Consumers mediate the effects of experimental ocean acidification and warming on primary producers

It is well known that ocean acidification can have profound impacts on marine organisms. However, we know little about the direct and indirect effects of ocean acidification and also how these effects interact with other features of environmental change such as warming and declining consumer pressure. In this study, we tested whether the presence of consumers (invertebrate mesograzers) influenced the interactive effects of ocean acidification and warming on benthic microalgae in a seagrass community mesocosm experiment. Net effects of acidification and warming on benthic microalgal biomass and production, as assessed by analysis of variance, were relatively weak regardless of grazer presence. However, partitioning these net effects into direct and indirect effects using structural equation modeling revealed several strong relationships. In the absence of grazers, benthic microalgae were negatively and indirectly affected by sediment-associated microalgal grazers and macroalgal shading, but directly and positively affected by acidification and warming. Combining indirect and direct effects yielded no or weak net effects. In the presence of grazers, almost all direct and indirect climate effects were nonsignificant. Our analyses highlight that (i) indirect effects of climate change may be at least as strong as direct effects, (ii) grazers are crucial in mediating these effects, and (iii) effects of ocean acidification may be apparent only through indirect effects and in combination with other variables (e.g., warming). These findings highlight the importance of experimental designs and statistical analyses that allow us to separate and quantify the direct and indirect effects of multiple climate variables on natural communities.

Genome-Wide Analysis of the Response of Dickeya dadantii 3937 to Plant Antimicrobial Peptides

Antimicrobial peptides constitute an important factor in the defense of plants against pathogens, and bacterial resistance to these peptides have previously been shown to be an important virulence factor in Dickeya dadantii, the causal agent of soft-rot disease of vegetables. In order to understand the bacterial response to antimicrobial pep- tides, a transcriptional microarray analysis was performed upon treatment with sub-lethal concentration of thionins, a widespread plant peptide. In all, 36 genes were found to be overexpressed, and were classified according to their deduced function as i) transcriptional regulators, ii) transport, and iii) modification of the bacterial membrane. One gene encoding a uricase was found to be repressed. The majority of these genes are known to be under the control of the PhoP/PhoQ system. Five genes representing the different functions induced were selected for further analysis. The results obtained indicate that the presence of antimicrobial peptides induces a complex response which includes peptide-specific elements and general stress-response elements contributing differentially to the virulence in different hosts.

La arquitectura residencial en El Real Sitio de El Pardo (1885 - 1965): Orígenes, tipos y configuración del trazado urbano

