Regulation of gene expression in the dinoflagellate Lingulodinium polyedrum

Les dinoflagellés sont des eucaryotes unicellulaires que l’on retrouve autant en eau douce qu’en milieu marin. Ils sont particulièrement connus pour causer des fleurs d’algues toxiques nommées ‘marée-rouge’, ainsi que pour leur symbiose avec les coraux et pour leur importante contribution à la fixation du carbone dans les océans. Au point de vue moléculaire, ils sont aussi connus pour leur caractéristiques nucléaires uniques, car on retrouve généralement une quantité immense d’ADN dans leurs chromosomes et ceux-ci sont empaquetés et condensés sous une forme cristalline liquide au lieu de nucléosomes. Les gènes encodés par le noyau sont souvent présents en multiples copies et arrangés en tandem et aucun élément de régulation transcriptionnelle, y compris la boite TATA, n’a encore été observé. L’organisation unique de la chromatine des dinoflagellés suggère que différentes stratégies sont nécessaires pour contrôler l’expression des gènes de ces organismes. Dans cette étude, j’ai abordé ce problème en utilisant le dinoflagellé photosynthétique Lingulodinium polyedrum comme modèle. L. polyedrum est d’un intérêt particulier, car il a plusieurs rythmes circadiens (journalier). À ce jour, toutes les études sur l’expression des gènes lors des changements circadiens ont démontrées une régulation à un niveau traductionnel. Pour mes recherches, j’ai utilisé les approches transcriptomique, protéomique et phosphoprotéomique ainsi que des études biochimiques pour donner un aperçu de la mécanique de la régulation des gènes des dinoflagellés, ceci en mettant l’accent sur l’importance de la phosphorylation du système circadien de L. polyedrum. L’absence des protéines histones et des nucléosomes est une particularité des dinoflagellés. En utilisant la technologie RNA-Seq, j’ai trouvé des séquences complètes encodant des histones et des enzymes modifiant les histones. L polyedrum exprime donc des séquences conservées codantes pour les histones, mais le niveau d’expression protéique est plus faible que les limites de détection par immunodétection de type Western. Les données de séquençage RNA-Seq ont également été utilisées pour générer un transcriptome, qui est une liste des gènes exprimés par L. polyedrum. Une recherche par homologie de séquences a d’abord été effectuée pour classifier les transcrits en diverses catégories (Gene Ontology; GO). Cette analyse a révélé une faible abondance des facteurs de transcription et une surprenante prédominance, parmi ceux-ci, des séquences à domaine Cold Shock. Chez L. polyedrum, plusieurs gènes sont répétés en tandem. Un alignement des séquences obtenues par RNA-Seq avec les copies génomiques de gènes organisés en tandem a été réalisé pour examiner la présence de transcrits polycistroniques, une hypothèse formulée pour expliquer le manque d’élément promoteur dans la région intergénique de la séquence de ces gènes. Cette analyse a également démontré une très haute conservation des séquences codantes des gènes organisés en tandem. Le transcriptome a également été utilisé pour aider à l’identification de protéines après leur séquençage par spectrométrie de masse, et une fraction enrichie en phosphoprotéines a été déterminée comme particulièrement bien adapté aux approches d’analyse à haut débit. La comparaison des phosphoprotéomes provenant de deux périodes différentes de la journée a révélée qu’une grande partie des protéines pour lesquelles l’état de phosphorylation varie avec le temps est reliées aux catégories de liaison à l’ARN et de la traduction. Le transcriptome a aussi été utilisé pour définir le spectre des kinases présentes chez L. polyedrum, qui a ensuite été utilisé pour classifier les différents peptides phosphorylés qui sont potentiellement les cibles de ces kinases. Plusieurs peptides identifiés comme étant phosphorylés par la Casein Kinase 2 (CK2), une kinase connue pour être impliquée dans l’horloge circadienne des eucaryotes, proviennent de diverses protéines de liaison à l’ARN. Pour évaluer la possibilité que quelques-unes des multiples protéines à domaine Cold Shock identifiées dans le transcriptome puissent moduler l’expression des gènes de L. polyedrum, tel qu’observé chez plusieurs autres systèmes procaryotiques et eucaryotiques, la réponse des cellules à des températures froides a été examinée. Les températures froides ont permis d’induire rapidement un enkystement, condition dans laquelle ces cellules deviennent métaboliquement inactives afin de résister aux conditions environnementales défavorables. Les changements dans le profil des phosphoprotéines seraient le facteur majeur causant la formation de kystes. Les phosphosites prédits pour être phosphorylés par la CK2 sont la classe la plus fortement réduite dans les kystes, une découverte intéressante, car le rythme de la bioluminescence confirme que l’horloge a été arrêtée dans le kyste.

