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Die Region Baden-Wurttemberg war in der Vergangenheit Gegenstand zahlreicher Studien und wissenschaftlicher Untersuchungen. In dem vorliegenden Beitrag werden aus soziologischer Sicht die institutionellen Aspekte dieses regionalen Innovationssystems in den Vordergrund gestellt. Hierfur wird auf das neoinstitutionalistische Konzept des organisatorischen Feldes zuruckgegriffen. Die Ausgangsthese ist, dass auch im Zeitalter der modernen, globalisierten Wissensgesellschaft, die den Unternehmen ein hohes Mas an Flexibilitat und Fahigkeit zur kritischen Uberprufung tradierter Wissensinhalte und Verhaltensregeln abverlangt, regionale Institutionen eine wichtige Rolle spielen. Allerdings unterliegen diese Institutionen heutzutage einem erhohten Veranderungsdruck.

Par la voie d’economistes, de gestionnaires, de sociologues et de philosophes, l’echec entrepreneurial nourrit actuellement le debat intellectuel en France et a l’etranger. Le present article aborde ce theme dans une perspective sociologique, situant le phenomene par rapport au contexte social. Longtemps dissimule aux yeux du public, l’echec entrepreneurial devient de plus en plus visible dans les societes europeennes. On commence a lui reconnaitre des vertus. Il reunit des situations tres heterogenes, tout en resultant toujours d’un ensemble de classifications sociales. Face a l’ampleur du phenomene, les politiques publiques sont appelees a s’emparer du sujet et a proposer des solutions telles que la securisation des parcours entrepreneuriaux. Quant aux dirigeants d’entreprises etablies, leur implication a l’egard des jeunes entrepreneurs s’avere cruciale.

Cette recherche étudie le rôle des différents types de proximité dans les dynamiques collaboratives des espaces de coworking. À partir d’entretiens avec des acteurs des espaces de coworking situés dans des villes petites et moyennes, l’étude met en évidence que la proximité géographique ne suffit pas pour générer des collaborations et que d’autres formes de proximité sont nécessaires. Elle révèle aussi que la proximité électronique est importante pour s’affranchir des distances et que la proximité d’opportunité favorise la collaboration interorganisationnelle.

International audience

A l’instar du label « capitale europeenne de la culture », de nombreuses villes europeennes cherchent a redynamiser leur territoire au moyen de l’implantation ou du transfert d’equipements culturels. Ainsi, au cours des deux dernieres decennies, une extraordinaire multiplication de projets de creation de musees a eu lieu, souvent inities en commun par des decideurs politiques et culturels. La question se pose alors de leur ancrage dans un systeme sectoriel d’innovation de la culture, visant autant des nouveautes conceptuelles qu’organisationnelles. L’article presentera une analyse de l’implantation du Centre Pompidou-Metz, demontrant l’importance de la formation et de l’institutionnalisation d’un champ d’action strategique autour du projet, celui-ci produisant a la fois un cadre institutionnel favorable a sa realisation et liant des acteurs a interets variables autour des enjeux communs. Selon l’hypothese avancee, le succes et les retombees economiques a venir dependront fortement de l’etat, de l’evolution et de l’etendue de ce champ.

International audience; The present article analyses the process of the creation of a second Centre Pompidououtside Paris, based on the hypothesis that the success of the project depends on its socialembeddedness. It is argued that a strong indicator of this embeddedness is the emergence of anew strategic action field—crystallizing the meeting and linking of different actors with divergentinterests, but shared understandings about the common stakes. The process starts from acoincidence of cultural, political and economic motives that have their origins in related fields.These different interests are promoted by a few key protagonists who negotiate a preliminary, butstill fragile social order, mixing idealistic ideas and values with political and economic issues.The analysis reveals that social institutions of French culture exert a strong influence, whileinternal governance units in Paris retain significant powers of control. Although new networkconnections seem to form smoothly around the Centre Pompidou-Metz globally in the region, inboth the cultural and the economic domain they have developed quite unevenly.

