A genomic approach to gene fusion technology

Gene expression profiling provides powerful analyses of transcriptional responses to cellular perturbation. In contrast to DNA array-based methods, reporter gene technology has been underused for this application. Here we describe a genomewide, genome-registered collection of Escherichia coli bioluminescent reporter gene fusions. DNA sequences from plasmid-borne, random fusions of E. coli chromosomal DNA to a Photorhabdus luminescens luxCDABE reporter allowed precise mapping of each fusion. The utility of this collection covering about 30% of the transcriptional units was tested by analyzing individual fusions representative of heat shock, SOS, OxyR, SoxRS, and cya/crp stress-responsive regulons. Each fusion strain responded as anticipated to environmental conditions known to activate the corresponding regulatory circuit. Thus, the collection mirrors E. coli's transcriptional wiring diagram. This genomewide collection of gene fusions provides an independent test of results from other gene expression analyses. Accordingly, a DNA microarray-based analysis of mitomycin C-treated E. coli indicated elevated expression of expected and unanticipated genes. Selected luxCDABE fusions corresponding to these up-regulated genes were used to confirm or contradict the DNA microarray results. The power of partnering gene fusion and DNA microarray technology to discover promoters and define operons was demonstrated when data from both suggested that a cluster of 20 genes encoding production of type I extracellular polysaccharide in E. coli form a single operon.

Genome-wide gene expression profiles of the developing mouse hippocampus

We have analyzed the developmental molecular programs of the mouse hippocampus, a cortical structure critical for learning and memory, by means of large-scale DNA microarray techniques. Of 11,000 genes and expressed sequence tags examined, 1,926 showed dynamic changes during hippocampal development from embryonic day 16 to postnatal day 30. Gene-cluster analysis was used to group these genes into 16 distinct clusters with striking patterns that appear to correlate with major developmental hallmarks and cellular events. These include genes involved in neuronal proliferation, differentiation, and synapse formation. A complete list of the transcriptional changes has been compiled into a comprehensive gene profile database (http://BrainGenomics.Princeton.edu), which should prove valuable in advancing our understanding of the molecular and genetic programs underlying both the development and the functions of the mammalian brain.

Detection of a major gene for resistance to fusiform rust disease in loblolly pine by genomic mapping.

Genomic mapping has been used to identify a region of the host genome that determines resistance to fusiform rust disease in loblolly pine where no discrete, simply inherited resistance factors had been previously found by conventional genetic analysis over four decades. A resistance locus, behaving as a single dominant gene, was mapped by association with genetic markers, even though the disease phenotype deviated from the expected Mendelian ratio. The complexity of forest pathosystems and the limitations of genetic analysis, based solely on phenotype, had led to an assumption that effective long-term disease resistance in trees should be polygenic. However, our data show that effective long-term resistance can be obtained from a single qualitative resistance gene, despite the presence of virulence in the pathogen population. Therefore, disease resistance in this endemic coevolved forest pathosystem is not exclusively polygenic. Genomic mapping now provides a powerful tool for characterizing the genetic basis of host pathogen interactions in forest trees and other undomesticated, organisms, where conventional genetic analysis often is limited or not feasible.

Fate of a redundant gamma-globin gene in the atelid clade of New World monkeys: implications concerning fetal globin gene expression.

Conclusive evidence was provided that gamma 1, the upstream of the two linked simian gamma-globin loci (5'-gamma 1-gamma 2-3'), is a pseudogene in a major group of New World monkeys. Sequence analysis of PCR-amplified genomic fragments of predicted sizes revealed that all extant genera of the platyrrhine family Atelidae [Lagothrix (woolly monkeys), Brachyteles (woolly spider monkeys), Ateles (spider monkeys), and Alouatta (howler monkeys)] share a large deletion that removed most of exon 2, all of intron 2 and exon 3, and much of the 3' flanking sequence of gamma 1. The fact that two functional gamma-globin genes were not present in early ancestors of the Atelidae (and that gamma 1 was the dispensible gene) suggests that for much or even all of their evolution, platyrrhines have had gamma 2 as the primary fetally expressed gamma-globin gene, in contrast to catarrhines (e.g., humans and chimpanzees) that have gamma 1 as the primary fetally expressed gamma-globin gene. Results from promoter sequences further suggest that all three platyrrhine families (Atelidae, Cebidae, and Pitheciidae) have gamma 2 rather than gamma 1 as their primary fetally expressed gamma-globin gene. The implications of this suggestion were explored in terms of how gene redundancy, regulatory mutations, and distance of each gamma-globin gene from the locus control region were possibly involved in the acquisition and maintenance of fetal, rather than embryonic, expression.