Enzyme reactions as index of freshness of Fish and Shellfish

An attempt has been made in this study to screen some fish muscle enzymes to assess their potential worth in testing the degree of freshness of fish. A problem with routine enzyme activity determinations is the complexity of the method of enzyme assay. Hence, in the present study as far as possible simple assay techniques were adopted. Several species were screened to assess the possibility of employing this procedure on a large scale. It is hoped that findings of this study will lead to the development of meaningful criteria in testing the freshness of fish. This thesis has been divided into five chapters

Transcriptomic analysis of rhizobium leguminosarum biovar viciae in symbiosis with host plants pisum sativum and Vicia cracca

Rhizobium leguminosarum bv. viciae forms nitrogen-fixing nodules on several legumes, including pea (Pisum sativum) and vetch (Vicia cracca), and has been widely used as a model to study nodule biochemistry. To understand the complex biochemical and developmental changes undergone by R. leguminosarum bv. viciae during bacteroid development, microarray experiments were first performed with cultured bacteria grown on a variety of carbon substrates (glucose, pyruvate, succinate, inositol, acetate, and acetoacetate) and then compared to bacteroids. Bacteroid metabolism is essentially that of dicarboxylate-grown cells (i.e., induction of dicarboxylate transport, gluconeogenesis and alanine synthesis, and repression of sugar utilization). The decarboxylating arm of the tricarboxylic acid cycle is highly induced, as is gamma-aminobutyrate metabolism, particularly in bacteroids from early (7-day) nodules. To investigate bacteroid development, gene expression in bacteroids was analyzed at 7, 15, and 21 days postinoculation of peas. This revealed that bacterial rRNA isolated from pea, but not vetch, is extensively processed in mature bacteroids. In early development (7 days), there were large changes in the expression of regulators, exported and cell surface molecules, multidrug exporters, and heat and cold shock proteins. fix genes were induced early but continued to increase in mature bacteroids, while nif genes were induced strongly in older bacteroids. Mutation of 37 genes that were strongly upregulated in mature bacteroids revealed that none were essential for nitrogen fixation. However, screening of 3,072 mini-Tn5 mutants on peas revealed previously uncharacterized genes essential for nitrogen fixation. These encoded a potential magnesium transporter, an AAA domain protein, and proteins involved in cytochrome synthesis.

Characterization of Caulobacter crescentus response to low temperature and identification of genes involved in freezing resistance

Free-living bacteria must respond to a wide range of temperature changes, and have developed specific mechanisms to survive in extreme environments. In this work we describe a remarkable resistance of mesophilic bacterium Caulobacter crescentus to several cycles of freezing at -80 degrees C, which was able to grow at low temperatures. Exponentially growing cells and late stationary-phase cells presented higher freezing resistance at both -20 and -80 degrees C than early stationary-phase cells. Cryotolerance was observed when log-phase cultures grown at 30 degrees C were preincubated at 5, 15 or 20 degrees C before freezing at -20 degrees C. A transposon library was screened to identify mutants sensitive to freezing at -80 degrees C and three strains presenting < 10% survival were isolated. Identification of genes disrupted in each mutant showed that they encoded an AddA family DNA helicase, a DEAD/DEAH box RNA helicase and a putative RND (resistance, nodulation, cell division) efflux system component. These strains showed longer generation times than wild-type cells when growing at 15 degrees C, with the RNA helicase mutant presenting a severe growth defect. These analyses suggest that the singular intrinsic resistance to freezing of C. crescentus is in fact a consequence of several independent traits, especially the maintenance of a proper degree of supercoiling of nucleic acids.

