Rfc5, a small subunit of replication factor C complex, couples DNA replication and mitosis in budding yeast.

The inhibition of DNA synthesis prevents mitotic entry through the action of the S phase checkpoint. In the yeast Saccharomyces cerevisiae, an essential protein kinase, Spk1/Mec2/Rad53/Sad1, controls the coupling of S phase to mitosis. In an attempt to identify genes that genetically interact with Spk1, we have isolated a temperature-sensitive mutation, rfc5-1, that can be suppressed by overexpression of SPK1. The RFC5 gene encodes a small subunit of replication factor C complex. At the restrictive temperature, rfc5-1 mutant cells entered mitosis with unevenly separated or fragmented chromosomes, resulting in loss of viability. Thus, the rfc5 mutation defective for DNA replication is also impaired in the S phase checkpoint. Overexpression of POL30, which encodes the proliferating cell nuclear antigen, suppressed the replication defect of the rfc5 mutant but not its checkpoint defect. Taken together, these results suggested that replication factor C has a direct role in sensing the state of DNA replication and transmitting the signal to the checkpoint machinery.

Isoform-specific interaction of the alpha1A subunits of brain Ca2+ channels with the presynaptic proteins syntaxin and SNAP-25.

Presynaptic Ca2+ channels are crucial elements in neuronal excitation-secretion coupling. In addition to mediating Ca2+ entry to initiate transmitter release, they are thought to interact directly with proteins of the synaptic vesicle docking/fusion machinery. Here we report isoform-specific, stoichiometric interaction of the BI and rbA isoforms of the alpha1A subunit of P/Q-type Ca2+ channels with the presynaptic membrane proteins syntaxin and SNAP-25 in vitro and in rat brain membranes. The BI isoform binds to both proteins, while only interaction with SNAP-25 can be detected in vitro for the rbA isoform. The synaptic protein interaction ("synprint") site involves two adjacent segments of the intracellular loop connecting domains II and III between amino acid residues 722 and 1036 of the BI sequence. This interaction is competitively blocked by the corresponding region of the N-type Ca2+ channel, indicating that these two channels bind to overlapping regions of syntaxin and SNAP-25. Our results provide a molecular basis for a physical link between Ca2+ influx into nerve terminals and subsequent exocytosis of neurotransmitters at synapses that have presynaptic Ca2+ channels containing alpha1A subunits.

Identification of functional domains and evolution of Tc1-like transposable elements.

Tc1-like transposable elements from teleost fish have been phylogenetically examined to determine the mechanisms involved in their evolution and conserved domains of function. We identified two new functional domains in these elements. The first is a bipartite nuclear localization signal, indicating that transposons can take advantage of the transport machinery of host cells for nuclear uptake of their transposases. The second is a novel combination of a paired domain-related protein motif juxtaposed to a leucine zipper-like domain located in the putative DNA-binding regions of the transposases. This domain coexists with a special inverted repeat structure in certain transposons in such phylogenetically distant hosts as fish and insects. Our data indicate that reassortment of functional domains and horizontal transmission between species are involved in the formation and spread of new types of transposable elements.

Domains with transcriptional regulatory activity within the ALL1 and AF4 proteins involved in acute leukemia.

The ALLI gene, located at chromosome band 11q23, is involved in acute leukemia through a series of chromosome translocations and fusion to a variety of genes, most frequently to A4 and AF9. The fused genes encode chimeric proteins proteins. Because the Drosophila homologue of ALL1, trithorax, is a positive regulator of homeotic genes and acts at the level of transcription, it is conceivable that alterations in ALL1 transcriptional activity may underlie its action in malignant transformation. To begin studying this, we examined the All1, AF4, AF9, and AF17 proteins for the presence of potential transcriptional regulatory domains. This was done by fusing regions of the proteins to the yeast GAL4 DNA binding domain and assaying their effect on transcription of a reporter gene. A domain of 55 residues positioned at amino acids 2829-2883 of ALL1 was identified as a very strong activator. Further analysis of this domain by in vitro mutagenesis pointed to a core of hydrophobic and acidic residues as critical for the activity. An ALL1 domain that repressed transcription of the reporter gene coincided with the sequence homologous to a segment of DNA methyltransferase. An AF4 polypeptide containing residues 480-560 showed strong activation potential. The C-terminal segment of AF9 spanning amino acids 478-568 transactivated transcription of the reporter gene in HeLa but not in NIH 3T3 cells. These results suggest that ALL1, AF4, and probably AF9 interact with the transcriptional machinery of the cell.

