Exploring the DNA-binding specificities of zinc fingers with DNA microarrays

A key step in the regulation of networks that control gene expression is the sequence-specific binding of transcription factors to their DNA recognition sites. A more complete understanding of these DNA–protein interactions will permit a more comprehensive and quantitative mapping of the regulatory pathways within cells, as well as a deeper understanding of the potential functions of individual genes regulated by newly identified DNA-binding sites. Here we describe a DNA microarray-based method to characterize sequence-specific DNA recognition by zinc-finger proteins. A phage display library, prepared by randomizing critical amino acid residues in the second of three fingers of the mouse Zif268 domain, provided a rich source of zinc-finger proteins with variant DNA-binding specificities. Microarrays containing all possible 3-bp binding sites for the variable zinc fingers permitted the quantitation of the binding site preferences of the entire library, pools of zinc fingers corresponding to different rounds of selection from this library, as well as individual Zif268 variants that were isolated from the library by using specific DNA sequences. The results demonstrate the feasibility of using DNA microarrays for genome-wide identification of putative transcription factor-binding sites.

Developmental Expression and Substrate Specificities of Alfalfa Caffeic Acid 3-O-Methyltransferase and Caffeoyl Coenzyme A 3-O-Methyltransferase in Relation to Lignification1

The biosynthesis of monolignols can potentially occur via two parallel pathways involving free acids or their coenzyme A (CoA) esters. Caffeic acid 3-O-methyltransferase (COMT) and caffeoyl CoA 3-O-methyltransferase (CCOMT) catalyze functionally identical reactions in these two pathways, resulting in the formation of mono- or dimethoxylated lignin precursors. The activities of the two enzymes increase from the first to the sixth internode in stems of alfalfa (Medicago sativa L.), preceding the deposition of lignin. Alfalfa CCOMT is highly similar at the amino acid sequence level to the CCOMT from parsley, although it contains a six-amino acid insertion near the N terminus. Transcripts encoding both COMT and CCOMT are primarily localized to vascular tissue in alfalfa stems. Alfalfa CCOMT expressed in Escherichia coli catalyzes O-methylation of caffeoyl and 5-hydroxyferuloyl CoA, with preference for caffeoyl CoA. It has low activity against the free acids. COMT expressed in E. coli is active against both caffeic and 5-hydroxyferulic acids, with preference for the latter compound. Surprisingly, very little extractable O-methyltransferase activity versus 5-hydroxyferuloyl CoA is present in alfalfa stem internodes, in which relative O-methyltransferase activity against 5-hy-droxyferulic acid increases with increasing maturity, correlating with increased lignin methoxyl content.

A novel strategy for generating monoclonal antibodies from single, isolated lymphocytes producing antibodies of defined specificities.

We report a novel approach to the generation of monoclonal antibodies based on the molecular cloning and expression of immunoglobulin variable region cDNAs generated from single rabbit or murine lymphocytes that were selected for the production of specific antibodies. Single cells secreting antibodies for a specific peptide either from gp116 of the human cytomegalovirus or from gp120 of HIV-1 or for sheep red blood cells were selected using antigen-specific hemolytic plaque assays. Sheep red blood cells were coated with specific peptides in a procedure applicable to any antigen that can be biotinylated. Heavy- and light-chain variable region cDNAs were rescued from single cells by reverse transcription-PCR and expressed in the context of human immunoglobulin constant regions. These chimeric murine and rabbit monoclonal antibodies replicated the target specificities of the original antibody-forming cells. The selected lymphocyte antibody method exploits the in vivo mechanisms that generate high-affinity antibodies. This method can use lymphocytes from peripheral blood, can exploit a variety of procedures that identify individual lymphocytes producing a particular antibody, and is applicable to the generation of monoclonal antibodies from many species, including humans.

Mapping the protein regions responsible for the functional specificities of the Arabidopsis MADS domain organ-identity proteins.

The Arabidopsis MADS domain proteins AP1, AP3, PI, and AG specify floral organ identity. All of these proteins contain a MADS domain required for DNA binding and dimerization; a region termed L (linker between MADS domain and K domain), which plays an important role in dimerization specificity; the K domain, named for its similarity to the coiled-coil domain of keratin; and a C-terminal region of unknown function. To determine which regions of these proteins are responsible for their abilities to specify different organs, we have made a number of chimeric MADS box genes. The in vivo function of these chimeric genes was investigated by ectopic expression in transgenic Arabidopsis plants. The four proteins fall into two classes on the basis of regions responsible for their functional specificities. The L region and K domain define the functional specificities of AP3 and PI, while the MADS domain and L region define the functional specificities of AP1 and AG.

In vivo selection of basic region-leucine zipper proteins with altered DNA-binding specificities.

