Estudi d'un receptor quinasa atípic (Mark) i de les proteïnes que interaccionen amb el seu domini intracel·lular. Transducció del senyal i desenvolupament en blat de moro (Zea mays)

Aquesta tesi doctoral s'engloba dins d'un projecte general d'estudi de gens implicats en l'embriogènesi del blat de moro. L'embriogènesi del blat de moro, i en general la de totes les plantes superiors, es dóna en tres etapes: una primera etapa on es diferencien tots els diversos teixits que formaran l'embrió, una segona etapa on l'embrió acumula productes de reserva i un tercer període, la dormància, que finalitza quan les condicions ambientals són les idònies per a la germinació. En el laboratori estàvem interessats, concretament, en l'estudi de gens implicats en la primera etapa morfogenètica, on els diferents teixits i estructures embrionàries queden definides. Per tal d'estudiar gens que s'expressaven en aquest període, una de les estratègies que es va realitzar fou un crivellat diferencial entre teixit embrionari i teixit de planta adulta. D'entre els diferents clons obtinguts, un corresponia a un clon parcial que presentava similitud amb receptors quinasa i que fou objecte d'estudi. A partir d'aquest clon es va obtenir el clon complet i es va anomenar MARK (per Maize Atypical Receptor Kinase). MARK presenta una estructura típica d'un receptor quinasa amb un domini extracel.lular, que conté 6 còpies imperfectes de LRR (Leucine- Rich Repeats), un únic domini transmembrana i un domini quinasa intracel.lular. El domini quinasa de MARK presenta, però, algunes variacions en els residus aminoacídics que es consideren claus per a la funció catalítica dels dominis quinasa. En concret cinc dels aminoàcids considerats essencials per a la fosforilació es troben substituits en el domini quinasa de MARK (DK-MARK). Els experiments de fosforilació in vitro que es van realitzar al laboratori, van mostrar com MARK era incapaç de fosforilar in vitro. Aquesta característica no és, però, exclusiva de MARK. Una búsqueda en les bases de dades ens van permetre identificar altres seqüències que també presentaven els mateixos o altres canvis en aquestes posicions aminoacídiques. En les bases de dades de plantes es van identificar un conjunt de seqüències genòmiques o ESTs amb aquestes característiques i només una d'elles, la proteïna TMKL1 d'Arabidopsis, ha sigut descrita com un receptor quinasa incapaç de fosforilar in vitro. Respecte a la búsqueda de receptors similars a MARK en les bases de dades d'animals, es van identificar també un conjunt de proteïnes que, en alguns casos, s'ha descrit que no tenen activitat quinasa in vivo. Per exemple, un dels casos més ben estudiats és el del receptor erbB3 que forma part de la família de receptors del EGF (Epidermal Growth Factor). Aquesta família de receptors està formada per 4 receptors: erbB1, erbB2, erbB3 i erbB4, dels quals només l'erbB3 no presenta activitat catalítica. S'ha descrit que erbB3 és capaç, tot i no fosforilar in vivo, de participar activament en la transducció del senyal formant heterodímers amb els altres membres de la família. Així, erbB3 és fosforilat pel seu partner i pot iniciar la cascada de transducció del senyal. La participació d'erbB3 en la transducció del senyal és essencial ja que embrions de ratolí knock-out pel gen erbB3 són inviables. Així doncs, el fet que receptors quinasa catalíticament inactius participin en les cascades de transducció del senyal, suggereix l'existència de nous mecanismes d'acció per a la transducció del senyal. Per tant, l'objectiu d'aquest treball fou l'estudi del mecanisme d'acció de MARK mitjançant la caracterització les proteïnes capaces d'interaccionar amb el seu domini quinasa. Per tal d'assolir aquest objectiu, es va realitzar un crivellat de doble-híbrid amb una llibreria de cDNA d'embrions de blat de moro de 7 DAP. D'aquest crivellat es va obtenir un conjunt de possibles clons positius que foren seqüenciats i entre els quals es van escollir per un estudi més detallat aquells que s'havien obtingut més vegades com a clons independents. Aquests clons codificaven per: una SAMDC (S-Adenosil Descarboxilasa), una eIF5 (Eukaryotic translation initiation), una hypothetical protein, una unknown protein, una gamma-adaptina i una MAP4K. Amb aquests 6 clons es van fer estudis in vitro i in vivo per tal de confirmar al seva interacció amb DK-MARK. Els estudis in vivo es van realitzar amb la soca de llevat AH109, una soca més astringent que la utilitzada en el crivellat, ja que presenta tres gens marcadors: Histidina, Adenina i Lacz. Els resultats obtinguts van mostrar que els clons codificants per SAMDC i eIF5 no van créixer en un medi selectiu per His i Ade i, per tant o es tracta de falsos positius del sistema o la seva interacció amb DK-MARK és dèbil. D'altra banda, la resta dels clons analitzats (proteïna hipotètica, una proteïna de funció desconeguda, la gamma-adaptina i una MAP4K) van créixer en medis en absència de Histidina i Adenina. Els assatjos de b-galactosidasa van