Le present article traite de l’influence des environnements institutionnels sur la formation et l’utilisation du capital social dans un domaine particulier : celui de la valorisation de la recherche et du transfert des technologies. Il etudie la creation d’entreprises a orientation scientifique ou technologique comme mode de transfert des connaissances, reliant recherche academique et entreprises. A l’instar du systeme d’innovation du Bade-Wurtemberg, caracterise par son riche paysage institutionnel, on remarque la force d’orientation des institutions sur les strategies relationnelles des acteurs. Ainsi, les relations professionnelles se nouent plus facilement la ou il existe des institutions, ces dernieres orientant les comportements des acteurs. Les activites de transfert de technologies et de valorisation des nouvelles connaissances scientifiques sont enracinees dans des reseaux formes a partir des secteurs technologiques bien institutionnalises (ceux des moyennes a hautes technologies). En revanche, les liens professionnels se developpent plus difficilement dans les secteurs faiblement institutionnalises. A ce titre, l’article presente l’exemple du secteur des biotechnologies, qui a fait l’objet d’une politique inedite de developpement de nouveaux clusters regionaux de haute technologie. Celui-ci montre les grandes difficultes de mise en place d’une telle strategie visant le rapprochement et l’intensification des echanges entre recherche et entreprises qui, malgre l’existence d’un fort potentiel de recherche, d’excellence, dans la region, et les soutiens politiques et economiques apportes a cette initiative necessite beaucoup de temps dans sa mise en œuvre.Codes JEL : M13, O14, O13, R11, L1, L2, Z13

Cet article presente une analyse sociologique des echecs organisationnels de jeunes entreprises pionnieres dans des secteurs economiques emergents, a partir d’une etude de cinq cas. Il interprete les projets pionniers comme visant la creation ou la modification d’institutions sociales. Dans cette perspective, l’echec peut resulter de l’incompatibilite avec un ordre institutionnel bien etabli, de l’inexistence d’un tel ordre, ou de l’insuffisance des ressources sociales dont disposent les entrepreneurs.

The autonomously replicating sequence-binding factor 1 (ABF1) from Sacchramoyces cerevisiae is known as a multifunctional DNA binding protein that is involved in transcriptional regulation, DNA-replication, and in restructuring of chromatin via nucleosome remodelling. ABF1 binds to DNA sequences found in ARS elements and in various transcriptional regulatory elements. This led to the early definition of the consensus motive 5′-CGTnnnnnnnGA(G/C)-3′. We have used a SELEX approach to expand and better characterize the DNA sequence requirements of ABF1. Starting from a pool of oligonucleotides randomized at a sequence of 30 nucleotides, we used EMSA to select for sequences with high affinity for ABF1. We obtained the sequences of 106 aptamers after the 15th SELEX round. A 16 nucleotide consensus was derived from this pool by analysis with the motif search programme MEME. Quantitative EMSA experiments verified our experimental approach since binding sequences which were bound with high affinity occurred more often in the pool and resembled the derived consensus to a higher degree. We found DNA sequences that are bound by ABF1 with nearly two-magnitude higher affinity as compared to the hitherto accepted ABF1 consensus sequence. This led us to postulate a strong recognition motive: 5′-TnnCGTnnnnnnTGAT-3′.

Baden-Wurttemberg's innovation system, which is characterised by a high degree of institutionalisation as well as a predominance of incremental innovations and medium technology finds itself in a profound process of transition. The paper discusses whether Baden-Wurttemberg's strength as an industrial district dominated by mature industries is an impediment or rather an asset with regard to developing new promising technological sectors in the region. The paper focuses on two newly emerging sectors: multimedia and biotechnology. Because of the strong orientation of actors in Baden-Wurttemberg to established companies in the core industries and the dominance of established organizational networks there is a lack of stimuli for developing new industries. Thus, institutional innovation and new networks are required to speed up the growth of the new industries. Baden-Wurttemberg can be expected to cope with the transition to the extent that it builds a close connection between its existing sectoral strengths and the new growth industries.