A transcription interference assay was used to generate mutant basic region-leucine zipper proteins with altered DNA-binding specificities. A library of mutants of a CCAAT/enhancer binding protein was constructed by randomizing five DNA-contacting amino acids in the basic region Asn-18, Ala-15, Val-14, Ser-11, and Arg-10. These mutants were then selected for their ability to bind mutant recognition sequences containing substitutions at the 2 and 3 positions of the wild-type sequence 5'-A5T4T3G2C1G1'C2'A3A4'T5'-3'. Mutants containing the sequence Leu-18Tyr-15Xaa-14Tyr-11Arg-10, in which four of the five contact residues are altered, were identified that recognize the palindromic sequence 5'-ATCYCGY'GAT-3' (Xaa = asparagine when Y = G; Xaa = methionine when Y = A). Moreover, in a selection against the sequence 5'-ATTACGTAAT-3', mutants were obtained containing substitutions not only in the basic region but also in the hinge region between the basic and leucine zipper regions. The mutant proteins showed high specificity in a functional transcription interference assay. A model for the interaction of these mutants with the target DNA sequences is discussed.

Proline-rich sequences that bind to Src homology 3 domains with individual specificities.

To study the binding specificity of Src homology 3 (SH3) domains, we have screened a mouse embryonic expression library for peptide fragments that interact with them. Several clones were identified that express fragments of proteins which, through proline-rich binding sites, exhibit differential binding specificity to various SH3 domains. Src-SH3-specific binding uses a sequence of 7 aa of the consensus RPLPXXP, in which the N-terminal arginine is very important. The SH3 domains of the Src-related kinases Fyn, Lyn, and Hck bind to this sequence with the same affinity as that of the Src SH3. In contrast, a quite different proline-rich sequence from the Btk protein kinase binds to the Fyn, Lyn, and Hck SH3 domains, but not to the Src SH3. Specific binding of the Abl SH3 requires a longer, more proline-rich sequence but no arginine. One clone that binds to both Src and Abl SH3 domains through a common site exhibits reversed binding orientation, in that an arginine indispensable for binding to all tested SH3 domains occurs at the C terminus. Another clone contains overlapping yet distinct Src and Abl SH3 binding sites. Binding to the SH3 domains is mediated by a common PXXP amino acid sequence motif present on all ligands, and specificity comes about from other interactions, often ones involving arginine. The rules governing in vivo usage of particular sites by particular SH3 domains are not clear, but one binding orientation may be more specific than another.

Preliminary identification of dehalogenation specificities exhibited by dehalorespiring bacteria from a marine microbial community

The ability of a previously PCB-enriched microbial culture from Venice Lagoon marine sediments to dechlorinate pentachlorophenol (PCP) and 2,3,5-trichlorophenol (2,3,5-TCP) was confirmed under anaerobic conditions in microcosms consisting of site water and sediment. Dechlorination activities against Aroclor 1254 PCB mixture were also confirmed as control. Pentachlorophenol was degraded to 2,4,6-TCP (75.92±0.85 mol%), 3,5-DCP (6.40±0.75 mol%), and phenol (15.40±0.87 mol%). From the distribution of the different dechlorination products accumulated in the PCP-spiked cultures over time, two dechlorination pathways for PCP were proposed: (i) PCP to 2,3,4,6-TeCP, then to 2,4,6-TCP through the removal of both meta double-flanked chlorine substituents (main pathway); (ii) alternately, PCP to 2,3,5,6-TeCP, 2,3,5-TCP, 3,5-DCP, then phenol, through the removal of the para double-flanked chlorine, followed by ortho single-flanked chlorines, and finally meta unflanked chlorines (minor pathway). Removal of meta double-flanked chlorines is thus preferred over all other substituents. 2,3,5-TCP, that completely lacks double-flanked chlorines, was degraded to 3,5-DCP through removal of the ortho single-flanked chlorine, with a 99.6% reduction in initial concentration of 2,3,5-TCP by week 14. 16S rRNA PCR-DGGE using Chloroflexi-specific primers revealed a different role of the two microorganisms VLD-1 and VLD-2, previously identified as dechlorinators in the Aroclor 1254 PCB-enriched community, in the dehalogenation of chlorophenols. VLD-1 was observed both in PCP- and TCP-dechlorinating communities, whereas VLD-2 only in TCP-dechlorinating communities. This indicates that VLD-1 and VLD-2 may both dechlorinate ortho single-flanked chlorines, but only VLD-1 is able to remove double-flanked meta or para chlorines.