ser tots positius a excepció de la proteïna hipotètica suggerint que potser aquesta interacció sigui més feble. D'altra banda també es van realitzar estudis in vitro amb la tècnica del pull-down. Els resultats obtinguts amb aquesta tècnica van recolzar els obtinguts en cèl.lules de llevat, ja que tots els clons analitzats a excepció dels codificants per SAMDC i eIF5 van donar un resultat d'interacció amb KD-MARK in vitro positiu. Davant aquests resultats ens vam centrar en l'estudi de la proteïna similar a MAP4K, doncs algunes proteïnes de la seva família s'han relacionat amb receptors de membrana. Els clons que es va obtenir del crivellat codificaven per una proteïna similar amb el domini C-terminal a les proteïnes BnMAP4Ka1 i a2 de Brassica napus. Aquestes proteïnes presenten una forta similitud de seqüència amb proteïnes de la família GCK/SPS1 que formen part d'un grup particular de MAPK relacionades amb la proteïna Ste20 (sterile 20 protein) de llevat. Ste20p activa la MAP3K de llevat Ste11 directament per fosforilació, transduint d'aquesta manera el senyal del receptor de feromones de creuament de les cèl.lules de llevat i es pot, doncs, considerar com una proteïna del tipus MAP4K (mitogen-activated protein kinase kinase kinase kinase). En els darrers anys, s'han identificat un gran nombre de proteïnes similars a Ste20: fins a una trentena en mamífers, en Drosophila, en Caenorhabditis elegans i en altres organismes. Segons la seva estructura aminoacídica, la família Ste20 s'ha classificat en dues subfamílies: les proteïnes STE20/PAK (p21-activated kinases) i la subfamília GCK/SPS1 (germinal center kinases). Les dues subfamílies estan formades per proteïnes que contenen un domini quinasa i un domini regulador, però, mentre que les proteïnes PAK presenten el domini quinasa en la part C-terminal, les GCKs el presenten en la regió N terminal. Les proteïnes GCK presenten una elevada diversitat estructural en el domini regulador permetent la seva classificació en 6 subfamílies. Mitjançant la tècnica del RACE es va obtenir el clon de cDNA complet que es va anomenar MIK (MARK Interacting Kinase). Amb la tècnica del Southern blot es va poder determinar que el gen MIK és un gen de còpia única en el genoma de blat de moro. Per tal d'analitzar la possible interacció entre DK-MARK i MIK, es va estudiar tant el patró d'expressió d'ambdós gens com el seu patró d'acumulació d'ambdues proteïnes durant l'embriogènesi del blat de moro. El patró d'expressió, analitzat per Northen blot va mostrar uns patrons coincidents al llarg de l'embriogènesi des del seu inici fins als 20 DAP amb una acumulació màxima de mRNA en embrions de 15 DAP. D'altra banda per tal d'estudiar el patró d'acumulació de la proteïna MIK així com per comparar-lo amb el de MARK, es van realitzar estudis de Westerns blot. Els resultats també van mostrar una coincidència en el temps de l'acumulació de les proteïnes MARK i MIK durant l'embriogènesi de blat de moro amb una major acumulació en embrions de 15 i 20 DAP. Es van dur a terme també estudis d'immunolocalitzacions sobre embrions de blat de moro de 15 DAP per tal d'estudiar en quins teixits s'acumulaven ambdues proteïnes. Les immunolocalitzacions van mostrar una major acumulació tant de MARK com de MIK en les zones meristemàtiques i en el teixit vascular sobretot del coleòptil on s'aprecia una forta co-localització de MARK i MIK. Totes aquestes dades són compatibles, doncs, amb una possible interacció de les proteïnes MARK i MIK, tot i que no la demostren. Per tal de demostrar la interacció es van realitzar experiments d'immunoprecipitació in vivo a partir d'extractes d'embrions. Malauradament, els resultats no són clars i en aquests moments en el laboratori s'estan posant a punt aquests experiments. També es van realitzar estudis comparatius de seqüència amb diferents proteïnes de la família GCK, mostrant una major similitud amb les proteïnes de la subfamília GCK-III. La subfamília GCK-III ha estat molt poc estudiada i en formen part un conjunt de proteïnes amb funcions molt diverses des de l'apoptosi, la citoquinesi o l'anòxia cel.lular. Per tant, la similitud de seqüència possiblement fa referència a una conservació en el mecanisme d'acció més que no pas a una conservació funcional. La possible interacció de MARK amb el domini C-terminal de MIK (el domini regulador) podria activar aquesta última iniciant una cascada de transducció del senyal en un model en el que una proteïna del tipus GCK-III faria de lligam directa entre un receptor de membrana i una cascada de senyalització intracel.lular. Aquest tipus de lligam entre un recepctor de membrana i mòduls intracel.lulars de senyalització s'ha descrit per a altres proteïnes GCK, si bé no directament sinó a través de proteïnes adaptadores. D'altra banda, la interacció directa de MARK, un receptor quinasa atípic que no té activitat catalítica, amb MIK suggereix un mecanisme on receptors atípics podrien interaccionar en la transducció del senyal activant la via de les MAPK.