Dissociation between changes in cytoplasmic free Ca2+ concentration and insulin secretion as evidenced from measurements in mouse single pancreatic islets.

Simultaneous measurements of cytosolic free Ca2+ concentration and insulin release, in mouse single pancreatic islets, revealed a direct correlation only initially after stimulation with glucose or K+. Later, there is an apparent dissociation between these two parameters, with translocation of alpha and epsilon isoenzymes of protein kinase C to membranes and simultaneous desensitization of insulin release in response to glucose. Recovery of insulin release, without any concomitant changes in cytosolic free Ca2+ concentration, after addition of phorbol 12-myristate 13-acetate, okadaic acid, and forskolin supports the notion that the desensitization process is accounted for by dephosphorylation of key regulatory sites of the insulin exocytotic machinery.

Identification and characterization of a TFIID-like multiprotein complex from Saccharomyces cerevisiae.

Although the mechanisms of transcriptional regulation by RNA polymerase II are apparently highly conserved from yeast to man, the identification of a yeast TATA-binding protein (TBP)-TBP-associated factor (TAFII) complex comparable to the metazoan TFIID component of the basal transcriptional machinery has remained elusive. Here, we report the isolation of a yeast TBP-TAFII complex which can mediate transcriptional activation by GAL4-VP16 in a highly purified yeast in vitro transcription system. We have cloned and sequenced the genes encoding four of the multiple yeast TAFII proteins comprising the TBP-TAFII multisubunit complex and find that they are similar at the amino acid level to both human and Drosophila TFIID subunits. Using epitope-tagging and immunoprecipitation experiments, we demonstrate that these genes encode bona fide TAF proteins and show that the yeast TBP-TAFII complex is minimally composed of TBP and seven distinct yTAFII proteins ranging in size from M(r) = 150,000 to M(r) = 25,000. In addition, by constructing null alleles of the cloned TAF-encoding genes, we show that normal function of the TAF-encoding genes is essential for yeast cell viability.

Interactions between the retinoid X receptor and a conserved region of the TATA-binding protein mediate hormone-dependent transactivation.

The retinoid X receptor (RXR) participates in a wide array of hormonal signaling pathways, either as a homodimer or as a heterodimer, with other members of the steroid and thyroid hormone receptor superfamily. In this report the ligand-dependent transactivation function of RXR has been characterized, and the ability of RXR to interact with components of the basal transcription machinery has been examined. In vivo and in vitro experiments indicate the RXR ligand-binding domain makes a direct, specific, and ligand-dependent contact with a highly conserved region of the TATA-binding protein. The ability of mutations that reduce ligand-dependent transcription by RXR to disrupt the RXR-TATA-binding protein interaction in vivo and in vitro suggests that RXR makes direct contact with the basal transcription machinery to achieve activation.

Expression of an Arabidopsis cryptochrome gene in transgenic tobacco results in hypersensitivity to blue, UV-A, and green light.

The Arabidopsis HY4 gene, required for blue-light-induced inhibition of hypocotyl elongation, encodes a 75-kDa flavoprotein (CRY1) with characteristics of a blue-light photoreceptor. To investigate the mechanism by which this photoreceptor mediates blue-light responses in vivo, we have expressed the Arabidopsis HY4 gene in transgenic tobacco. The transgenic plants exhibited a short-hypocotyl phenotype under blue, UV-A, and green light, whereas they showed no difference from the wild-type plant under red/far-red light or in the dark. This phenotype was found to cosegregate with overexpression of the HY4 transgene and to be fluence dependent. We concluded that the short-hypocotyl phenotype of transgenic tobacco plants was due to hypersensitivity to blue, UV-A, and green light, resulting from over-expression of the photoreceptor. These observations are consistent with the broad action spectrum for responses mediated by this cryptochrome in Arabidopsis and indicate that the machinery for signal, transduction required by the CRY1 protein is conserved among different plant species. Furthermore, the level of these photoresponses is seen to be determined by the cellular concentration of this photoreceptor.

Unsaturation of the membrane lipids of chloroplasts stabilizes the photosynthetic machinery against low-temperature photoinhibition in transgenic tobacco plants.