Innovation dynamics in rural areas: contributions to capture and measure innovation, valuing territorial specificities

Doutoramento em Gestão Interdisciplinar da Paisagem - Instituto Superior de Agronomia / Universidade dos Açores / Universidade de Évora

The fingerprinting of the materials used in portuguese illuminated manuscripts: origin, production and specificities of the 16th century Anttiphonary paints from the Biblioteca Pública de Évora

This thesis focuses on the characterization of materials utilized within the illuminations of Codex 116c of Manizola, a large 16th century antiphonal housed in the Biblioteca Pública de Évora (BPE). Using various spectroscopic techniques (XRF, FTIR, Raman and SEM-EDS), a selection of illuminations were analyzed for pigment and binder identification. The manuscript was further analyzed using fiber optic reflectance spectroscopy (FORS), a non-invasive and portable analysis method ideal for use in illuminations. Using historical documentation and results gained from more extensive analysis of the manuscript, a collection of reference paint samples were created to be analyzed using this method. These samples serve as a reference not only to assist in the identification of pigments used within the manuscript, but also for future studies on similar materials allowing for a better understanding of manuscript production during the 16th century; RESUMO: O presente trabalho é dedicado à caracterização dos materiais utilizados na produção das iluminuras do Codex 116c da Manziola do espólio da Biblioteca Pública de Évora (BPE). Trata-se de um antifonário de grandes dimensões produzido no séc XVI que deverá ter pertencido à Livraria de São Bento de Cástris. A identificação dos materiais utilizados na produção das iluminuras pode ser feita através de análises científicas. No entanto, alguns dos componentes das tintas utilizadas, especialmente os pigmentos orgânicos (lacas) e algumas misturas, apresentam obstáculos à sua identificação por métodos não invasivos. Através de várias técnicas espectroscópicas (XRF, FTIR, Raman e SEM-EDS), foi analisado um conjunto representativo de iluminuras, de modo a identificar os pigmentos e os ligantes presentes nas tintas. O manuscrito foi também analisado por FORS, um método portátil e não invasivo, ideal para a análise de iluminuras. Com base em documentos históricos e nos resultados analíticos, foi criado um conjunto de amostras de referência para ser analisado com FORS. Com esta abordagem, pretende-se que estas amostras, especialmente as de lacas, sirvam de referência não só na identificação dos pigmentos no manuscrito como em estudos sobre materiais semelhantes, contribuindo para um conhecimento mais aprofundado sobre a produção de manuscritos no séc XVI.

Homogeneidade e heterogeneidade nos sistemas educacionais: Argentina, Brasil, Chile e México

This study focus on the reconfiguration of educational management in Argentina, Brazil, Chile and Mexico, which was promoted by the new logic of social regulation and the new role attributed to the State, considering each country's own historicity. So, the cultural factors that interfere on the dynamics of the 90' school reform are analyzed. Aspects that show the homogeneity or heterogeneity of these reforms in the region, as well as local specificities that block out the concretization of the reform are underlined. It is shown that the historicity that characterizes the educational reform has taken, in each country, a form that can be called, in Mexico, conservative rupture; in Chile, conservative continuity; in Brazil, conservative renovation; and, in Argentina, interrupted rupture. Some conclusions about the impact of educational reform in the selected countries are recuperated through the analysis of 186 academic texts on the subject.

Formação de conceitos em crianças cegas: questões teóricas e implicações educacionais

Concept formation depends on language and thought, that promote the integration of information coming from the senses. It is postulated that changes in the person, the objects and events to be known suggest flexible models of concept teaching. It is assumed that the same considerations apply to teaching concepts to blind pupils. Specificities of this process are discussed, including the role of touch as resource, although not as a direct substitute to vision, and the notion of representation as a basis for the elaboration of pedagogical resources for the blind student.

Crescimento e desempenho motor em pre-escolares de Itapira SP : um enfoque bio-social-cultural

Universidade Estadual de Campinas. Faculdade de Educação Física

Gestos nos cantos da Lagoa Azul : crianças brincantes produtoras de cultura

Universidade Estadual de Campinas . Faculdade de Educação Física

A "reeducação" de adolescentes em uma comunidade terapêutica: o tratamento da drogadição em uma instituição religiosa

O abuso de drogas por adolescentes é, atualmente, um grave problema de saúde pública. Nessa área, a questão do tratamento destaca-se como um grande desafio, principalmente pelo fato de a maioria das intervenções não estar adaptada às especificidades desse público. Neste trabalho, serão apresentados os resultados de uma investigação que visou analisar o tratamento destinado a adolescentes em uma modalidade de atenção distinta das abordagens tradicionais da área da saúde: a comunidade terapêutica. Buscou-se compreender as concepções e práticas de atenção da instituição pesquisada, comparando-as com o prescrito pelas políticas públicas do setor. Os métodos utilizados foram a descrição etnográfica e a análise de conteúdo. Os resultados sugerem uma defasagem entre o prescrito pelas políticas públicas e a realidade da comunidade terapêutica pesquisada, a qual, apesar de proporcionar acolhimento às adolescentes, dificulta o desenvolvimento de suas singularidades e potencialidades devido à ênfase no "modelo moral" de tratamento da drogadição.