Organic geochemistry of sediment samples from Prydz Bay

High molecular weight aliphatic hydrocarbons were extracted from sediments at two sites (741 and 742) drilled during Ocean Drilling Program Leg 119 in Prydz Bay, a major embayment on the continental shelf of East Antarctica. The distributions of n-alkanes and triterpenoid and steroid hydrocarbons suggest that the n-alkanes and steranes are mainly of terrestrial origin and that the hydrocarbons are immature to slightly mature in the Lower Cretaceous sediments and immature to mature in the Tertiary sediments. At Site 741, the Lower Cretaceous depositional sequence, which is generally characterized by immature hydrocarbons, is interrupted by sediment having more mature components, suggesting a change of source during part of Early Cretaceous time. At Site 742, the mature geochemical parameters of a Pliocene sample correlate with results reported elsewhere for Site 739. In all but one of the other Tertiary samples, the geochemical parameters indicate intermediate maturity. The Lower Cretaceous and Pliocene sediments average about 1.9% organic carbon, a value of interest from the point of view of potential sources of petroleum offshore from Antarctica.

Radiocarbon ages and major n-alkane homologues of peat cores LVPS4 and LVPS5B, Russian Arctic

Coupled analyses of n-alkane biomarkers and plant macrofossils from a peat plateau deposit in the northeast European Russian Arctic were carried out to assess the effects of past hydrology on the molecular contributions of plants to the peat. The n-alkane biomarkers accumulated over 9.6 kyr of local paleohydrological changes in this complex peat profile in which a succession of vegetation changes occurred during a transition from a wet fen to a relatively dry peat plateau bog. This study shows that the contribution of the n-C31 alkane from rootlets to peat layers rich in fine and dark roots is important. The results further indicate that the n-alkane Paq and n-C23/n-C29 biomarker proxies that have been useful to reconstruct past water table levels in many peat deposits can be misleading when the contributions of Betula and Sphagnum fuscum to the peat are large. Under these conditions, the C23/(C27 + C31) n-alkane ratio seems to correct for the presence of Betula and S. fuscum and provides a better description for the relative amounts of moisture. The average chain length (ACL) n-alkane proxy also appears to be a good paleohydrology proxy in having larger values during dry and cold conditions in this Arctic bog setting.

Impact of elevated pCO2 on paralytic shellfish poisoning toxin content and composition in Alexandrium tamarense

Ocean acidification is considered a major threat to marine ecosystems and may particularly affect primary producers. Here we investigated the impact of elevated pCO2 on paralytic shellfish poisoning toxin (PST) content and composition in two strains of Alexandrium tamarense, Alex5 and Alex2. Experiments were carried out as dilute batch to keep carbonate chemistry unaltered over time. We observed only minor changes with respect to growth and elemental composition in response to elevated pCO2. For both strains, the cellular PST content, and in particular the associated cellular toxicity, was lower in the high CO2 treatments. In addition, Alex5 showed a shift in its PST composition from a nonsulfated analogue towards less toxic sulfated analogues with increasing pCO2. Transcriptomic analyses suggest that the ability of A. tamarense to maintain cellular homeostasis is predominantly regulated on the post-translational level rather than on the transcriptomic level. Furthermore, genes associated to secondary metabolite and amino acid metabolism in Alex5 were down-regulated in the high CO2 treatment, which may explain the lower PST content. Elevated pCO2 also induced up-regulation of a putative sulfotransferase sxtN homologue and a substantial down-regulation of several sulfatases. Such changes in sulfur metabolism may explain the shift in PST composition towards more sulfated analogues. All in all, our results indicate that elevated pCO2 will have minor consequences for growth and elemental composition, but may potentially reduce the cellular toxicity of A. tamarense.