CspC and CspD are essential for Caulobacter crescentus stationary phase survival

The cold shock response in bacteria involves the expression of low-molecular weight cold shock proteins (CSPs) containing a nucleic acid-binding cold shock domain (CSD), which are known to destabilize secondary structures on mRNAs, facilitating translation at low temperatures. Caulobacter crescentus cspA and cspB are induced upon cold shock, while cspC and cspD are induced during stationary phase. In this work, we determined a new coding sequence for the cspC gene, revealing that it encodes a protein containing two CSDs. The phenotypes of C. crescentus csp mutants were analyzed, and we found that cspC is important for cells to maintain viability during extended periods in stationary phase. Also, cspC and cspCD strains presented altered morphology, with frequent non-viable filamentous cells, and cspCD also showed a pronounced cell death at late stationary phase. In contrast, the cspAB mutant presented increased viability in this phase, which is accompanied by an altered expression of both cspC and cspD, but the triple cspABD mutant loses this characteristic. Taken together, our results suggest that there is a hierarchy of importance among the csp genes regarding stationary phase viability, which is probably achieved by a fine tune balance of the levels of these proteins.

SpdR, a Response Regulator Required for Stationary-Phase Induction of Caulobacter crescentus cspD

The cold shock protein (CSP) family includes small polypeptides that are induced upon temperature downshift and stationary phase. The genome of the alphaproteobacterium Caulobacter crescentus encodes four CSPs, with two being induced by cold shock and two at the onset of stationary phase. In order to identify the environmental signals and cell factors that are involved in cspD expression at stationary phase, we have analyzed cspD transcription during growth under several nutrient conditions. The results showed that expression of cspD was affected by the medium composition and was inversely proportional to the growth rate. The maximum levels of expression were decreased in a spoT mutant, indicating that ppGpp may be involved in the signalization for carbon starvation induction of cspD. A Tn5 mutant library was screened for mutants with reduced cspD expression, and 10 clones that showed at least a 50% reduction in expression were identified. Among these, a strain with a transposon insertion into a response regulator of a two-component system showed no induction of cspD at stationary phase. This protein (SpdR) was able to acquire a phosphate group from its cognate histidine kinase, and gel mobility shift assay and DNase I footprinting experiments showed that it binds to an inverted repeat sequence of the cspD regulatory region. A mutated SpdR with a substitution of the conserved aspartyl residue that is the probable phosphorylation site is unable to bind to the cspD regulatory region and to complement the spdR mutant phenotype.

Coordinate and time-dependent diffusion dynamics in protein folding

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

The Origin of Nonmonotonic Complex Behavior and the Effects of Nonnative Interactions on the Diffusive Properties of Protein Folding

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Estudo do coeficiente de difusão no enovelamento de proteína

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Indução à triploidia no tambaqui Colossoma macropomum, pacu Piaractus mesopotamicus e o respectivo híbrido tambacu

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Comparison of apoptotic cells between cryopreserved ejaculated sperm and epididymal sperm in stallions

The development of a reliable technique to freeze epididymal semen would provide a unique opportunity to preserve valuable genetic material from unexpectedly lost stallions. The aim of this study was to compare the apoptotic indices of sperm obtained from ejaculate, sperm recently recovered from the epididymides (EP), and sperm recovered from epididymides stored at 5 C for 24 hours (EP-stored). For the first category, two ejaculates from seven stallions were collected and then submitted to cryopreservation using an egg yolk-based extender. One week after the last semen collection, the stallions were submitted to bilateral orchiectomy, and sperm from one of the cauda epididymis was harvested immediately after castration (EP). The remaining testicle was stored in a passive refrigeration container at 5 C for 24 hours before the cauda epididymal sperm was harvested (EP-stored). Sperm harvesting from the epididymis for EP and EP-stored was performed by retrograde flushing of the caudal portion of the epididymis using a skim milk-based extender. The recovered sperm was then cryopreserved using the egg yolk-based extender. Sperm motility parameters were studied by computerassisted semen analysis, and apoptosis was estimated by measuring caspase activity and membrane phospholipid translocation using epifluorescence microscopy. The samples were evaluated immediately (0 hour) and 8 hours after thawing. At 0 hour, no differences in sperm parameters were observed among the groups, but after 8 hours, significant statistical differences were observed in sperm motility parameters and plasma membrane integrity among the treatment groups. In addition, viable cells with no apoptotic signs were more prevalent in EP and EP-stored, suggesting that epididymal sperm is less sensitive to the cold shock caused by sperm cryopreservation.

Moraxella catarrhalis - pathogen or commensal?

