Cloning and characterization of the full length cDNA and promoter of mouse tissue transglutaminase

Transglutaminases are a family of enzymes that catalyze the covalent cross-linking of proteins through the formation of $\varepsilon$-($\gamma$-glutaminyl)-lysyl isopeptide bonds. Tissue transglutaminase (Tgase) is an intracellular enzyme which is expressed in terminally differentiated and senescent cells and also in cells undergoing apoptotic cell death. To characterize this enzyme and examine its relationship with other members of the transglutaminase family, cDNAs, the first two exons of the gene and 2 kb of the 5$\sp\prime$ flanking region, including the promoter, were isolated. The full length Tgase transcript consists of 66 bp of 5$\sp\prime$-UTR (untranslated) sequence, an open reading frame which encodes 686 amino acids and 1400 bp of 3$\sp\prime$-UTR sequence. Alignment of the deduced Tgase protein sequence with that of other transglutaminases revealed regions of strong homology, particularly in the active site region.^ The Tgase cDNA was used to isolate and characterize a genomic clone encompassing the 5$\sp\prime$ end of the mouse Tgase gene. The transcription start site was defined using genomic and cDNA clones coupled with S1 protection analysis and anchored PCR. This clone includes 2.3 kb upstream of the transcription start site and two exons that contain the first 256 nucleotides of the mouse Tgase cDNA sequence. The exon intron boundaries have been mapped and compared with the exon intron boundaries of three members of the transglutaminase family: human factor XIIIa, the human keratinocyte transglutaminase and human erythrocyte band 4.1. Tissue Tgase exon II is similar to comparable exons of these genes. However, exon I bears no resemblance with any of the other transglutaminase amino terminus exons.^ Previous work in our laboratory has shown that the transcription of the Tgase gene is directly controlled by retinoic acid and retinoic acid receptors. To identify the region of the Tgase gene responsible for regulating its expression, fragments of the Tgase promoter and 5$\sp\prime$-flanking region were cloned into the chloramphenicol actetyl transferase (CAT) reporter constructs. Transient transfection experiments with these constructs demonstrated that the upstream region of Tgase is a functional promoter which contains a retinoid response element within a 1573 nucleotide region spanning nucleotides $-$252 to $-$1825. ^

Regulation of tyrosine hydroxylase gene expression by mRNA stabilization

Tyrosine hydroxylase (TH) expression increases in adrenal chromaffin cells treated with the nicotinic agonist, dimethylphenylpiperazinium (DMPP; 1 μM). We are using this response as a model of the changes in TH level that occur during increased cholinergic neural activity. Here we report a 4-fold increase in TH mRNA half-life in DMPP-treated chromaffin cells that is apparent when using a pulse-chase analysis to measure TH mRNA half-life. No increase is apparent using actinomycin D to measure half-life, indicating a requirement for ongoing transcription. Characterization of protein binding to the TH 3′UTR using RNA electro-mobility shift assays show the presence of two complexes both of which are increased by DMPP-treatment. The faster migrating complex (FMC) increases 2.5-fold and the slower migrating complex (SMC) increases 1.5-fold. Separation of UV crosslinked RNA-protein complexes on SDS polyacrylamide gels shows FMC to contain a single protein whereas SMC contains two proteins. Northwesterns yielded similar results. Transfection studies reveal an increase in expression of the full-length TH transcript due to DMPP-treatment similar to that of endogenous TH mRNA. This finding suggests the increased expression is due primarily to mRNA stabilization. Transfection of luciferase reporter constructs containing regions of the TH 3′UTR reveal only the full-length 3′UTR influenced the expression level of reporter transcripts. ^

Efficient local unfolding with ancestor stacks for full prolog

The integration of powerful partial evaluation methods into practical compilers for logic programs is still far from reality. This is related both to 1) efficiency issues and to 2) the complications of dealing with practical programs. Regarding efnciency, the most successful unfolding rules used nowadays are based on structural orders applied over (covering) ancestors, i.e., a subsequence of the atoms selected during a derivation. Unfortunately, maintaining the structure of the ancestor relation during unfolding introduces significant overhead. We propose an efficient, practical local unfolding rule based on the notion of covering ancestors which can be used in combination with any structural order and allows a stack-based implementation without losing any opportunities for specialization. Regarding the second issue, we propose assertion-based techniques which allow our approach to deal with real programs that include (Prolog) built-ins and external predicates in a very extensible manner. Finally, we report on our implementation of these techniques in a practical partial evaluator, embedded in a state of the art compiler which uses global analysis extensively (the Ciao compiler and, specifically, its preprocessor CiaoPP). The performance analysis of the resulting system shows that our techniques, in addition to dealing with practical programs, are also significantly more efficient in time and somewhat more efficient in memory than traditional tree-based implementations.