Nuevos mecanismos moleculares de tolerancia a sequía y otros tipos de estrés abiótico en especies arbóreas de interés económico

Las masas forestales tienen una importancia colosal para nuestra sociedad y el conjunto de la biosfera. Estudios recientes a escala mundial indican que la sequía es el factor abiótico que más afecta a su crecimiento y supervivencia, seguida por las temperaturas extremas y la salinidad. Aunque comprender los mecanismos con que las especies arbóreas toleran estas formas de estrés tiene un interés aplicado evidente, dichos mecanismos se han estudiado mucho más en especies herbáceas modelo o de interés agronómico. Existen sin embargo diferencias notables entre ellas, como se demuestra en esta tesis y en otros trabajos recientes. Nuestro estudio se centra concretamente en la respuesta molecular del chopo –el sistema modelo forestal más desarrollado– al estrés abiótico, con particular énfasis en la sequía. Utilizando una estrategia proteómica y tratamientos controlados, hemos identificado componentes mayoritarios de dicha respuesta. Su participación en la misma se ha validado mediante análisis transcripcionales detallados utilizando tecnología qRT-PCR (PCR cuantitativa en tiempo real). Hemos identificado proteínas cuyo nexo funcional con mecanismos de tolerancia ya era conocido, como chaperonas moleculares sHSP o enzimas que atenúan el estrés oxidativo, pero también proteínas cuya relación funcional con el estrés es menos clara o incluso novedosa, como polifenol oxidasas (PPO), deshidrogenasas/reductasas de cadena corta (SDR), o bicupinas (BIC), entre otras. El cuerpo central de la tesis consiste en la caracterización detallada de una PPO inusual, cuya inducción por estrés hídrico se describe por vez primera. Estas enzimas están ampliamente distribuidas en plantas, si bien su número es muy variable de unas especies a otras. Algunas, como nogal, tienen un único gen, mientras que Arabidopsis no tiene ninguno. En la última versión del genoma de chopo hemos identificado un total de 12 miembros bona fide, corrigiendo trabajos previos, y hemos caracterizado su expresión individual ante diferentes situaciones de estrés controlado y tratamientos hormonales. La isoforma antedicha es el único miembro de la familia que responde claramente a la deshidratación. También responde a salinidad y a la mayor parte de tratamientos hormonales ensayados, pero no a daño mecánico o tratamientos con metil jasmonato. Esto la diferencia de enzimas homólogas presentes en otras especies de plantas, que se han relacionado experimentalmente con estrés biótico. Los patrones de acumulación de transcritos en árboles adultos son compatibles con un papel protector frente a la sequía. La integración de nuestros estudios funcionales y filogenéticos sugiere que la familia ha sufrido un proceso reciente de diversificación y neofuncionalización, siendo la protección frente a deshidratación su papel primigenio. Aunque se conoce la actividad bioquímica in vitro de este tipo de enzimas, sus sustratos naturales son esencialmente una incógnita. Mediante expresión heteróloga en Escherichia coli BL21(DE3) hemos detectado que la enzima de chopo es capaz de oxidar L-DOPA a dopaquinona, siendo menos activa frente a otros sustratos. Por otra parte, hemos demostrado su localización cloroplástica mediante transformación transitoria de protoplastos con fusiones a la proteína fluorescente YFP. Mediante la obtención de plantas transgénicas de A. thaliana hemos demostrado que la enzima de chopo aumenta considerablemente la tolerancia in vivo frente a la deshidratación y al estrés salino. El análisis fenotípico detallado de las líneas transgénicas, combinando múltiples metodologías, nos ha permitido sustanciar que la tolerancia tiene una base compleja. Esta incluye una mayor protección del sistema fotosintético, una capacidad antioxidante muy incrementada y la acumulación de solutos osmoprotectores como la prolina. Los análisis metabolómicos nos han permitido asociar la expresión de la proteína a la síntesis de un flavano no descrito previamente en A. thaliana, vinculando la enzima de chopo con la síntesis de fenilpropanoides. También hemos observado alteraciones en los niveles hormonales que podrían subyacer a efectos pleiotrópicos con interés aplicado, como un aumento consistente del tamaño de la planta o el acortamiento del ciclo de crecimiento. Además de aportar datos novedosos sobre la funcionalidad