Tras la denominación de Real Sitio a mediados del siglo XVIII, bajo el reinado de Fernando VI, su sucesor Carlos III procedió a la incorporación a su Patrimonio de todos los Montes y Bosques de El Pardo. Comenzó entonces el proceso de planeamiento urbano y de construcción arquitectónica que finalizó en torno al año 1800. En lo sucesivo, no sólo se mantiene el curso de la conservación y consolidación de los edificios principales, sino que se realiza obra nueva de índole civil. Algunos edificios cambiaron de propiedad y de uso hasta que tras la Guerra Civil se procedió a la mayor transformación vivida por el Real Sitio. El intervalo que aquí se trata (1885 a 1965), no ha suscitado, en los estudios sobre El Pardo, atención suficiente al no acontecer obra nueva de carácter patrimonial ni ha sido objeto de análisis el trazado y la fisonomía del centro urbano residencial del pueblo que Carlos III configuró. Sin embargo se estima relevante analizar los cambios en la actividad residencial; en primer lugar porque coexiste con la arquitectura oficial y, por tanto, se entiende necesario un análisis global del conjunto y en segundo lugar porque facilita la comprensión sobre la imagen original de carácter histórico del conjunto de finales del siglo XVIII. Este marco temporal determina tres partes principales de estudio que estructuran la presente tesis, cuyas fechas establecen los intervalos históricos clave: Actuaciones sobre el núcleo urbano consolidado (1885-1931). Cese de la actividad constructiva (1931-1939). Propuestas regeneradoras y crecimiento acelerado (1939-1965). Dentro de ellos se establecen, a su vez, dos subcapítulos diferenciados con la finalidad de explicar los sucesos que pautaron los cambios trascendentales en la historia de El Pardo. En el estudio del estado de la cuestión se observa que en El Pardo, al igual que sucede en otros Reales Sitios, se investigan los edificios destacados como el Palacio, la Casita del Príncipe, la Casa de Oficios y la Casa de Infantes desde el punto de vista de su historia pero no desde la arquitectura ni de cómo esta afecta al desarrollo del trazado y por tanto al contexto urbano. Se manifiestan determinadas carencias de tratamiento gráfico que facilitarían la comprensión histórica mediante el análisis de la forma y cómo esta ha ido variando sustancialmente. El concepto de escala y orientación reordena el estudio, no sólo de estos edificios protagonistas sino de los que se entretejen a su alrededor y componen el conjunto histórico, lo cual aporta nuevas conclusiones al estado de la cuestión que aquí compete. El principal objetivo de la tesis es, por tanto, contribuir a la dimensión patrimonial mediante el estudio de la arquitectura residencial del pueblo de El Pardo y en cómo esta ha ido conformando y consolidando el entramado urbano original en torno a edificios de la realeza y corte. Analizar aquellos edificios que perduran, los que fueron reconstruidos, rehabilitados, y apuntar acontecimientos históricos que formularon la actual fisonomía. Sistematizar y reordenar sobre la traza actual los edificios que desaparecieron, nos da las pistas sobre las modificaciones en concepto de escala arquitectónica y urbana. El estudio de las fuentes y establecer una metodología de conexión de estas, ayuda a detectar dónde no se han dirigido aún los focos de interés así como las lagunas que han quedado por explorar con el fin de responder a nuevas hipótesis, conceder conclusiones y abrir otras líneas de investigación. Como conclusiones generales, la tesis aporta documentación nueva sobre el objeto de estudio, no solicitada, digitalizada o publicada con anterioridad. En ella se analizan los procesos de configuración, consolidación y transformación en el Real Sitio mediante la sistematización de estados comparativos. Con respecto al estudio de los diferentes contextos natural y urbano la tesis analiza cómo los accidentes naturales, el desarrollo de infraestructuras y el impulso de la agronomía afectaron a El Pardo a partir del siglo XIX, y estudia los procesos de configuración, consolidación y transformación en el Real Sitio mediante la sistematización de la documentación encontrada de manera gráfica y escrita. En relación al marco patrimonial arquitectónico, la tesis analiza los procesos edificatorios históricos. Se estudian, a su vez, cambios de ocupación o uso que derivaron en reformas, ampliaciones, obras de nueva planta e incluso en derribos, así como los proyectos no materializados o que se llevaron a cabo de manera parcial. Con respecto al análisis del momento histórico, la tesis analiza las posibles afectaciones, políticas, sociales y económicas en las etapas de Monarquía, Segunda República, Guerra Civil y Posguerra. Por último, la tesis abre cuatro vías de investigación (que ya se han tratado y avanzado en parte pero que escapan a los límites de este trabajo) que pueden plantear nuevas hipótesis, reportando así respuestas sobre objetos de estudio complementarios y paralelos al presente. Estas refieren a análisis más concretos sobre El Palacio Real de El Pardo y la Casa de Oficios, el Camino Real de Madrid a El Pardo desde la Puerta de Hierro, los cuarteles, puertas y portilleras del Monte de El Pardo y los proyectos desarrollados por el arquitecto Diego Méndez en los Reales Sitios para el Patrimonio Nacional. ABSTRACT Following the Royal Site denomination being granted in the mid-18th Century, during the reign of Ferdinand