The proteins XPA and RPA are assumed to be involved in primary damage recognition of global genome nucleotide excision repair. XPA as well as RPA have been each reported to specifically bind DNA lesions, and ternary complex formation with damaged DNA has also been shown. We employed fluorescence anisotropy measurements to study the DNA-binding properties of XPA and RPA under true equilibrium conditions using damaged DNA probes carrying a terminal fluorescein modification as a reporter. XPA binds with low affinity and in a strongly salt-dependent manner to DNA containing a 1,3-d(GTG) intrastrand adduct of the anticancer drug cisplatin or a 6-nt mismatch (K(D) = 400 nM) with 3-fold preference for damaged vs undamaged DNA. At near physiological salt conditions binding is very weak (K(D)2 microM). RPA binds to damaged DNA probes with dissociation constants in the range of 20 nM and a nearly 15-fold preference over undamaged DNA. The presence of a cisplatin modification weakens the affinity of RPA for single-stranded DNA by more than 1 order of magnitude indicating that binding to the lesion itself is not a driving force in damage recognition. Our fluorescence anisotropy assays also show that the presence of XPA does not enhance the affinity of RPA for damaged DNA although both proteins interact. In contrast, cooperative binding of XPA and RPA is observed in EMSA. Our results point to a damage-sensing function of the XPA-RPA complex with RPA mediating the important DNA contacts.

In Germany, in the second half of the nineties, a dynamic development of commercial biotechnology took place, undertaken by a considerable number of new firms. The following article deals with the foundation of biotechnological firms in the BioRegion Rhine-Neckar Triangle (with the center in Heidelberg). For a long time, the development of biotechnology in Germany and in the Rhine-Neckar Triangle was hindered by institutional barriers. The main reasons were the narrowing of company strategies of big chemical and pharmaceutical firms, the low impulses to found biotechnology companies out of academic research, the institutional surroundings not aligned to the needs of biotechnology (e.g. no venture capital market) and finally government restraints concerning the specific and particularly regional concentrated support of biotechnology. These particularities are also reflected by the structure of the population of young companies in the Rhine-Neckar Triangle. The main emphasis of biotechnology firms is placed on technological services (research on demand) and platform technologies, different sub-areas of therapeutics research (pharmaceutical basic materials, finding active substances), on the production for the diagnostics industry and on bioinformatics. These companies do not co-operate much, national and international relations predominate. Only a few scientists of regional research institutions want to found a biotechnology firm, although the region seems to attract a certain number of company founders from other regions.

DNA methyltransferases flip their target bases out of the DNA double helix for catalysis. Base flipping ofC 5-cytosine DNA methyltransferases was directly observed in the protein-DNA cocrystal structures of M.HhaI and M.HaeIII. Indirect structural evidence for base flipping of N 6-adenine andN 4-cytosine DNA methyltransferases was obtained by modeling DNA into the three-dimensional structures of M.TaqI and M.PvuII in complex with the cofactor. In addition, biochemical evidence of base flipping was reported for different N 6-adenine DNA methyltransferases. As no protein-DNA cocrystal structure for the related N 6-adenine andN 4-cytosine DNA methyltransferases is available, we used light-induced photochemical cross-linking to identify the binding site of the extrahelical target bases. TheN 6-adenine DNA methyltransferases M.TaqI and M.CviBIII, which both methylate adenine within the double-stranded 5′-TCGA-3′ DNA sequence, were photo-cross-linked to duplex oligodeoxyribonucleotides containing 5-iodouracil at the target position in 50–60% and almost quantitative yield, respectively. Proteolytic fragmentation of the M.CviBIII-DNA complex followed by Edman degradation and electrospray ionization mass spectrometry indicates photo-cross-linking to tyrosine 122. In addition, the mutant methyltransferases M.TaqI/Y108A and M.TaqI/F196A were photo-cross-linked with 6-fold and 2-fold reduced efficiency, respectively, which suggests that tyrosine 108 is the primary site of modification in M.TaqI. Our results indicate a close proximity between the extrahelical target base and tyrosine 122 in M.CviBIII or tyrosine 108 in M.TaqI. As both residues belong to the conserved motif IV ((N/D/S)(P/I)P(Y/F/W)) found in all N 6-adenine andN 4-cytosine DNA as well as inN 6-adenine RNA methyltransferases, a similar spatial relationship between the target bases and the aromatic amino acid residue within motif IV is expected for all these methyltransferases.