in vivo de esta familia de oxidasas, los resultados de esta tesis demuestran que los árboles son sistemas de estudio interesantes para caracterizar nuevas estrategias de tolerancia al estrés abiótico con potencial aplicado. ABSTRACT Forests masses have an extraordinary importance for our society and the biosphere. Recent worldwide studies indicate that drought is the abiotic factor that affects more their growing and survival, followed by extreme temperatures and salinity. The understanding of how the arboreal species tolerate the stress has an evident practical interest, but their mechanisms have been studied much more in herbaceous species or with agronomic interest. However, considerable differences exist between them, as this thesis and recent studies show. Our study is focused on the molecular response of the poplar –the more developed forestry model system- to abiotic stress, specifically focused in the drought. Using a proteomic strategy and controlled treatments, we have identified main components in such response. Its participation has been validated through transcriptional analysis using qRT-PCR technology. We have identified proteins whose functional connection with tolerance mechanisms were already known, as molecular chaperones sHSP or enzymes that attenuate the oxidative stress, but also some proteins whose functional relationship with the stress is less clear or even novel, as polifenol oxidases (PPO), short chain deshidrogenases/reductases (SDR), or bicupines (BIC), among others. The central body of the thesis consists of the detailed characterization of an unsual PPO, whose induction due to drought stress is first described. These enzymes are thoroughly distributed in plants, but their number of members is very variable among species. Some of them, as the walnut tree, have a single gene, while Arabidopsis has none. We have identified a total of 12 members in the last version of the poplar genome, correcting previous works, and have characterized their individual expression against different situations of controlled stress and hormone treatments. The aforementioned isoform is the only member of the family that responds clearly to the drought. It also reacts to salinity and the majority of hormonal treatments tested, but it does not respond to mechanical damage or treatments with methyl jasmonate. This is the difference with homologue enzymes present in other plant species, which have been related experimentally with abiotic stress. The accumulation patterns of transcripts in adult trees are compatible with a protector role against drought. The integration of our functional and phylogenetic studies suggests that the family has suffered a recent process of diversification and neofunctionalization, being the protection against drought their original role. Although the in vitro biochemistry activity of this kind of enzymes is already known, their natural substracts are essentially a mystery. By means of heterologous expression of Escherichia coli BL21(DE3) we have detected that the enzyme of poplar is able to oxidize L-DOPA to dopaquinone, being less active against other substrates. Additionally, we have proven its chloroplastic location with transitory transformation of protoplasts with YFP protein fusion. By means of getting transgenic plants of A. thaliana, we have demonstrated that the poplar enzyme increases notably the in vivo tolerance against the drought and salinity stresses. The phenotypic analysis of the transgenic lines, and the use of multiple methodologies, allowed us to test the complexity of the tolerance. This includes a major protection of the photosynthetic system, a very increased antioxidant capacity and the accumulation of osmoprotectant solutes as the proline. The metabolic analysis has allowed to associate the protein expression with the synthesis of a Flavan non described previously in A. thalaiana, linking the enzyme of poplar with the synthesis of phenylpropanoids. We have observed alterations in the hormonal levels that could underlie pleiotropic effects with applied interest, as a consistent increase of the size of the plant and the reduction of the growth cycle. The results of this thesis, in addition to provide novel data about the in vivo functionality of the oxidase family, demonstrate that the trees are interesting systems of study to characterize new strategies of tolerance against abiotic stress with applied potential.