VI, his successor Charles III proceeded to include all the Forests and Woodlands of El Pardo in his heritage. That then gave rise to the process of town planning and architectural construction that was completed around 1800. Thereafter, not only the process of conservation and consolidation of the main buildings has been maintained, but new civil engineering works have also been carried out. Some buildings changed ownership and use until, after the Civil War, the greatest transformation experienced by the Royal Site was undertaken. The time frame this paper concerns (1885 to 1965), has not attracted sufficient attention in studies of El Pardo due to there having been no new works with heritage status, nor has there been an analysis of the layout and external appearance of the residential centre in the town once conceived by Charles III. However, it is considered relevant to analyse the changes in residential activity, firstly, because it coexists with the official architecture and, thus, it is considered necessary to perform a global analysis of the complex and, secondly, because it facilitates a historical understanding of the original appearance of the complex at the end of the 18th Century. This time framework defines three main parts of the study that provide the structure of this thesis, the dates of which establish the key historical time frames: Actions in the consolidated town centre (1885-1931). Cessation of construction works (1931-1939). Proposals of regeneration and accelerated growth (1939-1965). Two distinct sub-chapters are also established within these, in order to explain the events that marked the transcendental changes in the history of El Pardo. When studying the subject matter, it is noted that in El Pardo, as is the case in other Royal Sites, outstanding buildings such as the Palace, the Prince's Cottage, the Trades House and the Infantes House are usually researched strictly from the point of view of their history, but not from an architectural perspective, nor analysing how that affects the development of the site layout and thus the urban area. Specific shortcomings are evident in the graphic treatment that would have otherwise facilitated a historical understanding through the analysis of the shape and the way it has gradually undergone substantial variation. The concept of scale and orientation reorganises the study, not only of these key buildings, but also of those that are woven around them and make up the historic complex, allowing entirely new conclusions concerning the subject matter analysed herein. Therefore, the main purpose of this thesis is to outline our heritage through the study of the residential architecture of the town of El Pardo and the analysis of the way the original town has been built up and consolidated around the buildings erected by royalty and the court; to analyse the buildings that still remain, those that were rebuilt, refurbished, and to note historic events that shaped its current appearance. To this end, a systematic classification and reorganisation on the current urban layout of the buildings that have disappeared will give us the key to understand changes in the concept of architectural and urban scale. Studying the sources and establishing a methodology to connect them will help us detect those areas where the focus of interest has not concentrated yet, and will also reveal the gaps that remain unexplored, in order to respond to new hypotheses, reach new conclusions and open up new lines of research. As general conclusions, this thesis provides new documentation on the subject matter that had not been requested, digitized or published before. There we find an analysis of the processes of configuration, consolidation and transformation of the Royal Site through a systematic classification of comparative states. With regard to the study of the multiple natural and urban environments, this thesis analyses the way natural features, development of infrastructures and agricultural driving forces affected El Pardo as of the 19th Century, and it studies the processes of configuration, consolidation and transformation of the Royal Site by systematically classifying the documentation found in graphic and written documents. In relation to the architectural heritage framework, this thesis analyses historical building processes. Likewise, a study is also performed on the changes in land occupation or use that led to reforms, extensions, new buildings and even to demolitions, as well as on unrealized projects, or even on those that were partially implemented. As for the analysis of the historical time period, this thesis assesses the potential political, social and economic effects of the Monarchy, Second Republic, Civil War and Post-War Periods. Finally, this thesis opens up four lines of investigation (that have already been discussed and partially advanced, but which fall beyond the scope of this work) that could pose new hypotheses, thus giving answer to other subject matters parallel and complementary to the one assessed herein. These refer to more specific analyses of El Palacio Real de El Pardo (Royal Palace of El Pardo) and the Casa de Oficios (Trades House), the Royal Highway from Madrid to El Pardo from Puerta de Hierro, the barracks, gates and entrances to estates in the Woodlands of El Pardo and the projects developed on the Royal Sites by the architect Diego Méndez for the National Heritage.