Moraxella catarrhalis is an exclusively human commensal and mucosal pathogen. Its role as a disease-causing organism has long been questioned. Today, it is recognized as one of the major causes of acute otitis media in children, and its relative frequency of isolation from both the nasopharynx and the middle ear cavity has increased since the introduction of the heptavalent pneumococcal conjugate vaccine, which is associated with a shift in the composition of the nasopharyngeal flora in infants and young children. Although otitis media caused by M. catarrhalis is generally believed to be mild in comparison with pneumococcal disease, numerous putative virulence factors have now been identified and it has been shown that several surface components of M. catarrhalis induce mucosal inflammation. In adults with chronic obstructive pulmonary disease (COPD), M. catarrhalis is now a well-established trigger of approximately 10% of acute inflammatory exacerbations.Although the so-called cold shock response is a well-described bacterial stress response in species such as Escherichia coli, Bacillus subtilis or - more recently - Staphylococcus aureus, M. catarrhalis is the only typical nasopharyngeal pathogen in which this response has been investigated. Indeed, a 3-h 26°C cold shock, which may occur physiologically, when humans inspire cold air for prolonged periods of time, increases epithelial cell adherence and enhances proinflammatory host responses and may thus contribute to the symptoms referred to as common cold, which typically are attributed to viral infections.

Tab. 1: Summary of available data about antifreeze peptides of polar fish

In the blood of Antarctic notothenioid and Arctic gadiform fishes, freezing is inhibited by antifreeze glycopeptide macromolecules (AFGP). These antifreeze molecules are built up of repeating tripeptide units (Ala-Ala-Thr)n, to which the disaccharide fl-D-galactosyl-(1->3)a-N-acetyl-D-galactosamine is linked through the hydroxyl oxygen of the threonyl residue. Species of Liparididae, Zoarcidae, Cottidae and Pleuronectidae synthezise only unglycosylated antifreeze peptides (AFP). It could be demonstrated for the Antarctic silverfish Pleuragramma antarcticum that the synthesis of AFGP is not constitutive but rather regulated by water temperature. Moreover a novel glycopeptid was isolated and characterised from P. antarcticum, the Pleuragramma-antifreeze glycopeptid (PAGP). The level of antifreeze concentration was dependent on the ambient water temperature, the depth of distribution, the life cycle and the evolution of the species. Surprisingly, detectable AFGPs in perciform fish of the Antarctic and gadiform fish of the Arctic and Antarctic could illustrate, that before the continental drift occurred a precursor glycopeptid existed, and that the existence of freezing resistance in some species reflects the past glaciation. The wide distribution and high heterogeneity of AFPs point to the assumption that these peptides are results of cold shock stress responses.

Respuesta transcripcional de Populus a bifenilos policlorados: Aspectos aplicados a la fitorremediación