A cluster of oppositely imprinted transcripts at the Gnas locus in the distal imprinting region of mouse chromosome 2

Imprinted genes tend to occur in clusters. We have identified a cluster in distal mouse chromosome (Chr) 2, known from early genetic studies to contain both maternally and paternally imprinted, but unspecified, genes. Subsequently, one was identified as Gnas, which encodes a G protein α subunit, and there is clinical and biochemical evidence that the human homologue GNAS1, mutated in patients with Albright hereditary osteodystrophy, is also imprinted. We have used representational difference analysis, based on parent-of-origin methylation differences, to isolate candidate imprinted genes in distal Chr 2 and found two oppositely imprinted genes, Gnasxl and Nesp. Gnasxl determines a variant G protein α subunit associated with the trans-Golgi network and Nesp encodes a secreted protein of neuroendocrine tissues. Gnasxl is maternally methylated in genomic DNA and encodes a paternal-specific transcript, whereas Nesp is paternally methylated with maternal-specific expression. Their reciprocal imprinting may offer insight into the distal Chr 2 imprinting phenotypes. Remarkably, Gnasxl, Nesp, and Gnas are all part of the same transcription unit; transcripts for Gnasxl and Nesp are alternatively spliced onto exon 2 of Gnas. This demonstrates an imprinting mechanism in which two oppositely imprinted genes share the same downstream exons.

Ubiquitin-dependent degradation of IκBα is mediated by a ubiquitin ligase Skp1/Cul 1/F-box protein FWD1

Activation of the transcription factor nuclear factor kappa B (NF-κB) is controlled by proteolysis of its inhibitory subunit (IκB) via the ubiquitin-proteasome pathway. Signal-induced phosphorylation of IκBα by a large multisubunit complex containing IκB kinases is a prerequisite for ubiquitination. Here, we show that FWD1 (a mouse homologue of Slimb/βTrCP), a member of the F-box/WD40-repeat proteins, is associated specifically with IκBα only when IκBα is phosphorylated. The introduction of FWD1 into cells significantly promotes ubiquitination and degradation of IκBα in concert with IκB kinases, resulting in nuclear translocation of NF-κB. In addition, FWD1 strikingly evoked the ubiquitination of IκBα in the in vitro system. In contrast, a dominant-negative form of FWD1 inhibits the ubiquitination, leading to stabilization of IκBα. These results suggest that the substrate-specific degradation of IκBα is mediated by a Skp1/Cull 1/F-box protein (SCF) FWD1 ubiquitin-ligase complex and that FWD1 serves as an intracellular receptor for phosphorylated IκBα. Skp1/Cullin/F-box protein FWD1 might play a critical role in transcriptional regulation of NF-κB through control of IκB protein stability.

The p42 variant of ETS1 protein rescues defective Fas-induced apoptosis in colon carcinoma cells

ETS1 is a cellular homologue of the product of the viral ets oncogene of the E26 virus, and it functions as a tissue-specific transcription factor. It plays an important role in cell proliferation, differentiation, lymphoid cell development, transformation, angiogenesis, and apoptosis. ETS1 controls the expression of critical genes involved in these processes by binding to ets binding sites present in the transcriptional regulatory regions. The ETS1 gene generates two proteins, p51 and a spliced variant, p42, lacking exon VII. In this paper we show that p42-ETS1 expression bypasses the damaged Fas-induced apoptotic pathway in DLD1 colon carcinoma cells by up-regulating interleukin 1β-converting enzyme (ICE)/caspase-1 and causes these cancer cells to become susceptible to the effects of the normal apoptosis activation system. ICE/caspase-1 is a redundant system in many cells and tissues, and here we demonstrate that it is important in activating apoptosis in cells where the normal apoptosis pathway is blocked. Blocking ICE/caspase-1 activity by using specific inhibitors of this protease prevents the p42-ETS1-induced apoptosis from occurring, indicating that the induced ICE/caspase-1 enzyme is responsible for killing the cancer cells. p42-ETS1 activates a critical alternative apoptosis pathway in cancer cells that are resistant to normal immune attack, and thus it may be useful as an anticancer therapeutic.