About the origin of low wafer performance and crystal defect generation on seed-cast growth of industrial mono-like silicon ingots

The era of the seed-cast grown monocrystalline-based silicon ingots is coming. Mono-like, pseudomono or quasimono wafers are product labels that can be nowadays found in the market, as a critical innovation for the photovoltaic industry. They integrate some of the most favorable features of the conventional silicon substrates for solar cells, so far, such as the high solar cell efficiency offered by the monocrystalline Czochralski-Si (Cz-Si) wafers and the lower cost, high productivity and full square-shape that characterize the well-known multicrystalline casting growth method. Nevertheless, this innovative crystal growth approach still faces a number of mass scale problems that need to be resolved, in order to gain a deep, 100% reliable and worldwide market: (i) extended defects formation during the growth process; (ii) optimization of the seed recycling; and (iii) parts of the ingots giving low solar cells performance, which directly affect the production costs and yield of this approach. Therefore, this paper presents a series of casting crystal growth experiments and characterization studies from ingots, wafers and cells manufactured in an industrial approach, showing the main sources of crystal defect formation, impurity enrichment and potential consequences at solar cell level. The previously mentioned technological drawbacks are directly addressed, proposing industrial actions to pave the way of this new wafer technology to high efficiency solar cells.

Cardioprotection from ischemia by a brief exposure to physiological levels of ethanol: Role of epsilon protein kinase C

Recent epidemiological studies indicate beneficial effects of moderate ethanol consumption in ischemic heart disease. Most studies, however, focus on the effect of long-term consumption of ethanol. In this study, we determined whether brief exposure to ethanol immediately before ischemia also produces cardioprotection. In addition, because protein kinase C (PKC) has been shown to mediate protection of the heart from ischemia, we determined the role of specific PKC isozymes in ethanol-induced protection. We demonstrated that (i) brief exposure of isolated adult rat cardiac myocytes to 10–50 mM ethanol protected against damage induced by prolonged ischemia; (ii) an isozyme-selective ɛPKC inhibitor developed in our laboratory inhibited the cardioprotective effect of acute ethanol exposure; (iii) protection of isolated intact adult rat heart also occurred after incubation with 10 mM ethanol 20 min before global ischemia; and (iv) ethanol-induced cardioprotection depended on PKC activation because it was blocked by chelerythrine and GF109203X, two PKC inhibitors. Consumption of 1–2 alcoholic beverages in humans leads to blood alcohol levels of ≈10 mM. Therefore, our work demonstrates that exposure to physiologically attainable ethanol levels minutes before ischemia provides cardioprotection that is mediated by direct activation of ɛPKC in the cardiac myocytes. The potential clinical implications of our findings are discussed.

Striking sequence similarity in inter- and intra-specific comparisons of class I SLG alleles from Brassica oleracea and Brassica campestris: Implications for the evolution and recognition mechanism

Self-incompatibility in Brassica is controlled by a single multi-allelic locus (S locus), which contains at least two highly polymorphic genes expressed in the stigma: an S glycoprotein gene (SLG) and an S receptor kinase gene (SRK). The putative ligand-binding domain of SRK exhibits high homology to the secretory protein SLG, and it is believed that SLG and SRK form an active receptor kinase complex with a self-pollen ligand, which leads to the rejection of self-pollen. Here, we report 31 novel SLG sequences of Brassica oleracea and Brassica campestris. Sequence comparisons of a large number of SLG alleles and SLG-related genes revealed the following points. (i) The striking sequence similarity observed in an inter-specific comparison (95.6% identity between SLG14 of B. oleracea and SLG25 of B. campestris in deduced amino acid sequence) suggests that SLG diversification predates speciation. (ii) A perfect match of the sequences in hypervariable regions, which are thought to determine S specificity in an intra-specific comparison (SLG8 and SLG46 of B. campestris) and the observation that the hypervariable regions of SLG and SRK of the same S haplotype were not necessarily highly similar suggests that SLG and SRK bind different sites of the pollen ligand and that they together determine S specificity. (iii) Comparison of the hypervariable regions of SLG alleles suggests that intragenic recombination, together with point mutations, has contributed to the generation of the high level of sequence variation in SLG alleles. Models for the evolution of SLG/SRK are presented.