Identificazione di miRNA correlati ad EGFR: nuovi potenziali biomarcatori di risposta alla terapia di 2a-3a linea in pazienti con NSCLC metastatico

La diagnosi di neoplasia epiteliale maligna polmonare è legata tradizionalmente alla distinzione tra carcinoma a piccole cellule (small-cell lung cancer, SCLC) e carcinoma non-a piccole cellule del polmone (non-small-cell lung cancer, NSCLC). Nell’ambito del NSCLC attualmente è importante di-stinguere l’esatto istotipo (adenocarcinoma, carcinoma squamocellulare e carcinoma neuroendocrino) perchè l’approccio terapeutico cambia a seconda dell’istotipo del tumore e la chemioterapia si dimostra molto spesso inefficace. Attualmente alcuni nuovi farmaci a bersaglio molecolare per il gene EGFR, come Erlotinib e Gefitinib, sono utilizzati per i pazienti refrattari al trattamento chemioterapico tradizionale, che non hanno risposto a uno o più cicli di chemioterapia o che siano progrediti dopo questa. I test per la rilevazione di specifiche mutazioni nel gene EGFR permettono di utilizzare al meglio questi nuovi farmaci, applicandoli anche nella prima linea di trattamento sui pazienti che hanno una maggiore probabilità di risposta alla terapia. Sfortunatamente, non tutti i pazienti rispondono allo stesso modo quando trattati con farmaci anti-EGFR. Di conseguenza, l'individuazione di biomarcatori predittivi di risposta alla terapia sarebbe di notevole importanza per aumentare l'efficacia dei questi farmaci a target molecolare e trattare con farmaci diversi i pazienti che con elevata probabilità non risponderebbero ad essi. I miRNAs sono piccole molecole di RNA endogene, a singolo filamento di 20-22 nucleotidi che svolgono diverse funzioni, una delle più importanti è la regolazione dell’espressione genica. I miRNAs possono determinare una repressione dell'espressione genica in due modi: 1-legandosi a sequenze target di mRNA, causando così un silenziamento del gene (mancata traduzione in proteina), 2- causando la degradazione dello specifico mRNA. Lo scopo della ricerca era di individuare biomarcatori capaci di identificare precocemente i soggetti in grado di rispondere alla terapia con Erlotinib, aumentando così l'efficacia del farmaco ed evitan-do/riducendo possibili fenomeni di tossicità e il trattamento di pazienti che probabilmente non ri-sponderebbero alla terapia offrendo loro altre opzioni prima possibile. In particolare, il lavoro si è fo-calizzato sul determinare se esistesse una correlazione tra la risposta all'Erlotinib ed i livelli di espressione di miRNAs coinvolti nella via di segnalazione di EGFR in campioni di NSCLC prima dell’inizio della terapia. Sono stati identificati 7 microRNA coinvolti nel pathway di EGFR: miR-7, -21, 128b, 133a, -133b, 146a, 146b. Sono stati analizzati i livelli di espressione dei miRNA mediante Real-Time q-PCR in campioni di NSCLC in una coorte di pazienti con NSCLC metastatico trattati con Erlotinib dal 1° gennaio 2009 al 31 dicembre 2014 in 2°-3° linea dopo fallimento di almeno un ciclo di chemioterapia. I pazienti sottoposti a trattamento con erlotinib per almeno 6 mesi senza presentare progressione alla malattia sono stati definiti “responders” (n=8), gli altri “non-responders” (n=25). I risultati hanno mostrato che miR-7, -133b e -146a potrebbero essere coinvolti nella risposta al trat-tamento con Erlotinib. Le indagini funzionali sono state quindi concentrate su miR-133b, che ha mo-strato la maggiore espressione differenziale tra i due gruppi di pazienti. E 'stata quindi studiata la capacità di miR-133b di regolare l'espressione di EGFR in due linee di cellule del cancro del polmone (A549 e H1299). Sono stati determinati gli effetti di miR-133b sulla crescita cellulare. E’ stato anche analizzato il rapporto tra miR-133b e sensibilità a Erlotinib nelle cellule NSCLC. L'aumento di espressione di miR-133b ha portato ad una down-regolazione del recettore di EGF e del pathway di EGFR relativo alla linea cellulare A549. La linea cellulare H1299 era meno sensibili al miR-133b up-regulation, probabilmente a causa dell'esistenza di possibili meccanismi di resistenza e/o di com-pensazione. La combinazione di miR-133b ed Erlotinib ha aumentato l'efficacia del trattamento solo nella linea cellulare A549. Nel complesso, questi risultati indicano che miR-133b potrebbe aumentare / ripristinare la sensibilità di Erlotinib in una frazione di pazienti.