Lentiviral transactivator (Tat) proteins are essential for viral replication. Tat proteins of human immunodeficiency virus type 1 and bovine immunodeficiency virus form complexes with their respective RNA targets (Tat responsive element, TAR), and specific binding of the equine anemia virus (EIAV) Tat protein to a target TAR RNA is suggested by mutational analysis of the TAR RNA. Structural data on equine infectious anemia virus Tat protein reveal a helix-loop-helix-turn-helix limit structure very similar to homeobox domains that are known to bind specifically to DNA. Here we report results of gel-shift and footprinting analysis as well as fluorescence and nuclear magnetic resonance spectroscopy experiments that clearly show that EIAV Tat protein binds to DNA specifically at the long terminal repeat Pu.1 (GTTCCTGTTTT) and AP-1 (TGACGCG) sites, and thus suggest a common mechanism for the action of some of the known lentiviral Tat proteins via the AP-1 initiator site. Complex formation with DNA induces specific shifts of the proton NMR resonances originating from amino acids in the core and basic domains of the protein.

Adenylosuccinate synthetase from Saccharomyces cerevisiae was investigated in order to find a structural explanation for its ability to bind specifically to single-stranded ARS elements (autonomously replicating sequences). Using the E. coli enzyme as template, a model for the structure of adenylosuccinate synthetase from S. cerevisiae was generated and subsequently refined by molecular dynamics techniques. The resulting three-dimensional structure offers an explanation for the DNA binding activity of the yeast enzyme by revealing a distinct basic region that is not present in the homologous enzymes from other organisms. The model is also in good agreement with biochemical data available for a mutant protein in which Glycine 252 is replaced by Aspartate. On the basis of the model a significant structural distortion near the catalytic center was predicted for this mutant, corresponding well to the enzymatic inactivity observed. The mutant enzyme shows larger structural fluctuations than the wild-type protein according to the results of two independent molecular dynamics simulations.

A fast breakdown of glycogen is observed in muscles of stress-susceptible pigs leading to pale, soft and exudative (PSE) meat. We report a comparative study of pyruvate kinase from muscles of normal and PSE-prone pigs. Compared with enzyme from normal muscle, pyruvate kinase isolated from PSE-muscle shows a five times lower K(m) for phosphoenol pyruvate and a more than ten times higher k (cat)K (m) value. The pH-dependency of the enzymatic activity is shifted to more acidic values for pyruvate kinase from PSE muscles. According to isoelectric focusing, pyruvate kinase from PSE muscle consists of three isoforms, while only two isoforms are detectable in pyruvate kinase preparations from normal pigs. The various isoforms were isolated by preparative isoelectric focusing and their steady-state properties were compared. Isoform 3, which is found only in PSE muscle, shows a 10-fold higher specific activity, a 30-fold lower K(m) value and a 100-fold increased k (cat)K (m) value for phosphoenol pyruvate compared to isoform 1. The presence of isoform 3 in PSE-muscle appears to be responsible for the high activity of this enzyme under the more acidic conditions prevailing in PSE-muscle. In vitro phosphorylation and dephosphorylation experiments using total enzyme and purified isoenzyme 1 suggest that isoforms 2 and 3 arise from isoform 1 by phosphorylation. Thus protein phosphorylation seems to be responsible for the shift in activity of pyruvate kinase, a key enzyme of glycolysis, under the acidic conditions of PSE-muscles.

Adenylosuccinate synthase (ASS) from Saccharomyces cerevisiae has been shown to bind specifically to the T-rich side of the autonomously replicating sequence (ARS) core consensus sequence [Zeidler, R., Hobert, O., Johannes, L., Faulhammer, H. & Krauss, G. (1993) J. Biol. Chem. 268, 20191–20197]. We have cloned and sequenced the gene for ASS and have studied in detail the enzymatic properties and DNA-binding activity of ASS. The deduced amino acid sequence of the yeast ASS is highly similar to the same enzymes from other sources from which it is however distinguished by its more basic nature. We show that the enzymatic activity of ASS is inhibited in a highly specific manner by the binding of a 44-base DNA oligonucleotide carrying the ARS core consensus sequence. Other nucleic acids, rNTP and dNTP are not able to mimic the specific inhibitory effect. Single-base substitutions in the ARS core sequence lead to a tenfold reduction in inhibition. The inhibition data corroborate the earlier report on the DNA-binding specificity of this enzyme. The homologous enzymes from Escherichia coli and Dictyostelium discoideum do not show specific binding to single-stranded ARS sequences and their enzymatic activity is not influenced by the presence of a 44-base DNA oligonucleotide carrying the ARS core consensus sequence. Treatment of ASS with alkaline phosphatase leads to a loss of DNA binding and to a loss of the inhibition by DNA of the enzymatic activity which suggests that the DNA-binding activity but not the enzymatic activity may be regulated by the phosphorylation status of the protein.