ACE: And/or-parallel copying-based execution of logic programs

In this paper we present a novel execution model for parallel implementation of logic programs which is capable of exploiting both independent and-parallelism and or-parallelism in an efficient way. This model extends the stack copying approach, which has been successfully applied in the Muse system to implement or-parallelism, by integrating it with proven techniques used to support independent and-parallelism. We show how all solutions to non-deterministic andparallel goals are found without repetitions. This is done through recomputation as in Prolog (and in various and-parallel systems, like &-Prolog and DDAS), i.e., solutions of and-parallel goals are not shared. We propose a scheme for the efficient management of the address space in a way that is compatible with the apparently incompatible requirements of both and- and or-parallelism. We also show how the full Prolog language, with all its extra-logical features, can be supported in our and-or parallel system so that its sequential semantics is preserved. The resulting system retains the advantages of both purely or-parallel systems as well as purely and-parallel systems. The stack copying scheme together with our proposed memory management scheme can also be used to implement models that combine dependent and-parallelism and or-parallelism, such as Andorra and Prometheus.

A documentation generator for logic programming systems

We describe lpdoc, a tool which generates documentation manuals automatically from one or more logic program source files, written in ISO-Prolog, Ciao, and other (C)LP languages. It is particularly useful for documenting library modules, for which it automatically generates a rich description of the module interface. However, it can also be used quite successfully to document full applications. The documentation can be generated in many formats including t e x i n f o, dvi, ps, pdf, inf o, html/css, Unix nrof f/man, Windows help, etc., and can include bibliographic citations and images, lpdoc can also genérate "man" pages (Unix man page format), nicely formatted plain ascii "readme" files, installation scripts useful when the manuals are included in software distributions, brief descriptions in html/css or inf o formats suitable for inclusión in on-line Índices of manuals, and even complete WWW and inf o sites containing on-line catalogs of documents and software distributions. A fundamental advantage of using lpdoc is that it helps maintaining a true correspondence between the program and its documentation, and also identifying precisely to what versión of the program a given printed manual corresponds. The quality of the documentation generated can be greatly enhanced by including within the program text assertions (declarations with types, modes, etc. ...) for the predicates in the program, and machine-readable comments. These assertions and comments are written using the Ciao system assertion language. A simple compatibility library allows conventional (C)LP systems to ignore these assertions and comments and treat normally programs documented in this way. The lpdoc manual, all other Ciao system manuals, and most of this paper, are generated by lpdoc.

Photonic Logic Based on Molecular Reorientation of Nematic Liquid Crystals

A new type of photonic logic, based on the use of nematic liquid crystals is proposed. The system takes advantage of the refractive-index changes induced by laser beams. Examples of AND, OR and NOR functions are presented.

Coder/decoder with an optical programmable logic cell

A major research area is the representation of knowledge for a given application in a compact manner such that desired information relating to this knowledge is easily recoverable. A complicated procedure may be required to recover the information from the stored representation and convert it back to usable form. Coder/decoder are the devices dedicated to that task. In this paper the capabilities that an Optical Programmable Logic Cell offers as a basic building block for coding and decoding are analyzed. We have previously published an Optically Programmable Logic Cells (OPLC), for applications as a chaotic generator or as basic element for optical computing. In optical computing previous studies these cells have been analyzed as full-adder units, being this element a basic component for the arithmetic logic structure in computing. Another application of this unit is reported in this paper. Coder and decoder are basic elements in computers, for example, in connections between processors and memory addressing. Moreover, another main application is the generation of signals for machine controlling from a certain instruction. In this paper we describe the way to obtain a coder/decoder with the OPLC and which type of applications may be the best suitable for this type of cell.

The TRANSPLANTA collection of Arabidopsis lines: a resource for functional analysis of transcription factors based on their conditional over-expression

