carbodiimide) (EDC). EDC treatment generated covalently cross-linked products of ncd and alpha-tubulin and of ncd and beta-tubulin, indicating that the ncd motor domain interacts with both alpha- and beta-tubulin. When the Drosophila kinesin motor domain protein was substituted for the ncd motor domain, cross-linked products of kinesin and alpha-tubulin and of kinesin and beta-tubulin were produced. EDC treatment of mixtures of ncd motor domain and unassembled tubulin dimers or of kinesin motor domain and unassembled tubulin dimers produced the same motor-tubulin products generated in the presence of MTs. These results indicate that kinesin family motors of opposite polarity interact with both tubulin monomers and support a model in which some portion of each protein's motor domain overlaps adjacent alpha- and beta-tubulin subunits.

Dissection of transcription factor TFIIF functional domains required for initiation and elongation.

TFIIF is unique among the general transcription factors because of its ability to control the activity of RNA polymerase II at both the initiation and elongation stages of transcription. Mammalian TFIIF, a heterodimer of approximately 30-kDa (RAP30) and approximately 70-kDa (RAP74) subunits, assists TFIIB in recruiting RNA polymerase II into the preinitiation complex and activates the overall rate of RNA chain elongation by suppressing transient pausing by polymerase at many sites on DNA templates. A major objective of efforts to understand how TFIIF regulates transcription has been to establish the relationship between its initiation and elongation activities. Here we establish this relationship by demonstrating that TFIIF transcriptional activities are mediated by separable functional domains. To accomplish this, we sought and identified distinct classes of RAP30 mutations that selectively block TFIIF activity in transcription initiation and elongation. We propose that (i) TFIIF initiation activity is mediated at least in part by RAP30 C-terminal sequences that include a cryptic DNA-binding domain similar to conserved region 4 of bacterial sigma factors and (ii) TFIIF elongation activity is mediated in part by RAP30 sequences located immediately upstream of the C terminus in a region proposed to bind RNA polymerase II and by additional sequences located in the RAP30 N terminus.

Domains of transcription factor Sp1 required for synergistic activation with sterol regulatory element binding protein 1 of low density lipoprotein receptor promoter.

Feedback regulation of transcription from the low density lipoprotein (LDL) receptor gene is fundamentally important in the maintenance of intracellular sterol balance. The region of the LDL receptor promoter responsible for normal sterol regulation contains adjacent binding sites for the ubiquitous transcription factor Sp1 and the cholesterol-sensitive sterol regulatory element-binding proteins (SREBPs). Interestingly, both are essential for normal sterolmediated regulation of the promoter. The cooperation by Sp1 and SREBP-1 occurs at two steps in the activation process. SREBP-1 stimulates the binding of Sp1 to its adjacent recognition site in the promoter followed by enhanced stimulation of transcription after both proteins are bound to DNA. In the present report, we have defined the protein domains of Sp1 that are required for both synergistic DNA binding and transcriptional activation. The major activation domains of Sp1 that have previously been shown to be essential to activation of promoters containing multiple Sp1 sites are required for activation of the LDL receptor promoter. Additionally, the C domain is also crucial. This slightly acidic approximately 120-amino acid region is not required for efficient synergistic activation by multiple Sp1 sites or in combination with other recently characterized transcriptional regulators. We also show that Sp1 domain C is essential for full, enhanced DNA binding by SREBP-1. Taken together with other recent studies on the role of Sp1 in promoter activation, the current experiments suggest a unique combinatorial mechanism for promoter activation by two distinct transcription factors that are both essential to intracellular cholesterol homeostasis.

Chimeric tumor necrosis factor receptors with constitutive signaling activity.