Characterization of a gamma-aminobutyric acid transport system of rhizobium leguminosarum bv. viciae 3841

Spontaneous mutants of Rhizobium leguminosarum bv. viciae 3841 were isolated that grow faster than the wild type on gamma-aminobutyric acid (GABA) as the sole carbon and nitrogen source. These strains (RU1736 and RU1816) have frameshift mutations (gtsR101 and gtsR102, respectively) in a GntR-type regulator (GtsR) that result in a high rate of constitutive GABA transport. Tn5 mutagenesis and quantitative reverse transcription-PCR showed that GstR regulates expression of a large operon (pRL100242 to pRL100252) on the Sym plasmid that is required for GABA uptake. An ABC transport system, GtsABCD (for GABA transport system) (pRL100248-51), of the spermidine/putrescine family is part of this operon. GtsA is a periplasmic binding protein, GtsB and GtsC are integral membrane proteins, and GtsD is an ATP-binding subunit. Expression of gtsABCD from a lacZ promoter confirmed that it alone is responsible for high rates of GABA transport, enabling rapid growth of strain 3841 on GABA. Gts transports open-chain compounds with four or five carbon atoms with carboxyl and amino groups at, or close to, opposite termini. However, aromatic compounds with similar spacing between carboxyl and amino groups are excellent inhibitors of GABA uptake so they may also be transported. In addition to the ABC transporter, the operon contains two putative mono-oxygenases, a putative hydrolase, a putative aldehyde dehydrogenase, and a succinate semialdehyde dehydrogenase. This suggests the operon may be involved in the transport and breakdown of a more complex precursor to GABA. Gts is not expressed in pea bacteroids, and gtsB mutants are unaltered in their symbiotic phenotype, suggesting that Bra is the only GABA transport system available for amino acid cycling.

Excision of anabaena PCC 7120 nifD element in escherichia coli: growth kinetics and RecA regulated xisA expression and DNA rearrangement

Anabaena PCC 7120 nifHDK operon is interrupted by an 11 kb DNA element which is excised during the development of heterocysts by Excisase A, encoded by the xisA gene residing on the element. The excision is a site-specific recombination event that occurs at the I I base pair direct repeats flanking the element. Earlier work showed the excision of the I I kb element in Escherichia coli at a frequency 0.3%. We report here the excision of this element at 1.1% and 1.98% in E. coli DH5 alpha, and 1.9% and 10.9% in E. coli JM 101 when grown on Luria broth and minimal media, respectively. Excision of nifD element in isogenic recA(-) (RK1) and recA(+) (RK2) E. coli JM101 P1 transductants, showed similar results to that of E. coli JM101 and DH5 alpha, respectively. A plasmid pMX32, carrying a xisA defective 11 kb element, showed no excision in E. coli RK2 strain. In contrast to Anabaena PCC 7120, excision of nifD element did not increase in E. call DH5 alpha grown in iron-deficient conditions. A PxisA::lacZ transcriptional fusion, used to detect the expression of elusive xisA gene, showed maximal beta-galactosidase activity in the stationary phase. The results suggest that the excision event in E. coli may involve additional factors, such as RecA and that the physiological status can influence the excision of nifD element. (C) 2007 Elsevier Ltd. All rights reserved.