Using tobacco plants that had been transformed with the cDNA for glycerol-3-phosphate acyltransferase, we have demonstrated that chilling tolerance is affected by the levels of unsaturated membrane lipids. In the present study, we examined the effects of the transformation of tobacco plants with cDNA for glycerol-3-phosphate acyltransferase from squash on the unsaturation of fatty acids in thylakoid membrane lipids and the response of photosynthesis to various temperatures. Of the four major lipid classes isolated from the thylakoid membranes, phosphatidylglycerol showed the most conspicuous decrease in the level of unsaturation in the transformed plants. The isolated thylakoid membranes from wild-type and transgenic plants did not significantly differ from each other in terms of the sensitivity of photosystem II to high and low temperatures and also to photoinhibition. However, leaves of the transformed plants were more sensitive to photoinhibition than those of wild-type plants. Moreover, the recovery of photosynthesis from photoinhibition in leaves of wild-type plants was faster than that in leaves of the transgenic tobacco plants. These results suggest that unsaturation of fatty acids of phosphatidylglycerol in thylakoid membranes stabilizes the photosynthetic machinery against low-temperature photoinhibition by accelerating the recovery of the photosystem II protein complex.

Molecular cloning and expression of the 32-kDa subunit of human TFIID reveals interactions with VP16 and TFIIB that mediate transcriptional activation.

Transcription factor TFIID consists of TATA binding protein (TBP) and at least eight TBP-associated factors (TAFs). As TAFs are required for activated but not basal transcription, we have proposed that TAFs act as coactivators to mediate signals between activators and the basal transcription machinery. Here we report the cloning, expression, and biochemical characterization of the 32-kDa subunit of human (h) TFIID, termed hTAFII32. We find that hTAFII32 is the human homologue of Drosophila TAFII40. In vitro protein-protein interaction assays reveal that as observed with Drosophila TAFII40, hTAFII32 interacts with the C-terminal 39-amino acid activation domain of the acidic transactivator viral protein 16 (VP16) as well as with the general transcription factor TFIIB. Moreover, a partial recombinant TFIID complex containing hTAFII32 was capable of mediating in vitro transcriptional activation by the VP16 activation domain. These findings indicate that specific activator-coactivator interactions have been conserved between human and Drosophila and provide additional support for the function of these interactions in mediating transcriptional activation.

Protease footprinting reveals a surface on transcription factor TFIIB that serves as an interface for activators and coactivators.

Transcriptional stimulation by the model activator GAL4-VP16 (a chimeric protein consisting of the DNA-binding domain of the yeast activator GAL4 and the acidic activation domain of the herpes simplex virus protein VP16) involves a series of poorly understood protein-protein interactions between the VP16 activation domain and components of the RNA polymerase II general transcription machinery. One of these interactions is the VP16-mediated binding and recruitment of transcription factor TFIIB. However, TATA box-binding protein (TBP)-associated factors (TAFs), or coactivators, are required for this interaction to culminate in productive transcription complex assembly, and one such TAF, Drosophila TAF40, reportedly forms a ternary complex with VP16 and TFIIB. Due to TFIIB's central role in gene activation, we sought to directly visualize the surfaces of this protein that mediate formation of the ternary complex. We developed an approach called protease footprinting in which the broad-specificity proteases chymotrypsin and alkaline protease were used to probe binding of 32P-end-labeled TFIIB to GAL4-VP16 or TAF40. Analysis of the cleavage products revealed two regions of TFIIB protected by VP16 from protease attack, one of which overlapped with a region protected by TAF40. The close proximity of the VP16 and TAF40 binding sites on the surface of TFIIB suggests that this region could act as a regulatory interface mediating the effects of activators and coactivators on transcription complex assembly.

Omics for Investigating Chitosan as an Antifungal and Gene Modulator

Chitosan is a biopolymer with a wide range of applications. The use of chitosan in clinical medicine to control infections by fungal pathogens such as Candida spp. is one of its most promising applications in view of the reduced number of antifungals available. Chitosan increases intracellular oxidative stress, then permeabilizes the plasma membrane of sensitive filamentous fungus Neurospora crassa and yeast. Transcriptomics reveals plasma membrane homeostasis and oxidative metabolism genes as key players in the response of fungi to chitosan. A lipase and a monosaccharide transporter, both inner plasma membrane proteins, and a glutathione transferase are main chitosan targets in N. crassa. Biocontrol fungi such as Pochonia chlamydosporia have a low content of polyunsaturated free fatty acids in their plasma membranes and are resistant to chitosan. Genome sequencing of P. chlamydosporia reveals a wide gene machinery to degrade and assimilate chitosan. Chitosan increases P. chlamydosporia sporulation and enhances parasitism of plant parasitic nematodes by the fungus. Omics studies allow understanding the mode of action of chitosan and help its development as an antifungal and gene modulator.