Many hormone and cytokine receptors are crosslinked by their specific ligands, and multimerization is an essential step leading to the generation of a signal. In the case of the tumor necrosis factor (TNF) receptors (TNF-Rs), antibody-induced crosslinking is sufficient to trigger a cytolytic effect. However, the quaternary structural requirements for signaling--i.e., the formation of dimers, trimers, or higher-order multimers--have remained obscure. Moreover, it has not been clear whether the 55-kDa or 75-kDa TNF-R is responsible for initiation of cytolysis. We reasoned that an obligate receptor dimer, targeted to the plasma membrane, might continuously signal the presence of TNF despite the actual absence of the ligand. Such a molecule, inserted into an appropriate vector, could be used to project receptor-specific "TNF-like" activity to specific cells and tissues in vivo. Accordingly, we constructed sequences encoding chimeric receptors in which the extracellular domain of the mouse erythropoietin receptor (Epo-R) was fused to the "stem," transmembrane domain, and cytoplasmic domain of the two mouse TNF-Rs. Thus, the Epo-R group was used to drive dimerization of the TNF-R cytoplasmic domain. These chimeric proteins were well expressed in a variety of cell lines and bound erythropoietin at the cell surface. Both the 55-kDa and the 75-kDa Epo/TNF-R chimeras exerted a constitutive cytotoxic effect detected by cotransfection or clonogenic assay. Thus, despite the lack of structural homology between the cytoplasmic domains of the two TNF-Rs, a similar signaling endpoint was observed. Moreover, dimerization (rather than trimerization or higher-order multimerization) was sufficient for elicitation of a biological response.

Chimeric plant calcium/calmodulin-dependent protein kinase gene with a neural visinin-like calcium-binding domain.

Calcium, a universal second messenger, regulates diverse cellular processes in eukaryotes. Ca2+ and Ca2+/calmodulin-regulated protein phosphorylation play a pivotal role in amplifying and diversifying the action of Ca(2+)-binding domain was cloned and characterized from lily. The cDNA clone contains an open reading frame coding for a protein of 520 amino acids. The predicted structure of CCaMK contains a catalytic domain followed by two regulatory domains, a calmodulin-binding domain and a visinin-like Ca(2+)-binding domain. The amino-terminal region of CCaMK contains all 11 conserved subdomains characteristic of serine/threonine protein kinases. The calmodulin-binding region of CCaMK has high homology (79%) to alpha subunit of mammalian Ca2+/calmodulin-dependent protein kinase. The calmodulin-binding region is fused to a neural visinin-like domain that contains three Ca(2+)-binding EF-hand motifs and a biotin-binding site. The Escherichia coli-expressed protein (approximately 56 kDa) binds calmodulin in a Ca(2+)-dependent manner. Furthermore, 45Ca-binding assays revealed that CCaMK directly binds Ca2+. The CCaMK gene is preferentially expressed in developing anthers. Southern blot analysis revealed that CCaMK is encoded by a single gene. The structural features of the gene suggest that it has multiple regulatory controls and could play a unique role in Ca2+ signaling in plants.

Identification of receptor-binding domains on human interleukin 5 and design of an interleukin 5-derived receptor antagonist.

A detailed structure-function analysis of human interleukin 5 (hIL5) has been performed. The hIL5 receptor is composed of two different polypeptide chains, the alpha and beta subunits. The alpha subunit alone is sufficient for ligand binding, but association with the beta subunit leads to a 2- to 3-fold increase in binding affinity. The beta chain is shared with the receptors for IL3 and granulocyte/macrophage-colony-stimulating factor--hence the descriptor beta C (C for common). All hIL5 mutants were analyzed in a solid-phase binding assay for hIL5R alpha interaction and in a proliferation assay using IL5-dependent cell lines for receptor-complex activation. Most residues affecting binding to the receptor alpha subunit were clustered in a loop connecting beta-strand 1 and helix B (mutants H38A, K39A, and H41A), in beta-strand 2 (E89A and R91A; weaker effect for E90A) and close to the C terminus (T109A, E110A, W111S, and I112A). Mutations at one position, E13 (Glu13), caused a reduced activation of the hIL5 receptor complex. In the case of E13Q, only 0.05% bioactivity was detected on a hIL5-responsive subclone of the mouse promyelocytic cell line FDC-P1. Moreover, on hIL5-responsive TF1 cells, the same mutant was completely inactive and proved to have antagonistic properties. Interactions of this mutant with both receptor subunits were nevertheless indistinguishable from those of nonmutated hIL5 by crosslinking and Scatchard plot analysis of transfected COS-1 cells.

Proper placement of the Escherichia coli division site requires two functions that are associated with different domains of the MinE protein.