Activation of human monocytes induces differential resistance to apoptosis with rapid down regulation of caspase-8/FLICE

Cells of the monocyte/macrophage lineage play a central role in both innate and acquired immunity of the host. However, the acquisition of functional competence and the ability to respond to a variety of activating or modulating signals require maturation and differentiation of circulating monocytes and entail alterations in both biochemical and phenotypic profiles of the cells. The process of activation also confers survival signals essential for the functional integrity of monocytes enabling the cells to remain viable in microenvironments of immune or inflammatory lesions that are rich in cytotoxic inflammatory mediators and reactive free-radical species. However, the molecular mechanisms of activation-induced survival signals in monocytes remain obscure. To define the mechanistic basis of activation-induced resistance to apoptosis in human monocytes at the molecular level, we evaluated the modulation of expression profiles of genes associated with the cellular apoptotic pathways upon activation and demonstrate the following: (i) activation results in selective resistance to apoptosis particularly to that induced by signaling via death receptors and DNA damage; (ii) concurrent with activation, the most apical protease in the death receptor pathway, caspase-8/FLICE is rapidly down-regulated at the mRNA level representing a novel regulatory mechanism; and (iii) activation of monocytes also leads to dramatic induction of the Bfl-1 gene, an anti apoptotic member of the Bcl-2 family. Our findings thus provide a potential mechanistic basis for the activation-induced resistance to apoptosis in human monocytes.

Identification of functional domains on human interleukin 10

Interleukin 10 (IL-10) is a recently described natural endogenous immunosuppressive cytokine that has been identified in human, murine, and other organisms. Human IL-10 (hIL-10) has high homology with murine IL-10 (mIL-10) as well as with an Epstein–Barr virus genome product BCRFI. This viral IL-10 (vIL-10) shares a number of activities with hIL-10. IL-10 significantly affects chemokine biology, because human IL-10 inhibits chemokine production and is a specific chemotactic factor for CD8+ T cells. It suppresses the ability of CD4+ T cells, but not CD8+ T cells, to migrate in response to IL-8. A nonapeptide (IT9302) with complete homology to a sequence of hIL-10 located in the C-terminal portion (residues 152–160) of the cytokine was found to possess activities that mimic some of those of hIL-10. These are: (i) inhibition of IL-1β-induced IL-8 production by peripheral blood mononuclear cell, (ii) inhibition of spontaneous IL-8 production by cultured human monocytes, (iii) induction of IL-1 receptor antagonistic protein production by human monocytes, (iv) induction of chemotactic migration of CD8+ human T lymphocytes in vitro, (v) desensitization of human CD8+ T cells resulting in an unresponsiveness toward rhIL-10-induced chemotaxis, (vi) suppression of the chemotactic response of CD4+ T human lymphocytes toward IL-8, (vii) induction of IL-4 production by cultured normal human CD4+ T cells, (viii) down-regulation of tumor necrosis factor-α production by CD8+ T cells, and (ix) inhibition of class II major histocompatibility complex antigen expression on IFN-γ-stimulated human monocytes. Another nonapeptide (IT9403) close to the NH2-terminal part of hIL-10 did not reveal cytokine synthesis inhibitory properties, but proved to be a regulator of mast cell proliferation. In conclusion, we have identified two functional domains of IL-10 exerting different IL-10 like activities, an observation that suggests that relatively small segments of these signal proteins are responsible for particular biological functions.