Entre économie de la fonctionnalité et monopole : la United Shoe Machinery Company dans la première moitié du XXe siècle

L’histoire de la United Shoe Machinery Company (USMC) montre que la réalité ne s’adapte pas toujours aux simplifications de la théorie. Comme le signale l’économie de la fonctionnalité, la stratégie de l’entreprise de vendre l’usage plutôt que la machine fournit plusieurs avantages importants, mais elle a également contribué au fait que les usines de chaussures subissent une véritable dépendance technologique de cette compagnie et au fait que l’USMC soit parvenue à une domination monopolistique du marché. D’autre part, en remettant en cause les rudiments généraux de la théorie économique néoclassique, cette position de monopole n’a pas empêché que l’entreprise ait un fonctionnement efficace et ait facilité la modernisation technologique de l’industrie de la chaussure, aux États-Unis et dans d’autres pays.

Rehabilitating, repairing and restoring against demolishing, tearing down and destroying: the recovery of the military fort for the Santa Pola town museum

This communication develops the process of interventions of the Renaissance fortress of a new plant built in 1554–57 in Santa Pola. It is one of the earliest examples built with reference to military architecture theoretical treaties (XV–XVI) and best preserved. The study runs its own story from its initial military use, through the use of civil equipment until the final cultural and Museum Center. First, the project of Italian origin is examined and its use as barracks for troops for a duration of three centuries (1557–1850), pointing out the architectural constants of war machinery in a defense position and its origin as a rainwater collector and cistern: a perfect square with two bastions in which a plan of the uprising is preserved (1778). Secondly, we study the changes in the mentioned architecture throughout a century and a half (1850–1990) after its change of ownership (from the state to the municipality), and as a result of the new use as a city hall and public endowment: a market and health and leisure centre, which meant the demolition of defensive elements and the opening up to the outside of the inner parade ground. And thirdly, the new transfer of the municipal offices brings in the beginning of a project of transformations (1990–2015) that retrieves the demolished elements at the same time as it assigns the entire fort for a cultural centre: exhibition, research and history museum, promoting the identity between the citizens and the building which stands in the foundations of their city. The conclusions take us through an interesting route that goes from the approach of defensive tactics, its use as administrative headquarters to the current cultural policy of preservation. In addition, all the known plans of the fort are recovered (of military, civil and cultural use), some unpublished, as well as the project of the North wing that has guided the last operation and which has been set as a pattern of reference.

Effects of Economic Factors on Adoption of Robotics and Consequences of Automation for Productivity Growth of Dairy Farms. Factor Markets Working Paper No. 32, December 2012

In the long term, productivity and especially productivity growth are necessary conditions for the survival of a farm. This paper focuses on the technology choice of a dairy farm, i.e. the choice between a conventional and an automatic milking system. Its aim is to reveal the extent to which economic rationality explains investing in new technology. The adoption of robotics is further linked to farm productivity to show how capital-intensive technology has affected the overall productivity of milk production. The empirical analysis applies a probit model and an extended Cobb-Douglas-type production function to a Finnish farm-level dataset for the years 2000–10. The results show that very few economic factors on a dairy farm or in its economic environment can be identified to affect the switch to automatic milking. Existing machinery capital and investment allowances are among the significant factors. The results also indicate that the probability of investing in robotics responds elastically to a change in investment aids: an increase of 1% in aid would generate an increase of 2% in the probability of investing. Despite the presence of non-economic incentives, the switch to robotic milking is proven to promote productivity development on dairy farms. No productivity growth is observed on farms that keep conventional milking systems, whereas farms with robotic milking have a growth rate of 8.1% per year. The mean rate for farms that switch to robotic milking is 7.0% per year. The results show great progress in productivity growth, with the average of the sector at around 2% per year during the past two decades. In conclusion, investments in new technology as well as investment aids to boost investments are needed in low-productivity areas where investments in new technology still have great potential to increase productivity, and thus profitability and competitiveness, in the long run.