A family of promoter probe vectors incorporating autofluorescent and chromogenic reporter proteins for studying gene expression in Gram-negative bacteria

A series of promoter probe vectors for use in Gram-negative bacteria has been made in two broad-host-range vectors, pOT (pBBR replicon) and pJP2 (incP replicon). Reporter fusions can be made to gfpUV, gfprnut3.1, unstable gfpmut3.1 variants (LAA, LVA, AAV and ASV), gfp+, dsRed2, dsRedT3, dsRedT4, mRFP1, gusA or lacZ. The two vector families, pOT and pJP2, are compatible with one another and share the same polylinker for facile interchange of promoter regions. Vectors based on pJP2 have the advantage of being ultra-stable in the environment due to the presence of the parABCDE genes. As a confirmation of their usefulness, the dicarboxylic acid transport system promoter (dctA(p)) was cloned into a pOT (pRU1097)- and a pJP2 (pRU1156)-based vector and shown to be expressed by Rhizobium leguminosarum in infection threads of vetch. This indicates the presence of dicarboxylates at the earliest stages of nodule formation.

The activity of ribosome modulation factor during growth of Escherichia coli under acidic conditions

Expression of the gene encoding ribosome modulation factor (RMF), as measured using an rmf-lacZ gene fusion, increased with decreasing pH in exponential phase cultures of Escherichia coli. Expression was inversely proportional to the growth rate and independent of the acidifying agent used and it was concluded that expression of rmf was growth rate controlled in exponential phase under acid conditions. Increased rmf expression during exponential phase was not accompanied by the formation of ribosome dimers as occurs during stationary phase. Nor did it appear to have a significant effect on cell survival under acid stress since the vulnerability of an RMF-deficient mutant strain was similar to that of the parent strain. Ribosome degradation was increased in the mutant strain compared to the parent strain at pH 3.75. Also, the peptide elongation rate was reduced in the mutant strain but not the parent during growth under acid conditions. It is speculated that the function of RMF during stress-induced reduction in growth rate is two-fold: firstly to prevent reduced elongation efficiency by inactivating surplus ribosomes and thus limiting competition for available protein synthesis factors, and secondly to protect inactivated ribosomes from degradation.

CztR, a LysR-Type Transcriptional Regulator Involved in Zinc Homeostasis and Oxidative Stress Defense in Caulobacter crescentus

Caulobacter crescentus is a free-living alphaproteobacterium that has 11 predicted LysR-type transcriptional regulators (LTTRs). Previously, a C. crescentus mutant strain with a mini-Tn5lacZ transposon inserted into a gene encoding an LTTR was isolated; this mutant was sensitive to cadmium. In this work, a mutant strain with a deletion was obtained, and the role of this LTTR (called CztR here) was evaluated. The transcriptional start site of this gene was determined by primer extension analysis, and its promoter was cloned in front of a lacZ reporter gene. beta-Galactosidase activity assays, performed with the wild-type and mutant strains, indicated that this gene is 2-fold induced when cells enter stationary phase and that it is negatively autoregulated. Moreover, this regulator is essential for the expression of the divergent cztA gene at stationary phase, in minimal medium, and in response to zinc depletion. This gene encodes a hypothetical protein containing 10 predicted transmembrane segments, and its expression pattern suggests that it encodes a putative zinc transporter. The cztR strain was also shown to be sensitive to superoxide (generated by paraquat) and to hydrogen peroxide but not to tert-butyl hydroperoxide. The expression of katG and ahpC, but not that of the superoxide dismutase genes, was increased in the cztR mutant. A model is proposed to explain how CztR binding to the divergent regulatory regions could activate cztA expression and repress its own transcription.