Molecular basis for a link between complement and the vascular complications of diabetes

Activated terminal complement proteins C5b to C9 form the membrane attack complex (MAC) pore. Insertion of the MAC into endothelial cell membranes causes the release of growth factors that stimulate tissue growth and proliferation. The complement regulatory membrane protein CD59 restricts MAC formation. Because increased cell proliferation characterizes the major chronic vascular complications of human diabetes and because increased glucose levels in diabetes cause protein glycation and impairment of protein function, we investigated whether glycation could inhibit CD59. Glycation-inactivation of CD59 would cause increased MAC deposition and MAC-stimulated cell proliferation. Here, we report that (i) human CD59 is glycated in vivo, (ii) glycated human CD59 loses its MAC-inhibitory function, and (iii) inactivation of CD59 increases MAC-induced growth factor release from endothelial cells. We demonstrate by site-directed mutagenesis that residues K41 and H44 form a preferential glycation motif in human CD59. The presence of this glycation motif in human CD59, but not in CD59 of other species, may help explain the distinct propensity of humans to develop vascular proliferative complications of diabetes.

The pattern of gene expression in human CD15+ myeloid progenitor cells

We performed a genome-wide analysis of gene expression in primary human CD15+ myeloid progenitor cells. By using the serial analysis of gene expression (SAGE) technique, we obtained quantitative information for the expression of 37,519 unique SAGE-tag sequences. Of these unique tags, (i) 25% were detected at high and intermediate levels, whereas 75% were present as single copies, (ii) 53% of the tags matched known expressed sequences, 34% of which were matched to more than one known expressed sequence, and (iii) 47% of the tags had no matches and represent potentially novel genes. The correct genes were confirmed by application of the generation of longer cDNA fragments from SAGE tags for gene identification (GLGI) technique for high-copy tags with multiple matches. A set of genes known to be important in myeloid differentiation were expressed at various levels and used different spliced forms. This study provides a normal baseline for comparison of gene expression in myeloid diseases. The strategy of using SAGE and GLGI techniques in this study has broad applications to the genome-wide identification of expressed genes.

The spinal biology in humans and animals of pain states generated by persistent small afferent input

Behavioral models indicate that persistent small afferent input, as generated by tissue injury, results in a hyperalgesia at the site of injury and a tactile allodynia in areas adjacent to the injury site. Hyperalgesia reflects a sensitization of the peripheral terminal and a central facilitation evoked by the persistent small afferent input. The allodynia reflects a central sensitization. The spinal pharmacology of these pain states has been defined in the unanesthetized rat prepared with spinal catheters for injection and dialysis. After tissue injury, excitatory transmitters (e.g., glutamate and substance P) acting though N-methyl-d-aspartate (NMDA) and neurokinin 1 receptors initiate a cascade that evokes release of (i) NO, (ii) cyclooxygenase products, and (iii) activation of several kinases. Spinal dialysis show amino acid and prostanoid release after cutaneous injury. Spinal neurokinin 1, NMDA, and non-NMDA receptors enhance spinal prostaglandin E2 release. Spinal prostaglandins facilitate release of spinal amino acids and peptides. Activation by intrathecal injection of receptors on spinal C fiber terminals (μ,/∂ opiate, α2 adrenergic, neuropeptide Y) prevents release of primary afferent peptides and spinal amino acids and blocks acute and facilitated pain states. Conversely, consistent with their role in facilitated processing, NMDA, cyclooxygenase 2, and NO synthase inhibitors act to diminish only hyperalgesia. Importantly, spinal delivery of several of these agents diminishes human injury pain states. This efficacy emphasizes (i) the role of facilitated states in humans, (ii) shows the importance of spinal systems in human pain processing, and (iii) indicates that these preclinical mechanisms reflect processes that regulate the human pain experience.