Transcription factors (TFs) are key regulators of gene expression in all organisms. In eukaryotes, TFs are often represented by functionally redundant members of large gene families. Overexpression might prove a means to unveil the biological functions of redundant TFs; however, constitutive overexpression of TFs frequently causes severe developmental defects, preventing their functional characterization. Conditional overexpression strategies help to overcome this problem. Here, we report on the TRANSPLANTA collection of Arabidopsis lines, each expressing one of 949 TFs under the control of a β–estradiol-inducible promoter. Thus far, 1636 independent homozygous lines, representing an average of 2.6 lines for every TF, have been produced for the inducible expression of 634 TFs. Along with a GUS-GFP reporter, randomly selected TRANSPLANTA lines were tested and confirmed for conditional transgene expression upon β–estradiol treatment. As a proof of concept for the exploitation of this resource, β–estradiol-induced proliferation of root hairs, dark-induced senescence, anthocyanin accumulation and dwarfism were observed in lines conditionally expressing full-length cDNAs encoding RHD6, WRKY22, MYB123/TT2 and MYB26, respectively, in agreement with previously reported phenotypes conferred by these TFs. Further screening performed with other TRANSPLANTA lines allowed the identification of TFs involved in different plant biological processes, illustrating that the collection is a powerful resource for the functional characterization of TFs. For instance, ANAC058 and a TINY/AP2 TF were identified as modulators of ABA-mediated germination potential, and RAP2.10/DEAR4 was identified as a regulator of cell death in the hypocotyl–root transition zone. Seeds of TRANSPLANTA lines have been deposited at the Nottingham Arabidopsis Stock Centre for further distribution.

La paradoja de Mies : las simetrías invisibles a través del Pabellón de Barcelona

El vínculo de Mies van der Rohe con la simetría es un invariante que se intuye en toda su obra más allá de su pretendida invisibilidad. Partiendo del proyecto moderno como proceso paradójico, que Mies lo expresa en sus conocidos aforismos, como el célebre “menos es más”, la tesis pretende ser una aproximación a este concepto clave de la arquitectura a través de una de sus obras más importantes: el Pabellón Alemán para la Exposición Universal de 1.929 en Barcelona. Ejemplo de planta asimétrica según Bruno Zevi y un “auténtico caballo de Troya cargado de simetrías” como lo definió Robin Evans. El Pabellón representó para la modernidad, la culminación de una década que cambió radicalmente la visión de la arquitectura hasta ese momento, gracias al carácter inclusivo de lo paradójico y las innumerables conexiones que hubo entre distintas disciplinas, tan antagónicas, como el arte y la ciencia. De esta última, se propone una definición ampliada de la simetría como principio de equivalencia entre elementos desde la invariancia. En esta definición se incorpora el sentido recogido por Lederman como “expresión de igualdad”, así como el planteado por Hermann Weyl en su libro Simetría como “invariancia de una configuración bajo un grupo de automorfismos” (libro que Mies tenía en su biblioteca privada). Precisamente para Weyl, el espacio vacío tiene un alto grado de simetría. “Cada punto es igual que los otros, y en ninguno hay diferencias intrínsecas entre las diversas direcciones." A partir de este nuevo significado, la obra de Mies adquiere otro sentido encaminado a la materialización de ese espacio, que él pretendía que “reflejase” el espíritu de la época y cuya génesis se postula en el Teorema de Noether que establece que “por cada simetría continua de las leyes físicas ha de existir una ley de conservación”. Estas simetrías continúas son las simetrías invisibles del espacio vacío que se desvelan “aparentemente” como oposición a las estructuras de orden de las simetrías de la materia, de lo lleno, pero que participan de la misma lógica aporética miesiana, de considerarlo otro material, y que se definen como: (i)limitado, (in)grávido, (in)acabado e (in)material. Finalmente, una paradoja más: El “espacio universal” que buscó Mies, no lo encontró en América sino en este pabellón. Como bien lo han intuido arquitectos contemporáneos como Kazuyo Sejima + Ryue Nishizawa (SANAA) legítimos herederos del maestro alemán. ABSTRACT The relationship between Mies van der Rohe with the symmetry is an invariant which is intuited in his entire work beyond his intentional invisibility. Based on the modern project as a paradoxical process, which Mies expresses in his aphorisms know as the famous “less is more”, the thesis is intended to approach this key concept in architecture through one of his most important works: The German Pavilion for the World Expo in 1929 in Barcelona, an example of asymmetric floor according to Bruno Zevi and a “real Trojan horse loaded with symmetries”. As defined by Robin Evans. For modernity, this Pavilion represented the culmination of a decade which radically changed the vision of architecture so far, thanks to the inclusive character of the paradoxical and the innumerable connections that there were amongst the different disciplines, as antagonistic as Art and Science. Of the latter, an expanded definition of symmetry is proposed as the principle of equivalence between elements from the invariance. Incorporated into this definition is the sense defined by Leterman as “expression of equality,” like the one proposed by Hermann Weyl in his book Symmetry as “configuration invariance under a group of automorphisms” (a book which Mies had in his private library). Precisely for Weyl, the empty space has a high degree of symmetry. “Each point is equal to the other, and in none are there intrinsic differences among the diverse directions.” Based on this new meaning, Mies’ work acquires another meaning approaching the materialization of that space, which he intended to “reflect” the spirit of the time and whose genesis is postulated in the Noether’s theorem which establishes that “for every continuous symmetry of physical laws, there must be a law of conservation.” These continuous symmetries are the invisible empty space symmetries which reveal themselves “apparently” as opposition to the structures of matter symmetries, of those which are full, but which participate in the same Mies aporetic logic, if deemed other material, and which is defined as (un)limited, weight(less), (un)finished and (im)material. Finally, one more paradox: the “universal space” which Mies search for, he did not find it in America, but at this pavilion, just as the contemporary architects like Kazuyo Sejima + Ryue Nishizawa (SANAA) rightfully intuited, as legitimate heirs of the German master.