Environmental factors affecting transcription of the human L1 retrotransposon. I. Steroid hormone-like agents

The L1 retrotransposon has significantly shaped the structure of the human genome. At least 30% of human genome sequence can be attributed to L1 reverse transcriptase activity. There are 105 copies of the human L1 retrotransposon, L1Hs, most of which are defective, although ~8–9x103 are full length. L1Hs elements transpose through an RNA intermediate and transcription is thought to be the rate limiting step in retrotransposition. Because transcription of retrotransposons in a variety of organisms has been shown to respond to environmental stimuli, we investigated the influence of various agents on transcription from two different L1Hs promoters. The activity of the L1Hs promoters was analyzed by transfecting L1Hs-expressing cell lines with plasmids containing the L1Hs promoters fused to the LacZ reporter gene and monitoring expression with a ß-galactosidase assay. Small increases in ß-galactosidase activity were observed with both L1Hs promoters after treatment with serum, testosterone, dihydrotestosterone and organochloride pesticides, indicating that these agents can influence L1Hs transcription.

Environmental factors affecting transcription of the human L1 retrotransposon. II. Stressors

Retrotransposons have clearly molded the structure of the human genome. The reverse transcriptase coded for by long interspersed nuclear elements (LINEs) accounts for 35% of the human genome, with 8–9 x 105 copies of the most common human LINE element, L1Hs. Retrotransposons cycle through an RNA intermediate with transcription as the rate limiting step. Because various retrotransposons have been demonstrated to be induced by environmental stimuli, we investigated the response of the L1Hs promoter to various agents. L1Hs promoter activity was analyzed by transfecting an L1Hs-expressing cell line with plasmids containing one of two L1Hs promoters fused to the LacZ reporter gene. L1Hs promoter activity was then monitored with a ß-galactosidase assay. Treatment with UV light and heat shock resulted in a small increase in ß-galactosidase activity from one promoter, while treatment with tetradecanoylphorbol 13-acetate resulted in small increases in ß-galactosidase activity from both promoters. No increase in ß-galactosidase activity was observed after exposure to X-rays or hydrogen peroxide.

Adamts5, the gene encoding a proteoglycan-degrading metalloprotease, is expressed by specific cell lineages during mouse embryonic development and in adult tissues.

The secreted metalloprotease ADAMTS5 is implicated in destruction of the cartilage proteoglycan aggrecan in arthritis, but its physiological functions are unknown. Its expression profile during embryogenesis and in adult tissues is therefore of considerable interest. β-Galactosidase (β-gal) histochemistry, enabled by a LacZ cassette inserted in the Adamts5 locus, and validated by in situ hybridization with an Adamts5 cRNA probe and ADAMTS5 immunohistochemistry, was used to profile Adamts5 expression during mouse embryogenesis and in adult mouse tissues. Embryonic expression was scarce prior to 11.5 days of gestation (E11.5) and noted only in the floor plate of the developing brain at E9.5. After E11.5 there was continued expression in brain, especially in the choroid plexus, peripheral nerves, dorsal root ganglia, cranial nerve ganglia, spinal and cranial nerves, and neural plexuses of the gut. In addition to nerves, developing limbs have Adamts5 expression in skeletal muscle (from E13.5), tendons (from E16.5), and inter-digital mesenchyme of the developing autopod (E13.5–15.5). In adult tissues, there is constitutive Adamts5 expression in arterial smooth muscle cells, mesothelium lining the peritoneal, pericardial and pleural cavities, smooth muscle cells in bronchi and pancreatic ducts, glomerular mesangial cells in the kidney, dorsal root ganglia, and in Schwann cells of the peripheral and autonomic nervous system. Expression of Adamts5 during neuromuscular development and in smooth muscle cells coincides with the broadly distributed proteoglycan versican, an ADAMTS5 substrate. These observations suggest the major contexts in which developmental and physiological roles could be sought for this protease.

Identification and characterization of genes conferring salt tolerance to Escherichia coli from pond water metagenome

Metagenomics provides culture-independent access to gene pool of the whole microbial communities. To identify genes responsible for salt tolerance in unculturable bacteria, Escherichia coli clones were enriched with an ability to grow at inhibitory NaCl concentrations (750 mM) from a pond water metagenomic library. From two unique clones, genes encoding for proteins with similarity to a putative general stress protein (GspM) harbouring GsiB domain and a putative enoyl-CoA hydratase (EchM) were identified to be responsible for salt tolerance. The gspM was expressed by its native promoter whereas the echM was expressed from the lacZ promoter of the plasmid. EchM was overexpressed with a hexahistidyl tag. Purified EchM showed crotonyl-CoA hydratase activity. These genes have potential application in generating salt tolerant recombinant bacteria or transgenic plants.