The proper placement of the Escherichia coli division septum requires the MinE protein. MinE accomplishes this by imparting topological specificity to a division inhibitor coded by the minC and minD genes. As a result, the division inhibitor prevents septation at potential division sites that exist at the cell poles but permits septation at the normal division site at midcell. In this paper, we define two functions of MinE that are required for this effect and present evidence that different domains within the 88-amino acid MinE protein are responsible for each of these two functions. The first domain, responsible for the ability of MinE to counteract the activity of the MinCD division inhibitor, is located in a small region near the N terminus of the protein. The second domain, required for the topological specificity of MinE function, is located in the more distal region of the protein and affects the site specificity of placement of the division septum even when separated from the domain responsible for suppression of the activity of the division inhibitor.

Two nuclear localization signals present in the basic-helix 1 domains of MyoD promote its active nuclear translocation and can function independently.

MyoD, a member of the family of helix-loop-helix myogenic factors that plays a crucial role in skeletal muscle differentiation, is a nuclear phosphoprotein. Using microinjection of purified MyoD protein into rat fibroblasts, we show that the nuclear import of MyoD is a rapid and active process, being ATP and temperature dependent. Two nuclear localization signals (NLSs), one present in the basic region and the other in the helix 1 domain of MyoD protein, are demonstrated to be functional in promoting the active nuclear transport of MyoD. Synthetic peptides spanning these two NLSs and biochemically coupled to IgGs can promote the nuclear import of microinjected IgG conjugates in muscle and nonmuscle cells. Deletion analysis reveals that each sequence can function independently within the MyoD protein since concomittant deletion of both sequences is required to alter the nuclear import of this myogenic factor. In addition, the complete cytoplasmic retention of a beta-galactosidase-MyoD fusion mutant protein, double deleted at these two NLSs, argues against the existence of another functional NLS motif in MyoD.

Stathmin interaction with a putative kinase and coiled-coil-forming protein domains.

Stathmin is a ubiquitous, cytosolic 19-kDa protein, which is phosphorylated on up to four sites in response to many regulatory signals within cells. Its molecular characterization indicates a functional organization including an N-terminal regulatory domain that bears the phosphorylation sites, linked to a putative alpha-helical binding domain predicted to participate in coiled-coil, protein-protein interactions. We therefore proposed that stathmin may play the role of a relay integrating diverse intracellular regulatory pathways; its action on various target proteins would be a function of its combined phosphorylation state. To search for such target proteins, we used the two-hybrid screen in yeast, with stathmin as a "bait." We isolated and characterized four cDNAs encoding protein domains that interact with stathmin in vivo. One of the corresponding proteins was identified as BiP, a member of the hsp70 heat-shock protein family. Another is a previously unidentified, putative serine/threonine kinase, KIS, which might be regulated by stathmin or, more likely, be part of the kinases controlling its phosphorylation state. Finally, two clones code for subdomains of two proteins, CC1 and CC2, predicted to form alpha-helices participating in coiled-coil interacting structures. Their isolation by interaction screening further supports our model for the regulatory function of stathmin through coiled-coil interactions with diverse downstream targets via its presumed alpha-helical binding domain. The molecular and biological characterization of KIS, CC1, and CC2 proteins will give further insights into the molecular functions and mechanisms of action of stathmin as a relay of integrated intracellular regulatory pathways.

Calcium sensing receptor: molecular cloning in rat and localization to nerve terminals.

We have molecularly cloned a calcium sensing receptor (CaSR) from a rat striatal cDNA library. Rat CaSR displays 92% overall homology to its bovine counterpart with seven putative transmembrane domains characteristic of the superfamily of guanine nucleotide-binding proteins and significant homology with the metabotropic glutamate receptors. Northern blot analysis reveals two transcripts in thyroid, kidney, lung, ileum, and pituitary. In brain highest regional expression of the RNA occurs in the hypothalamus and the corpus striatum. Immunohistochemistry reveals discrete punctate localizations throughout the brain that appear to be associated with nerve terminals. No staining is evident in cell bodies of neurons or glia. Cerebral arteries display an intense network of CaSR immunoreactive fibers associated with vessel innervation. CaSR on nerve terminal membranes may regulate neurotransmitter disposition in response to Ca2+ levels in the synaptic space.

Two auxin-responsive domains interact positively to induce expression of the early indoleacetic acid-inducible gene PS-IAA4/5.