Kaum eine Methode hat die Analytik der Desoxyribonucleinsaure (DNA) so revolutioniert wie die Polymerase-Kettenreaktion (Polymerase Chain Reaction, PCR), eine DNA-Vervielfaltigungstechnik. Sie wird auf so verschiedenen Gebieten wie der Gerichtsmedizin, Gentechnologie, der medizinischen Diagnostik sowie der Palaontologie eingesetzt. Doch damit sind ihre Moglichkeiten noch lange nicht ausgeschopft.

The in vitro fidelity of reverse transcriptase from human immunodeficiency virus type I (HIV-1 RT) upon copying an RNA template was measured using the φXam16 reversion assay. A φX174 sequence harboring the amber 16 codon was cloned into a transcription vector. RNA obtained from transcription by bacteriophage T7 RNA polymerase was used as a template for RNA-directed DNA synthesis by HIV-1 RT. An imbalance of dNTP concentrations during the reverse transcription step served to distinguish between errors that arose from the transcription step and errors from reverse transcription. The frequency of dGTP · U mismatches was determined to be 1 360 , while dGTP · rA mismatches formed at a rate of 1 4600 . These are 20-fold and sevenfold higher, respectively, than the error rates determined for the same sequence with a DNA template. Due to a high background of errors in the RNA template originating from the transcription step only upper limits for the frequency of three other mismatches can be given. The data indicate that the reverse transcription step of the HIV-1 replication cycle contributes significantly to the generation of mutant viruses.

Die Region Baden-Wurttemberg war in der Vergangenheit Gegenstand zahlreicher Studien und wissenschaftlicher Untersuchungen. Die uber mehrere Jahrzehnte anhaltenden wirtschaftlichen Erfolge nach dem Krieg veranlassten eine Reihe von Wissenschaftlern, die Grunde des Erfolgsmodells Baden-Wurttemberg einer genaueren Betrachtung zu unterziehen. In den achtziger Jahren wurde die Region dann durch die Forschungsvorhaben einiger prominenter Wissenschaftler in den Mittelpunkt geruckt. Vor allem die Arbeiten renommierter auslandischer Wissenschaftler stimulierten die Diskussionen uber das badenwurttembergische Wirtschaftsmodell. Es handelte sich insbesondere um Arbeiten zur „flexiblen Spezialisierung“, zu den „industriellen Distrikten“ oder zur „diversifizierten Qualitatsproduktion“ (Sabel et al. 1989; Herrigel 1993; Streeck 1991). Diese Vorhaben stutzten sich zu einem grosen Teil auf Beobachtungen und Analysen von Unternehmen im Grosraum Stuttgart. Die wesentlichen Befunde wurden fur die Region insgesamt verallgemeinert, nicht zuletzt aufgrund der offensichtlichen institutionellen, sozialen, wirtschaftlichen und technologischen Koharenz des regionalen Modells.

Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated protein genes (cas genes) have been suggested to act as an immune system in archaea and bacteria mimicking the eukaryotic RNA interference (RNAi) system. We have investigated the properties of the protein SSO2001 from Sulfolobus solfataricus (Sso) P2, which is part of the cas gene cluster. This study shows that SSO2001 is an endonuclease specifically digesting double-stranded oligonucleotides and preferably cleaving at G:C pairs. Point mutations identify both highly conserved aspartate and glutamate residues as being crucial for the nuclease activity. The catalytic activity shows an optimum at neutral pH and pH 3.