Geoquímica multielementar de sedimentos de corrente no Estado de São Paulo: abordagem através da análise estatística multivariada

Este trabalho apresenta resultados geoquímicos multielementares de sedimentos de corrente no estado de São Paulo, obtidos através do projeto institucional do Serviço Geológico do Brasil denominado \"Levantamento Geoquímico de Baixa Densidade no Brasil\". Dados analíticos de 1422 amostras de sedimento de corrente obtidos por ICP-MS (Inductively Coupled Plasma Mass Spectrometry), para 32 elementos químicos (Al, Ba, Be, Ca, Ce, Co, Cr, Cs, Cu, Fe, Ga, Hf, K, La, Mg, Mn, Mo, Nb, Ni, P, Pb, Rb, Sc, Sn, Sr, Th, Ti, U, V, Y, Zn e Zr), foram processadas e abordadas através da análise estatística uni e multivariada. Os resultados do tratamento dos dados através de técnicas estatísticas univariadas forneceram os valores de background geoquímico (teor de fundo) dos 32 elementos para todo estado de São Paulo. A análise georreferenciada das distribuições geoquímicas unielementares evidenciaram a compartimentação geológica da área. As duas principais províncias geológicas do estado de São Paulo, Bacia do Paraná e Complexo Cristalino, se destacam claramente na maioria das distribuições geoquímicas. Unidades geológicas de maior expressão, como a Formação Serra Geral e o Grupo Bauru também foram claramente destacadas. Outras feições geoquímicas indicaram possíveis áreas contaminadas e unidades geológicas não cartografadas. Os resultados da aplicação de métodos estatísticos multivariados aos dados geoquímicos com 24 variáveis (Al, Ba, Ce, Co, Cr, Cs, Cu, Fe, Ga, La, Mn, Nb, Ni, Pb, Rb, Sc, Sr, Th, Ti, U, V, Y, Zn e Zr) permitiram definir as principais assinaturas e associações geoquímicas existentes em todo estado de São Paulo e correlacioná-las aos principais domínios litológicos. A análise de agrupamentos em modo Q forneceu oito grupos de amostras geoquimicamente correlacionáveis, que georreferenciadas reproduziram os principais compartimentos geológicos do estado: Complexo Cristalino, Grupos Itararé e Passa Dois, Formação Serra Geral e Grupos Bauru e Caiuá. A análise discriminante multigrupos comprovou, estatisticamente, a classificação dos grupos formados pela análise de agrupamentos e forneceu as principais variáveis discriminantes: Fe, Co, Sc, V e Cu. A análise de componentes principais, abordada em conjunto com a análise fatorial pelo método de rotação varimax, forneceram os principais fatores multivariados e suas respectivas associações elementares. O georreferenciamento dos valores de escores fatoriais multivariados delimitaram as áreas onde as associações elementares ocorrem e forneceram mapas multivariados para todo o estado. Por fim, conclui-se que os métodos estatísticos aplicados são indispensáveis no tratamento, apresentação e interpretação de dados geoquímicos. Ademais, com base em uma visão integrada dos resultados obtidos, este trabalho recomenda: (1) a execução dos levantamentos geoquímicos de baixa densidade em todo país em caráter de prioridade, pois são altamente eficazes na definição de backgrounds regionais e delimitação de províncias geoquímicas com interesse metalogenético e ambiental; (2) a execução do mapeamento geológico contínuo em escala adequada (maiores que 1:100.000) em áreas que apontam para possíveis existências de unidades não cartografadas nos mapas geológicos atuais.