The plant growth hormone indole-3-acetic acid (IAA) transcriptionally activates expression of several genes in plants. We have previously identified a 164-bp promoter region (-318 to -154) in the PS-IAA4/5 gene that confers IAA inducibility. Linker-scanning mutagenesis across the region has identified two positive domains: domain A (48 bp; -203 to -156) and domain B (44 bp; -299 to -256), responsible for transcriptional activation of PS-IAA4/5 by IAA. Domain A contains the highly conserved sequence 5'-TGTCCCAT-3' found among various IAA-inducible genes and behaves as the major auxin-responsive element. Domain B functions as an enhancer element which may also contain a less efficient auxin-responsive element. The two domains act cooperatively to stimulate transcription; however, tetramerization of domain A or B compensates for the loss of A or B function. The two domains can also mediate IAA-induced transcription from the heterologous cauliflower mosaic virus 35S promoter (-73 to +1). In vivo competition experiments with icosamers of domain A or B show that the domains interact specifically and with different affinities to low abundance, positive transcription factor(s). A model for transcriptional activation of PS-IAA4/5 by IAA is discussed.

Activation of YRP kinase by v-Src and protein kinase C-mediated signal transduction pathways.

We have previously reported that a serine(threonine) protein kinase that phosphorylates histone H1 in vitro is activated by tyrosine phosphorylation in v-Src-transformed rat 3Y1 fibroblasts. We now refer to this kinase as YRP kinase, for tyrosine-regulated protein kinase. Since YRP kinase may play a role in mediating the growth-stimulatory and morphology-altering effects of v-Src, we have further examined the signal transduction involved in the activation of YRP kinase. Although YRP kinase is constitutively activated in fibroblasts transformed by v-Src, activation of protein kinase C was also found to lead to activation of YRP kinase. Activation of YRP kinase by protein kinase C was found to be potentiated by vanadate treatment or overexpression of c-Src. The activation of YRP kinase by v-Src, however, does not appear to be mediated by protein kinase C, suggesting that YRP kinase can be activated by two separate signal transduction pathways. Transformation of fibroblasts by v-Ras or v-Mil did not result in activation of YRP kinase, indicating that the MAP kinase pathway does not mediate the activation of YRP kinase by v-Src or protein kinase C.

Robust fitting of Zernike polynomials to noisy point clouds defined over connected domains of arbitrary shape

A new method for fitting a series of Zernike polynomials to point clouds defined over connected domains of arbitrary shape defined within the unit circle is presented in this work. The method is based on the application of machine learning fitting techniques by constructing an extended training set in order to ensure the smooth variation of local curvature over the whole domain. Therefore this technique is best suited for fitting points corresponding to ophthalmic lenses surfaces, particularly progressive power ones, in non-regular domains. We have tested our method by fitting numerical and real surfaces reaching an accuracy of 1 micron in elevation and 0.1 D in local curvature in agreement with the customary tolerances in the ophthalmic manufacturing industry.

Lower semicontinuity of the feasible set mapping of linear systems relative to their domains

This paper deals with stability properties of the feasible set of linear inequality systems having a finite number of variables and an arbitrary number of constraints. Several types of perturbations preserving consistency are considered, affecting respectively, all of the data, the left-hand side data, or the right-hand side coefficients.

COMPENDIUM: a text summarisation tool for generating summaries of multiple purposes, domains, and genres

In this paper, we present a Text Summarisation tool, compendium, capable of generating the most common types of summaries. Regarding the input, single- and multi-document summaries can be produced; as the output, the summaries can be extractive or abstractive-oriented; and finally, concerning their purpose, the summaries can be generic, query-focused, or sentiment-based. The proposed architecture for compendium is divided in various stages, making a distinction between core and additional stages. The former constitute the backbone of the tool and are common for the generation of any type of summary, whereas the latter are used for enhancing the capabilities of the tool. The main contributions of compendium with respect to the state-of-the-art summarisation systems are that (i) it specifically deals with the problem of redundancy, by means of textual entailment; (ii) it combines statistical and cognitive-based techniques for determining relevant content; and (iii) it proposes an abstractive-oriented approach for facing the challenge of abstractive summarisation. The evaluation performed in different domains and textual genres, comprising traditional texts, as well as texts extracted from the Web 2.0, shows that compendium is very competitive and appropriate to be used as a tool for generating summaries.