Regulation of Denitrification Genes in Neisseria meningitidis by Nitric Oxide and the Repressor NsrR

The human pathogen Neisseria meningitidis is capable of growth using the denitrification of nitrite to nitrous oxide under microaerobic conditions. This process is catalyzed by two reductases: nitrite reductase (encoded by aniA) and nitric oxide (NO) reductase (encoded by norB). Here, we show that in N. meningitidis MC58 norB is regulated by nitric oxide via the product of gene NMB0437 which encodes NsrR. NsrR is a repressor in the absence of NO, but norB expression is derepressed by NO in an NsrR-dependent manner. nsrR-deficient mutants grow by denitrification more rapidly than wild-type N. meningitidis, and this is coincident with the upregulation of both NO reductase and nitrite reductase even under aerobic conditions in the absence of nitrite or NO. The NsrR-dependent repression of aniA (unlike that of norB) is not lifted in the presence of NO. The role of NsrR in the control of expression of aniA is linked to the function of the anaerobic activator protein FNR: analysis of nsrR and fnr single and nsrR fnr double mutants carrying an aniA promoter lacZ fusion indicates that the role of NsrR is to prevent FNR-dependent aniA expression under aerobic conditions, indicating that FNR in N. meningitidis retains considerable activity aerobically.

Caracterização molecular dos genes PSO2 e PSO4 de Saccharomyces cerevisiae envolvidos na reparação do DNA : análise funcional e identificação de interações proteína-proteína

O alelo mutante termo-condicionalmente letal pso4-1 do gene PRP19, que codifica uma proteína associada ao spliceossoma, permitiu investigar a influência deste gene no processamento de pré-mRNA, reparação do DNA e esporulação. Fenótipos relacionados a genes portadores de introns foram correlacionados à temperatura. Sistemas repórteres para processamento de pré-mRNA e RT-PCR mostraram que eficiência de processamento de mRNA no mutante pso4-1 é inversamente correlacionada com a temperatura de crescimento. Uma mutação pontual, substituindo uma leucina por uma serina foi identificada dentro da região codificadora N-terminal do alelo Pso4-1 e afeta as propriedades bioquímicas de Pso4-1p. Entre 24 clones isolados utilizando o sistema doishíbridos, 7 foram identificados como partes dos genes RAD2, RLF2 e DBR1. RAD2 codifica uma endonuclease indispensável para a via reparação por excisão de nucleotídeos (NER), RLF2 codifica a subunidade maior do fator de montagem da cromatina I, cuja deleção resulta em sinsibilidade à radiação UVC, enquanto que DBR1 codifica uma enzima que atua sobre substratos de RNA na forma lariat, degradando estruturas lariat em introns durante o processamento de mRNA. A caracterização dos fenótipos após tratamentos com mutágenos em linhagens mutantes simples e duplos de rad2∆, rlf2∆ e pso4-1 mostraram sensibilidade aumentada para para mutantes rad2∆/pso4-1 e rlf2∆/pso4-1, sugerindo uma interferência funcional destas proteínas no processo dereparação do DNA em Saccharomyces cerevisiae. O mecanismo exato de reparação de pontes inter-cadeia (ICL) em S. cerevisiae não é ainda totalmente conhecido. Identificando novos fenótipos e isolando proteínas potencialmente capazes de interagir com Pso2p através da técnica do sistema dois-híbridos, foi possível extender a caracterização deste gene específica para reparação de pontes intercadeia. Tratamentos com acetaldeído (ACA), um metabólito natural da via glicolítica, foi mais tóxico a linhagem mutante pso2 em comparação com a linhagem selvagem e também capaz de induzir a expressão da fusão contendo o promotor de PSO2 à lacZ (PSO2-lacZ) por um fator comparável à tratamentos com outros agentes indutores de ICLs, indicando que o metabólito natural ACA pode causar danos do tipo ICL em S. cerevisiae. A utilização do sistema dois-híbridos permitiu isolar partes de proteínas codificadas por nove diferentes genes, entre eles a proteína quinase Pak1p, um supressor de mutações termosensíveis da DNA Polimerase alfa. Pak1p interage com a extremidade C-terminal conservada de Pso2p, uma região da proteína recentemente nomeada β-CASP entre v ortólogos conhecidos do gene PSO2. A integridade do domínio β-CASP é essencial para a reparaçãode DNA proficiente como demonstrado em ensaios de complementação com mutantes pso2 ∆. Comparação da sobrevivência após tratamento com agentes mutagênicos de simples mutantes pso2 ∆ e pak1 ∆ assim com o dulpo mutante pso2 ∆/pak1 ∆ revelaram que o gene PAK1 é necessário para reparação do DNA proficiente como na linhagem selvagem. A interação epistática dos dois alelos mutantes na linhagem duplo mutante sugere que Pak1p atua na mesma via de reparação a qual PSO2 pertence e que PAK1 constitui um novo locus envolvido na reparação do DNA em S. cerevisiae.