Motivational profiles Spanish students of Compulsory Secondary Education: Differential analysis of academic self-attributions

Academic goals and academic self-attributions are relevant variables in school settings. The objective of this study is to identify whether there are combinations of multiple goals that lead to different motivational profiles and to determine whether there are significant differences between the groups obtained regarding causal attributions of success and failure (ability, effort, or external causes) in Mathematics and Language and Literature, and in overall academic performance. The Goal Achievement Tendencies Questionnaire (AGTQ) and the Sydney Attribution Scale (SAS) were administered to a sample of 2022 students of compulsory secondary education, ranging in age from 12 to 16 years (M = 13.81, SD = 1.35). Cluster analysis identified four motivational profiles: a group of students with a high generalized motivation profile, a group of students with low generalized motivation profile, a group of students with predominance of learning goals and achievement goals, and a final group of students with predominance of social reinforcement goals. Results revealed statistically significant differences between the profiles obtained in academic self-attributions.

Benthic foraminifera analysis of two sediments cores from the northern Cape Basin in the eastern South Atlantic Ocean

Two late Quaternary sediment cores from the northern Cape Basin in the eastern South Atlantic Ocean were analyzed for their benthic foraminiferal content and benthic stable carbon isotope composition. The locations of the cores were selected such that both of them presently are bathed by North Atlantic Deep Water (NADW) and past changes in deep water circulation should be recorded simultaneously at both locations. However, the areas are different in terms of primary production. One core was recovered from the nutrient-depleted Walvis Ridge area, whereas the other one is from the continental slope just below the coastal upwelling mixing area where present day organic matter fluxes are shown to be moderately high. Recent data served as the basis for the interpretation of the late Quaternary faunal fluctuations and the paleoceanographic reconstruction. During the last 450,000 years, NADW flux into the eastern South Atlantic Ocean has been restricted to interglacial periods, with the strongest dominance of a NADW-driven deep water circulation during interglacial stages 1, 9 and 11. At the continental margin, high productivity faunas and very low epibenthic d13C values indicate enhanced fluxes of organic matter during glacial periods. This can be attributed to a glacial increase and lateral extension of coastal upwelling. The long term glacial-interglacial paleoproductivity cycles are superimposed by high-frequency variations with a period of about 23,000 yr. Enhanced productivity in surface waters above the Walvis Ridge, far from the coast, is indicated during glacial stages 8, 10 and 12. During these periods, cold, nutrient-rich filaments from the mixing area were probably driven as far as to the southeastern flank of the Walvis Ridge.

Sedimentology of surface samples from the Weddell Sea, Antarctica

Mineralogical and granulometric properties of glacial-marine surface sediments of the Weddell Sea and adjoining areas were studied in order to decipher spatial variations of provenance and transport paths of terrigenous detritus from Antarctic sources. The silt fraction shows marked spatial differences in quartz contents. In the sand fractions heavy-mineral assemblages display low mineralogical maturity and are dominated by garnet, green hornblende, and various types of clinopyroxene. Cluster analysis yields distinct heavy-mineral assemblages, which can be attributed to specific source rocks of the Antarctic hinterland. The configuration of modern mineralogical provinces in the near-shore regions reflects the geological variety of the adjacent hinterland. In the distal parts of the study area, sand-sized heavy minerals are good tracers of ice-rafting. Granulometric characteristics and the distribution of heavy-mineral provinces reflect maxima of relative and absolute accumulation of ice-rafted detritus in accordance with major iceberg drift tracks in the course of the Weddell Gyre. Fine-grained and coarse-grained sediment fractions may have different origins. In the central Weddell Sea, coarse ice-rafted detritus basically derives from East Antarctic sources, while the fine-fraction is discharged from weak permanent bottom currents and/or episodic turbidity currents and shows affinities to southern Weddell Sea sources. Winnowing of quartz-rich sediments through intense bottom water formation in the southern Weddell Sea provides muddy suspensions enriched in quartz. The influence of quartz-rich suspensions moving within the Weddell Gyre contour current can be traced as far as the continental slope in the northwestern Weddell Sea. In general, the focusing of mud by currents significantly exceeds the relative and absolute contribution of ice-rafted detritus beyond the shelves of the study area.