MED1, a novel human methyl-CpG-binding endonuclease, interacts with DNA mismatch repair protein MLH1

The DNA mismatch repair (MMR) is a specialized system, highly conserved throughout evolution, involved in the maintenance of genomic integrity. To identify novel human genes that may function in MMR, we employed the yeast interaction trap. Using the MMR protein MLH1 as bait, we cloned MED1. The MED1 protein forms a complex with MLH1, binds to methyl-CpG-containing DNA, has homology to bacterial DNA repair glycosylases/lyases, and displays endonuclease activity. Transfection of a MED1 mutant lacking the methyl-CpG-binding domain (MBD) is associated with microsatellite instability (MSI). These findings suggest that MED1 is a novel human DNA repair protein that may be involved in MMR and, as such, may be a candidate eukaryotic homologue of the bacterial MMR endonuclease, MutH. In addition, these results suggest that cytosine methylation may play a role in human DNA repair.

Characterization of maize (Zea mays L.) Wee1 and its activity in developing endosperm

We report the characterization of a maize Wee1 homologue and its expression in developing endosperm. Using a 0.8-kb cDNA from an expressed sequence tag project, we isolated a 1.6-kb cDNA (ZmWee1), which encodes a protein of 403 aa with a calculated molecular size of 45.6 kDa. The deduced amino acid sequence shows 50% identity to the protein kinase domain of human Wee1. Overexpression of ZmWee1 in Schizosaccharomyces pombe inhibited cell division and caused the cells to enlarge significantly. Recombinant ZmWee1 obtained from Escherichia coli is able to inhibit the activity of p13suc1-adsorbed cyclin-dependent kinase from maize. ZmWee1 is encoded by a single gene at a locus on the long arm of chromosome 4. RNA gel blots showed the ZmWee1 transcript is about 2.4 kb in length and that its abundance reaches a maximum 15 days after pollination in endosperm tissue. High levels of expression of ZmWee1 at this stage of endosperm development imply that ZmWee1 plays a role in endoreduplication. Our results show that control of cyclin-dependent kinase activity by Wee1 is conserved among eukaryotes, from fungi to animals and plants.

Identification and characterization of the ARP1 gene, a target for the human acute leukemia ALL1 gene

ALL1, the human homologue of Drosophila trithorax, is directly involved in human acute leukemias associated with abnormalities at 11q23. Using the differential display method, we isolated a gene that is down-regulated in All1 double-knockout mouse embryonic stem (ES) cells. The gene, designated ARP1 (also termed RIEG, Ptx2, or Otlx2), is a member of a family of homeotic genes containing a short motif shared with several homeobox genes. Using a bacterially synthesized All1 polypeptide encompassing the AT-hook motifs, we identified a 0.5-kb ARP1 DNA fragment that preferentially bound to the polypeptide. Within this DNA, a region of ≈100 bp was protected by the polypeptide from digestion with ExoIII and DNase I. Whole-mount in situ hybridization to early mouse embryos of 9.5–10.5 days indicated a complex pattern of Arp1 expression spatially overlapping with the expression of All1. Although the ARP1 gene is expressed strongly in bone marrow cells, no transcripts were detected in six leukemia cell lines with 11q23 translocations. These results suggest that ARP1 is up-regulated by the All1 protein, possibly through direct interaction with an upstream DNA sequence of the former. The results are also consistent with the suggestion that ALL1 chimeric proteins resulting from 11q23 abnormalities act in a dominant negative fashion.

Protein kinase mutants of human ATR increase sensitivity to UV and ionizing radiation and abrogate cell cycle checkpoint control

In fission yeast, the rad3 gene product plays a critical role in sensing DNA structure defects and activating damage response pathways. A structural homologue of rad3 in humans (ATR) has been identified based on sequence similarity in the protein kinase domain. General information regarding ATR expression, protein kinase activity, and cellular localization is known, but its function in human cells remains undetermined. In the current study, the ATR protein was examined by gel filtration of protein extracts and was found to exist predominantly as part of a large protein complex. A kinase-inactivated form of the ATR gene was prepared by site-directed mutagenesis and was used in transfection experiments to probe the function of this complex. Introduction of this kinase-dead ATR into a normal fibroblast cell line, an ATM-deficient fibroblast line derived from a patient with ataxia–telangiectasia, or a p53 mutant cell line all resulted in significant losses in cell viability. Clones expressing the kinase-dead ATR displayed increased sensitivity to x-rays and UV and a loss of checkpoint control. We conclude that ATR functions as a critical part of a protein complex that mediates responses to ionizing and UV radiation in human cells. These responses include effects on cell viability and cell cycle checkpoint control.