Ectopic expression of the minimal whiE polyketide synthase generates a library of aromatic polyketides of diverse sizes and shapes

The single recombinant expressing the Streptomyces coelicolor minimal whiE (spore pigment) polyketide synthase (PKS) is uniquely capable of generating a large array of well more than 30 polyketides, many of which, so far, are novel to this recombinant. The characterized polyketides represent a diverse set of molecules that differ in size (chain length) and shape (cyclization pattern). This combinatorial biosynthetic library is, by far, the largest and most complex of its kind described to date and indicates that the minimal whiE PKS does not independently control polyketide chain length nor dictate the first cyclization event. Rather, the minimal PKS enzyme complex must rely on the stabilizing effects of additional subunits (i.e., the cyclase whiE-ORFVI) to ensure that the chain reaches the full 24 carbons and cyclizes correctly. This dramatic loss of control implies that the growing polyketide chain does not remain enzyme bound, resulting in the spontaneous cyclization of the methyl terminus. Among the six characterized dodecaketides, four different first-ring cyclization regiochemistries are represented, including C7/C12, C8/C13, C10/C15, and C13/C15. The dodecaketide TW93h possesses a unique 2,4-dioxaadamantane ring system and represents a new structural class of polyketides with no related structures isolated from natural or engineered organisms, thus supporting the claim that engineered biosynthesis is capable of producing novel chemotypes.

Soldier caste-specific gene expression in the mandibular glands of Hodotermopsis japonica (Isoptera: Termopsidae)

Although “polymorphic castes” in social insects are well known as one of the most important phenomena of polyphenism, few studies of caste-specific gene expressions have been performed in social insects. To identify genes specifically expressed in the soldier caste of the Japanese damp-wood termite Hodotermopsis japonica, we employed the differential-display method using oligo(dT) and arbitrary primers, compared mRNA from the heads of mature soldiers and pseudergates (worker caste), and identified a clone (PCR product) 329 bp in length termed SOL1. Northern blot analysis showed that the SOL1 mRNA is about 1.0 kb in length and is expressed specifically in mature soldiers, but not in pseudergates, even in the presoldier induction by juvenile hormone analogue, suggesting that the product is specific for terminally differentiated soldiers. By using the method of 5′- and 3′-rapid amplification of cDNA ends, we isolated the full length of SOL1 cDNA, which contained an ORF with a putative signal peptide at the N terminus. The sequence showed no significant homology with any other known protein sequences. In situ hybridization analysis showed that SOL1 is expressed specifically in the mandibular glands. These results strongly suggest that the SOL1 gene encodes a secretory protein specifically synthesized in the mandibular glands of the soldiers. Histological observations revealed that the gland actually develops during the differentiation into the soldier caste.

Cloning and expression of rat 25-hydroxyvitamin D3-1α-hydroxylase cDNA

A full-length cDNA for the rat kidney mitochondrial cytochrome P450 mixed function oxidase, 25-hydroxyvitamin D3-1α-hydroxylase (P4501α), was cloned from a vitamin D-deficient rat kidney cDNA library and subcloned into the mammalian expression vector pcDNA 3.1(+). When P4501α cDNA was transfected into COS-7 transformed monkey kidney cells, they expressed 25-hydroxyvitamin D3-1α-hydroxylase activity. The sequence analysis showed that P4501α was of 2,469 bp long and contained an ORF encoding 501 amino acids. The deduced amino acid sequence showed a 53% similarity and 44% identity to the vitamin D3-25-hydroxylase (CYP27), whereas it has 42.6% similarity and 34% identity with the 25-hydroxyvitamin D3-24-hydroxylase (CYP24). Thus, it composes a new subfamily of the CYP27 family. Further, it is more closely related to the CYP27 than to the CYP24. The expression of P4501α mRNA was greatly increased in the kidney of vitamin D-deficient rats. In rats with the enhanced renal production of 1α,25-dihydroxyvitamin D3 (rats fed a low Ca diet), P4501α mRNA was greatly increased in the renal proximal convoluted tubules.