Estratégias de Produção in vitro de Bioinseticida Viral: influências do Isolado, da Cinética e do Modo de operação

Societal concerns about environmental sustainability has lead to the development of ecologically-friendly alternatives to chemical insecticides for crop protection. One such alternative is biological pest control. In particular, baculoviruses are well suited as insect biopesticides due to their narrow host specificity and relative ease of propagation. In Brazil, the baculovirus Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) is the main biological control agent employed for the soybean pest, Anticarsia gemmatalis. This baculovirus biopesticide is currently produced using caterpillars, but increasing market demand for the product has encouraged the development of an in vitro manufacturing process, which can be scaled up to much higher virus productivities. In this study, three wild-type AgMNPV isolates (AgMNPV-2D, AgMNPV-MP2 and AgMNPV-MP5) and a recombinant form (vAgEGT-LacZ) were characterised in terms of occlusion body (OB) production and infection kinetics, to enable future optimisation of the in vitro production process. These viruses were propagated using a Spodoptera frugiperda (IPLB-SF21) insect cell line grown in shaker-flask batch cultures. Among the virus isolates tested, AgMNPV-MP5 was found to be the best producer, yielding (5.3±0.85)x108 OB/mL after 8 days post infection. The characterisation of vAgEGT-LacZ propagation in suspension cell cultures has not been previously reported in the literature; hence it became the main focus for this thesis. In particular, it was carried out a study on the effect of the multiplicity of infection (MOI) on OB production. Five successive batches were performed getting a final production (8.9±1.42)x1014 occlusion bodies, considering that production is related for a bioreactor with final volume of 10m3. A low MOI associated with a fed-batch process for vAgEGT-LacZ production was found to support a 3-fold higher OB yield when compared to the default batch process (1.8x107 and 5.3x107 OB/mL, respectively). This yield is competitive with regards to the production process.

Functional and Ultrastructural Analysis of Annexin A1 and Its Receptor in Extravasating Neutrophils during Acute Inflammation

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

Cold Shock Genes cspA and cspB from Caulobacter crescentus Are Posttranscriptionally Regulated and Important for Cold Adaptation

Cold shock proteins (CSPs) are nucleic acid binding chaperones, first described as being induced to solve the problem of mRNA stabilization after temperature downshift. Caulobacter crescentus has four CSPs: CspA and CspB, which are cold induced, and CspC and CspD, which are induced only in stationary phase. In this work we have determined that the synthesis of both CspA and CspB reaches the maximum levels early in the acclimation phase. The deletion of cspA causes a decrease in growth at low temperature, whereas the strain with a deletion of cspB has a very subtle and transient cold-related growth phenotype. The cspA cspB double mutant has a slightly more severe phenotype than that of the cspA mutant, suggesting that although CspA may be more important to cold adaptation than CspB, both proteins have a role in this process. Gene expression analyses were carried out using cspA and cspB regulatory fusions to the lacZ reporter gene and showed that both genes are regulated at the transcriptional and posttranscriptional levels. Deletion mapping of the long 5'-untranslated region (5'-UTR) of each gene identified a common region important for cold induction, probably via translation enhancement. In contrast to what was reported for other bacteria, these cold shock genes have no regulatory regions downstream from ATG that are important for cold induction. This work shows that the importance of CspA and CspB to C. crescentus cold adaptation, mechanisms of regulation, and pattern of expression during the acclimation phase apparently differs in many aspects from what has been described so far for other bacteria.