Human CUL1 forms an evolutionarily conserved ubiquitin ligase complex (SCF) with SKP1 and an F-box protein

The SCF ubiquitin ligase complex of budding yeast triggers DNA replication by catalyzing ubiquitination of the S phase cyclin-dependent kinase inhibitor SIC1. SCF is composed of three proteins—ySKP1, CDC53 (Cullin), and the F-box protein CDC4—that are conserved from yeast to humans. As part of an effort to identify components and substrates of a putative human SCF complex, we isolated hSKP1 in a two-hybrid screen with hCUL1, the closest human homologue of CDC53. Here, we show that hCUL1 associates with hSKP1 in vivo and directly interacts with both hSKP1 and the human F-box protein SKP2 in vitro, forming an SCF-like particle. Moreover, hCUL1 complements the growth defect of yeast cdc53ts mutants, associates with ubiquitination-promoting activity in human cell extracts, and can assemble into functional, chimeric ubiquitin ligase complexes with yeast SCF components. Taken together, these data suggest that hCUL1 functions as part of an SCF ubiquitin ligase complex in human cells. Further application of biochemical assays similar to those described here can now be used to identify regulators/components of hCUL1-based SCF complexes, to determine whether the hCUL2–hCUL5 proteins also are components of ubiquitin ligase complexes in human cells, and to screen for chemical compounds that modulate the activities of the hSKP1 and hCUL1 proteins.

Nucleolar localization of the Werner syndrome protein in human cells

Werner Syndrome (WS) is a human genetic disorder with many features of premature aging. The gene defective in WS (WRN) has been cloned and encodes a protein homologous to several helicases, including Escherichia coli RecQ, the human Bloom syndrome protein (BLM), and Saccharomyces cerevisiae Sgs1p. To better define the function of WRN protein we have determined its subcellular localization. Indirect immunofluorescence using polyclonal anti-human WRN shows a predominant nucleolar localization. Studies of WRN mutant cells lines confirmed the specificity of antibody recognition. No difference was seen in the subcellular localization of the WRN protein in a variety of normal and transformed human cell lines, including both carcinomas and sarcomas. The nucleolar localization of human WRN protein was supported by the finding that upon biochemical subcellular fractionation, WRN protein is present in an increased concentration in a subnuclear fraction enriched for nucleolar proteins. We have also determined the subcellular localization of the mouse WRN homologue (mWRN). In contrast to human WRN protein, mWRN protein is present diffusely throughout the nucleus. Understanding the function of WRN in these organisms of vastly differing lifespan may yield new insights into the mechanisms of lifespan determination.

Heteroduplex joint formation in Escherichia coli recombination is initiated by pairing of a 3′-ending strand

The formation of heteroduplex joints in Escherichia coli recombination is initiated by invasion of double-stranded DNA by a single-stranded homologue. To determine the polarity of the invasive strand, linear molecules with direct terminal repeats were released by in vivo restriction of infecting chimeric phage DNA and heteroduplex products of intramolecular recombination were analyzed. With this substrate, the invasive strand is expected to be incorporated into the circular crossover product and the complementary strand is expected to be incorporated into the reciprocal linear product. Strands of both polarities were incorporated into heteroduplex structures, but only strands ending 3′ at the break were incorporated into circular products. This result indicates that invasion of the 3′-ending strand initiates the heteroduplex joint formation and that the complementary 5′-ending strand is incorporated into heteroduplex structures in the process of reciprocal strand exchange. The polarity of the invasive strand was not affected by recD, recJ, or xonA mutations. However, xonA and recJ mutations increased the proportion of heteroduplexes containing 5′-ending strands. This observation suggests that RecJ exonuclease and exonuclease I may enhance recombination by degrading the displaced strands during branch migration and thereby causing strand exchange to be unidirectional.