Atomic force microscopy (AFM) has been used to examine the binding properties of the DNA-binding protein ORF80 to DNA. ORF80 is a 9.5 kDa protein that binds site-specifically to double-stranded DNA of the sequence TTAA-N(7)-TTAA. Direct sizing of the protein complexes on DNA fragments from the plasmid pRN1 with AFM shows that the protein ORF80 binds preferentially to two positions. These positions agree well with the ORF80 binding sites determined by footprinting analysis. The measurements allow an estimate of the stoichiometry of the DNA-protein complexes. In contrast to previous results, the single-molecule experiments suggest that only a low number of ORF80 molecules bind to a DNA-binding site.

The thermoacidophilic archaeon Sulfolobus solfataricus P2 encodes three hypothetic endo-β-glucanases, SSO1354, SSO1949 and SSO2534. We cloned and expressed the gene sso1949 encoding the 334 amino acids containing protein SSO1949, which can be classified as a member of glycoside hydrolase family 12. The purified recombinant enzyme hydrolyses carboxymethylcellulose as well as cello-oligomers, with cellobiose and cellotriose as main reaction products. By following the hydrolysis of a fluorescently labelled cellohexaoside under a wide variety of conditions, we show that SSO1949 is a unique extremophilic enzyme. This archaeal enzyme has a pH optimum of approx. pH 1.8 and a temperature optimum of approx. 80 °C. Furthermore, the enzyme is thermostable, with a half-life of approx. 8 h at 80 °C and pH 1.8. The thermostability is strongly pH-dependent. At neutral pH, the thermal inactivation rate is nearly two orders of magnitude higher than at pH 1.8. Homology modelling suggests that the catalytic domain of SSO1949 has a similar fold to other mesophilic, acidophilic and neutral cellulases. The presence of a signal peptide indicates that SSO1949 is a secreted protein, which enables S. solfataricus to use cellulose as an external carbon source. It appears that SSO1949 is perfectly adapted to the extreme environment in solfataric pools. A cellulolytic enzyme with such a combination of stability and activity at high temperatures and low pH has not been described so far and could be a valuable tool for the large-scale hydrolysis of cellulose under acidic conditions.

We report a kinetic and thermodynamic analysis of interactions between ssDNA and replication protein A (RPA) using surface plasmon resonance (SPR) and fluorescence correlation spectroscopy (FCS) at variable temperature. The two methods yield different values for the Gibbs free energy but nearly the same value for the reaction enthalpy of ssDNA-RPA complex formation. The Gibbs free energy was determined by SPR and FCS to be -62.6 and -54.7 kJ/mol, respectively. The values for the reaction enthalpy are -64.4 and -66.5 kJ/mol. It is concluded that the difference in Gibbs free energy measured by the two methods is due to different reaction entropies. The entropic contribution to the free energy at 25 degrees C is -1.8 kJ/mol for SPR and -11.8 kJ/mol for FCS. In SPR, the reaction is restricted to two dimensions because of immobilization of the DNA molecules to the sensor surface. In contrast, FCS is able to follow complex formation without spatial restrictions. In consequence, the reaction entropy determined from SPR experiments is lower than for FCS experiments.

Although DNA replication is a process common in all domains of life, primase and replicative DNA polymerase appear to have evolved independently in the bacterial domain versus the archaeal/eukaryal branch of life. Here, we report on a new type of replication protein that constitutes the first member of the DNA polymerase family E. The protein ORF904, encoded by the plasmid pRN1 from the thermoacidophile archaeon Sulfolobus islandicus, is a highly compact multifunctional enzyme with ATPase, primase and DNA polymerase activity. Recombinant purified ORF904 hydrolyses ATP in a DNA-dependent manner. Deoxynucleotides are preferentially used for the synthesis of primers approximately 8 nucleotides long. The DNA polymerase activity of ORF904 synthesizes replication products of up to several thousand nucleotides in length. The primase and DNA polymerase activity are located in the N-terminal half of the protein, which does not show homology to any known DNA polymerase or primase. ORF904 constitutes a new type of replication enzyme, which could have evolved independently from the eubacterial and archaeal/eukaryal proteins of DNA replication.