La degradación del suelo ha adquirido una magnitud preocupante. Los métodos tradicionales de descontaminación, son costosos e insuficientes. La fitorremediación representa una alternativa eficaz, de bajo coste, respetuosa con el medio ambiente, que además mejora las propiedades del suelo, si bien ha habido desarrollos relevantes en la última década. Desde el punto de vida científico, el reto principal es descifrar las rutas metabólicas implicadas en respuesta a contaminantes y comprender su regulación. Esta información es imprescindible si aspiramos a mejorar las capacidades naturales de algunas especies vegetales para remediar los suelos contaminados. Los estudios de esta Tesis se han centrado en Populus, el mejor modelo forestal disponible a raíz de la secuenciación de su genoma completo. Por otra parte, Populus tiene una gran capacidad natural para la degradación de contaminantes orgánicos, lo que explica su predominio en los programas forestales de fitorremediación que se desarrollan actualmente. Hemos elegido en concreto al híbrido Populus tremula x P. alba, por la facilidad con que se cultiva y su particular interés biotecnológico. La presente Tesis plantea un estudio comprehensivo de la respuesta molecular a bifenilos policlorados (PCBs), una familia de contaminantes orgánicos persistentes de particular relevancia a escala mundial. Se ha utilizado para ello una aproximación transcriptómica, basada en tecnología RNA-seq, para identificar los genes implicados en el metabolismo de los compuestos in planta y cuantificar sus niveles de activación en distintas situaciones controladas. La tesis pretende asimismo definir el control transcripcional subyacente a la respuesta bioquímica frente a este tipo de contaminantes. Resulta sorprendente que dicha respuesta sea prácticamente desconocida a nivel molecular, a pesar de su gran potencial aplicado en el contexto de la tecnología fitorremediadora. Para desarrollar este proyecto aplicamos a nuestros cultivos de chopo híbridos concentraciones diferentes de Aroclor 1221, una mezcla de PCBs muy utilizada a nivel comercial durante décadas, su uso está prohibido hoy internacionalmente. Y tomamos muestras de RNA a dos concentraciones y dos momentos distintos de exposición al contaminante, generando así una matriz de cuatro elementos con sus controles correspondientes. Con el fin de incrementar la especificidad de nuestro análisis, consideramos sobre todo los genes diferencialmente expresados más significativos según cuatro algoritmos estadísticos distintos. Por otra parte, realizamos análisis funcionales con herramientas bioinformáticas basadas en comparaciones de secuencias y en redes de co-expresión génica. La respuesta de los genes de particular interés fue validada mediante tecnología qRT-PCR (reacción de la polimerasa en cadena cuantitativa en tiempo real). Se trata del primer estudio comprehensivo de la respuesta de un organismo vegetal ante la presencia de PCBs. Este estudio nos ha permitido identificar una cantidad considerable de genes estructurales y reguladores, definiendo nuevos factores de transcripción cuya expresión es proporcional a la concentración de contaminante en el medio o al tiempo de exposición al mismo. Los análisis de correlación nos permiten afirmar en que la respuesta metabólica a PCBs, incluyendo posibles rutas degradadoras, participan en al menos quince factores de transcripción y unas cuarenta proteínas o enzimas que resultan particularmente inducidas. Entre las familias implicadas destacan los citocromos P450, la glutatión transferasas, las deshidrogenasas reductasas (short-chain dehydrogenase reductase) y las proteínas MDR (multi-drug resistance). Mientras que los factores de transcripción encontrados pertenecen a la familia de ZF-TF, MYBs, WRKYs entre otros. También identificamos proteínas de función desconocida que no se habían vinculado previamente a este tipo de respuestas en plantas, como la CSP (cold-shock domain proteins). Para estudiar su posible relación con la presencia de PCBs, se caracterizó un gen de esta familia detectado mediante espectrometría de masas en tándem (MS/MS) a partir de mapas IEF x SDS-PAGE (isoelectro focusing x sodium dodecyl sulphate- polyacrylamide gel electrophoresis) de alta resolución. Mediante qRT-PCR pudimos confirmar la inducción del gen correspondiente, ortólogo a PtCSP4 de P. trichocarpa (Potri.004g172600), en respuesta a Aroclor 1221. El análisis fenotípico de las líneas transgénicas de Arabidopsis thaliana que sobre-expresaba la proteína CSP de chopo híbrido confirmó un papel para la misma tolerancia a PCBs, posiblemente a través de mecanismos reguladores que activan proteínas MDR. Este trabajo, además de aportar datos novedosos sobre los mecanismos moleculares desencadenados por la presencia de un PCB en Populus, utilizado aquí como sistema modelo. Con ello se demuestra el potencial de las especies arbóreas no solo como agentes descontaminantes, ya explotado comercialmente, sino también como fuente potencial de genes interesantes. Entre los genes identificados en esta Tesis hay candidatos evidentes a participar en mecanismos de tolerancia al estrés inducido por la contaminación y también rutas metabólicas degradadores de PCBs. Precisamente la posibilidad de degradar al contaminante confiere particular interés a este tipo de estudios frente a la fitorremediación de metales pesados y otros contaminantes elementales. La comparación de los datos generados en este estudio con estudios análogos que se realicen en el futuro con otras especies y xenobióticos, contribuirán a definir mejor la respuesta de las plantas ante la contaminación orgánica y mejorar su potencial descontaminante. ABSTRACT Soil degradation has acquired a disturbing magnitude. Traditional methods of decontamination are expensive and insufficient. Phytoremediation represent an effective alternative, low cost, respectful of the environment, that also improves soil properties, although there have been relevant developments in the last decade. From a life scientist, the challenge is to decipher the major