Computer-modeling origin of a simple genetic apparatus

This computer simulation is based on a model of the origin of life proposed by H. Kuhn and J. Waser, where the evolution of short molecular strands is assumed to take place in a distinct spatiotemporal structured environment. In their model, the prebiotic situation is strongly simplified to grasp essential features of the evolution of the genetic apparatus without attempts to trace the historic path. With the tool of computer implementation confining to principle aspects and focused on critical features of the model, a deeper understanding of the model's premises is achieved. Each generation consists of three steps: (i) construction of devices (entities exposed to selection) presently available; (ii) selection; and (iii) multiplication of the isolated strands (R oligomers) by complementary copying with occasional variation by copying mismatch. In the beginning, the devices are single strands with random sequences; later, increasingly complex aggregates of strands form devices such as a hairpin-assembler device which develop in favorable cases. A monomers interlink by binding to the hairpin-assembler device, and a translation machinery, called the hairpin-assembler-enzyme device, emerges, which translates the sequence of R1 and R2 monomers in the assembler strand to the sequence of A1 and A2 monomers in the A oligomer, working as an enzyme.

Applications of pox virus vectors to vaccination: an update.

Recombinant pox viruses have been generated for vaccination against heterologous pathogens. Amongst these, the following are notable examples. (i) The engineering of the Copenhagen strain of vaccinia virus to express the rabies virus glycoprotein. When applied in baits, this recombinant has been shown to vaccinate the red fox in Europe and raccoons in the United States, stemming the spread of rabies virus infection in the wild. (ii) A fowlpox-based recombinant expressing the Newcastle disease virus fusion and hemagglutinin glycoproteins has been shown to protect commercial broiler chickens for their lifetime when the vaccine was administered at 1 day of age, even in the presence of maternal immunity against either the Newcastle disease virus or the pox vector. (iii) Recombinants of canarypox virus, which is restricted for replication to avian species, have provided protection against rabies virus challenge in cats and dogs, against canine distemper virus, feline leukemia virus, and equine influenza virus disease. In humans, canarypox virus-based recombinants expressing antigens from rabies virus, Japanese encephalitis virus, and HIV have been shown to be safe and immunogenic. (iv) A highly attenuated vaccinia derivative, NYVAC, has been engineered to express antigens from both animal and human pathogens. Safety and immunogenicity of NYVAC-based recombinants expressing the rabies virus glycoprotein, a polyprotein from Japanese encephalitis virus, and seven antigens from Plasmodium falciparum have been demonstrated to be safe and immunogenic in early human vaccine studies.

Identification of the coding region for a second poly(A) polymerase in Escherichia coli.

We had earlier identified the pcnB locus as the gene for the major Escherichia coli poly(A) polymerase (PAP I). In this report, we describe the disruption and identification of a candidate gene for a second poly(A) polymerase (PAP II) by an experimental strategy which was based on the assumption that the viability of E. coli depends on the presence of either PAP I or PAP II. The coding region thus identified is the open reading frame f310, located at about 87 min on the E. coli chromosome. The following lines of evidence support f310 as the gene for PAP II: (i) the deduced peptide encoded by f310 has a molecular weight of 36,300, similar to the molecular weight of 35,000 estimated by gel filtration of PAP II; (ii) the deduced f310 product is a relatively hydrophobic polypeptide with a pI of 9.4, consistent with the properties of partially purified PAP II; (iii) overexpression of f310 leads to the formation of inclusion bodies whose solubilization and renaturation yields poly(A) polymerase activity that corresponds to a 35-kDa protein as shown by enzyme blotting; and (iv) expression of a f310 fusion construct with hexahistidine at the N-terminus of the coding region allowed purification of a poly(A) polymerase fraction whose major component is a 36-kDa protein. E. coli PAP II has no significant sequence homology either to PAP I or to the viral and eukaryotic poly(A) polymerases, suggesting that the bacterial poly(A) polymerases have evolved independently. An interesting feature of the PAP II sequence is the presence of sets of two paired cysteine and histidine residues that resemble the RNA binding motifs seen in some other proteins.