Tsetse thrombin inhibitor: Bloodmeal-induced expression of an anticoagulant in salivary glands and gut tissue of Glossina morsitans morsitans

The tsetse thrombin inhibitor, a potent and specific low molecular mass (3,530 Da) anticoagulant peptide, was purified previously from salivary gland extracts of Glossina morsitans morsitans (Diptera: Glossinidae). A 303-bp coding sequence corresponding to the inhibitor has now been isolated from a tsetse salivary gland cDNA library by using degenerate oligonucleotide probes. The full-length cDNA contains a 26-bp untranslated segment at its 5′ end, followed by a 63-bp sequence corresponding to a putative secretory signal peptide. A 96-bp segment codes for the mature tsetse thrombin inhibitor, whose predicted molecular weight matches that of the purified native protein. Based on its lack of homology to any previously described family of molecules, the tsetse thrombin inhibitor appears to represent a unique class of naturally occurring protease inhibitors. Recombinant tsetse thrombin inhibitor expressed in Escherichia coli and the chemically synthesized peptide are both substantially less active than the purified native protein, suggesting that posttranslational modification(s) may be necessary for optimal inhibitory activity. The tsetse thrombin inhibitor gene, which is present as a single copy in the tsetse genome, is expressed at high levels in salivary glands and midguts of adult tsetse flies, suggesting a possible role for the anticoagulant in both feeding and processing of the bloodmeal.

Binding of factor VIIa to tissue factor induces alterations in gene expression in human fibroblast cells: Up-regulation of poly(A) polymerase

Tissue factor (TF) is the cellular receptor for an activated form of clotting factor VII (VIIa) and the binding of factor VII(a) to TF initiates the coagulation cascade. Sequence and structural patterns extracted from a global alignment of TF confers homology with interferon receptors of the cytokine receptor super family. Several recent studies suggested that TF could function as a genuine signal transducing receptor. However, it is unknown which biological function(s) of cells are altered upon the ligand, VIIa, binding to TF. In the present study, we examined the effect of VIIa binding to cell surface TF on cellular gene expression in fibroblasts. Differential mRNA display PCR technique was used to identify transcriptional changes in fibroblasts upon VIIa binding to TF. The display showed that VIIa binding to TF either up or down-regulated several mRNA species. The differential expression of one such transcript, VIIa-induced up-regulation, was confirmed by Northern blot analysis. Isolation of a full-length cDNA corresponding to the differentially expressed transcript revealed that VIIa-up-regulated gene was poly(A) polymerase. Northern blot analysis of various carcinomas and normal human tissues revealed an over expression of PAP in cancer tissues. Enhanced expression of PAP upon VIIa binding to tumor cell TF may potentially play an important role in tumor metastasis.

Gap Junctional Communication Modulates Gene Expression in Osteoblastic Cells

Bone-forming cells are organized in a multicellular network interconnected by gap junctions. In these cells, gap junctions are formed by connexin43 (Cx43) and connexin45 (Cx45). Cx43 gap junctions form pores that are more permeable to negatively charged dyes such as Lucifer yellow and calcein than are Cx45 pores. We studied whether altering gap junctional communication by manipulating the relative expression of Cx43 and Cx45 affects the osteoblast phenotype. Transfection of Cx45 in cells that express primarily Cx43 (ROS 17/2.8 and MC3T3-E1) decreased both dye transfer and expression of osteocalcin (OC) and bone sialoprotein (BSP), genes pivotal to bone matrix formation and calcification. Conversely, transfection of Cx43 into cells that express predominantly Cx45 (UMR 106–01) increased both cell coupling and expression of OC and BSP. Transient cotransfection of promoter–luciferase constructs and connexin expression vectors demonstrated that OC and BSP gene transcription was down-regulated by Cx45 cotransfection in ROS 17/2.8 and MC3T3-E1 cells, in association with a decrease in dye coupling. Conversely, cotransfection of Cx43 in UMR 106–01 cells up-regulated OC and BSP gene transcription. Activity of other less specific osteoblast promoters, such as osteopontin and osteonectin, was less sensitive to changes in gap junctional communication. Thus, altering gap junctional permeability by manipulating the expression of Cx43 and Cx45 in osteoblastic cells alters transcriptional activity of osteoblast-specific promoters, presumably via modulation of signals that can diffuse from cell to cell. A communicating intercellular network is required for the full elaboration of a differentiated osteoblastic phenotype.