metabolic pathways involved in response to pollutants and understand their regulation. This information is essential if we desire to enhance the natural abilities of some plant species to remediate contaminated soils. This thesis studies have focused on Populus, the best available forestry model following the sequencing of the entire genome. Moreover, Populus has a natural ability to degrade organic pollutants, which explains its predominance in phytoremediation forestry programs currently being developed. We have chosen specifically to hybrid Populus tremula x P. alba, the ease with which it is grown and its particular biotechnological interest. This thesis presents a comprehensive study of the molecular response to polychlorinated biphenyls (PCBs), a family of persistent organic pollutants of particular relevance worldwide. It has been used for a transcriptomic approach using RNA-seq technology, to identify genes involved in the metabolism of compounds in plant and quantify their levels of activation in different controlled situations. The thesis also aims to define the underlying transcriptional control the biochemical response to these pollutants. It is surprising that the response is virtually unknown at the molecular level, despite its great potential applied in the context of phytoremediation technology. To develop this project we applied our hybrid poplar crops different concentrations of Aroclor 1221, a mixture of PCBs widely used commercially for decades, its use is now banned internationally. And we RNA samples at two different concentrations and times of exposure to the pollutant, generating an array of four elements with their corresponding controls. In order to increase the specificity of our analysis, we consider mainly the most significant differentially expressed genes in four different statistical algorithms. Moreover, functional analyzes conducted with bioinformatics tools based on sequence comparisons and networks gene co-expression. The response of genes of particular interest was validated by qRT-PCR (polymerase reaction chain in real-time quantitative. This is the first comprehensive study of the response of a plant organism in the presence of PCBs. This study allowed us to identify a considerable amount of structural and regulatory genes, defining new transcription factors whose expression is proportional to the concentration of contaminant in the middle or at the time of exposure. Correlation analyzes allow us to affirm that the metabolic response to PCBs, including possible degradative pathways, at least fifteen involved in transcription factors and forty proteins or enzymes which are particularly induced. Among the families involved include cytochromes P450, the glutathione transferases, dehydrogenases reductases (short -chain dehydrogenase reductase) and MDR proteins (multi - drug resistance). While transcription factors belong to the family found ZF-TF, MYBs, WRKYs among others. We also identify proteins of unknown function that had not been previously linked to such responses in plants such as CSP (cold- shock domain proteins). To study their possible relationship with the presence of PCBs, a gene in this family was characterized and was detected by tandem mass spectrometry (MS/MS) from maps IEF x SDS -PAGE (sodium dodecyl isoelectro x sulphate- polyacrylamide gel electrophoresis) of high resolution. By qRT -PCR could confirm the induction of the corresponding gene, ortholog to PtCSP4 of P. trichocarpa (Potri.004g172600), in response to Aroclor 1221. Phenotypic analysis of transgenic Arabidopsis thaliana lines over- expressing the protein CSP poplar hybrid confirmed a role for PCBs same tolerance, possibly through regulatory mechanisms activated MDR proteins. This work, in addition to providing new data on the molecular mechanisms triggered by the presence of PCBs in Populus, used here as a model system. Thus the potential of tree species not only as decontamination agents, and commercially exploited, but also as a potential source of interesting genes is shown. Among the genes identified in this thesis there are evident candidates to participate in tolerance mechanisms to stress induced by pollution and degrading metabolic pathways of PCBs. Precisely the possibility of degrading the pollutant attaches particular interest to this type of study off the phytoremediation of heavy metals and other elemental pollutants. The comparison of the data generated in this study with similar studies carried out in the future with other species and xenobiotics contribute to better define the response of plants to organic pollution and improve their decontamination potential.

Novel folded protein domains generated by combinatorial shuffling of polypeptide segments

It has been proposed that the architecture of protein domains has evolved by the combinatorial assembly and/or exchange of smaller polypeptide segments. To investigate this proposal, we fused DNA encoding the N-terminal half of a β-barrel domain (from cold shock protein CspA) with fragmented genomic Escherichia coli DNA and cloned the repertoire of chimeric polypeptides for display on filamentous bacteriophage. Phage displaying folded polypeptides were selected by proteolysis; in most cases the protease-resistant chimeric polypeptides comprised genomic segments in their natural reading frames. Although the genomic segments appeared to have no sequence homologies with CspA, one of the originating proteins had the same fold as CspA, but another had a different fold. Four of the chimeric proteins were expressed as soluble polypeptides; they formed monomers and exhibited cooperative unfolding. Indeed, one of the chimeric proteins contained a set of very slowly exchanging amides and proved more stable than CspA itself. These results indicate that native-like proteins can be generated directly by combinatorial segment assembly from nonhomologous proteins, with implications for theories of the evolution of new protein folds, as well as providing a means of creating novel domains and architectures in vitro.