A novel homeobox gene PV.1 mediates induction of ventral mesoderm in Xenopus embryos.

The formation of ventral mesoderm has been traditionally viewed as a result of a lack of dorsal signaling and therefore assumed to be a default state of mesodermal development. The discovery that bone morphogenetic protein 4 (BMP4) can induce ventral mesoderm led to the suggestion that the induction of the ventral mesoderm requires a different signaling pathway than the induction of the dorsal mesoderm. However, the individual components of this pathway remained largely unknown. Here we report the identification of a novel Xenopus homeobox gene PV.1 (posterior-ventral 1) that is capable of mediating induction of ventral mesoderm. This gene is activated in blastula stage Xenopus embryos, its expression peaks during gastrulation and declines rapidly after neurulation is complete. PV.1 is expressed in the ventral marginal zone of blastulae and later in the posterior ventral area of gastrulae and neurulae. PV.1 is inducible in uncommited ectoderm by the ventralizing growth factor BMP4 and counteracts the dorsalizing effects of the dominant negative BMP4 receptor. Overexpression of PV.1 yields ventralized tadpoles and rescues embryos partially dorsalized by LiCl treatment. In animal caps, PV.1 ventralizes induction by activin and inhibits expression of dorsal specific genes. All of these effects mimic those previously reported for BMP4. These observations suggest that PV.1 is a critical component in the formation of ventral mesoderm and possibly mediates the effects of BMP4.

Discovery of adrenomedullin in rat ischemic cortex and evidence for its role in exacerbating focal brain ischemic damage.

Focal brain ischemia is the most common event leading to stroke in humans. To understand the molecular mechanisms associated with brain ischemia, we applied the technique of mRNA differential display and isolated a gene that encodes a recently discovered peptide, adrenomedullin (AM), which is a member of the calcitonin gene-related peptide (CGRP) family. Using the rat focal stroke model of middle cerebral artery occlusion (MCAO), we determined that AM mRNA expression was significantly increased in the ischemic cortex up to 17.4-fold at 3 h post-MCAO (P < 0.05) and 21.7-fold at 6 h post-MCAO (P < 0.05) and remained elevated for up to 15 days (9.6-fold increase; P < 0.05). Immunohistochemical studies localized AM to ischemic neuronal processes, and radioligand (125I-labeled CGRP) displacement revealed high-affinity (IC50 = 80.3 nmol) binding of AM to CGRP receptors in brain cortex. The cerebrovascular function of AM was studied using synthetic AM microinjected onto rat pial vessels using a cranial window or applied to canine basilar arteries in vitro. AM, applied abluminally, produced dose-dependent relaxation of preconstricted pial vessels (P < 0.05). Intracerebroventricular (but not systemic) AM administration at a high dose (8 nmol), prior to and after MCAO, increased the degree of focal ischemic injury (P < 0.05). The ischemia-induced expression of both AM mRNA and peptide in ischemic cortical neurons, the demonstration of the direct vasodilating effects of the peptide on cerebral vessels, and the ability of AM to exacerbate ischemic brain damage suggests that AM plays a significant role in focal ischemic brain injury.

Evidence against the exon theory of genes derived from the triose-phosphate isomerase gene.

The exon theory of genes proposes that the introns of protein-encoding nuclear genes are remnants of the DNA spacers between ancient minigenes. The discovery of an intron at a predicted position in the triose-phosphate isomerase (EC 5.3.1.1) gene of Culex mosquitoes has been hailed as an evidential pillar of the theory. We have found that that intron is also present in Aedes mosquitoes, which are closely related to Culex, but not in the phylogenetically more distant Anopheles, nor in the fly Calliphora vicina, nor in the moth Spodoptera littoralis. The presence of this intron in Culex and Aedes is parsimoniously explained as the result of an insertion in a recent common ancestor of these two species rather than as the remnant of an ancient intron. The absence of the intron in 19 species of very diverse organisms requires at least 10 independent evolutionary losses in order to be consistent with the exon theory.

Controle epigenético do gene imprinted SNRPN durante o desenvolvimento e reprogramação nuclear em equídeos

A tranferência nuclear de células somáticas (TNCS) está sendo utilizada para produzir cavalos de elite. No entanto, durante este procedimento pode ocorrer a perfuração da zona pelúcida, levando, ocasionalmente, à secção da massa celular interna, e conseqüente derivação de gêmeos monozigóticos. Além de serem relatadas alterações no processo de imprinting genômico, que conduzem ao desenvolvimento de doenças. Com a descoberta da possibilidade de reprogramar as células somáticas a um estado de pluripotência (iPSCs), estas células passaram a ser muito utilizadas em pesquisas de neurociência. Contudo, também ocorrem modificações epigenéticas durante esta reprogramação celular. Portanto, nossas hipóteses são que os gêmeos eqüinos gerados pela TNCS podem levar às irregularidades no desenvolvimento do sistema nervoso. O padrão de metilação do SNRPN nas estruturas dos fetos muares clonados, e as células iPSCs são diferentes dos padrões encontrados nos muares analisados. A expressão dos genes SNRPN, Necdin e UBE3A são maiores no cérebro, enquanto a expressão do H19 é maior nas membranas extra-embrionárias. Em nosso estudo, obtivemos duas gestações gemelares equinas derivadas da TNCS, que foram interrompidas com 40 e 60 dias de gestação, e comparados com gestações eqüinas únicas de idade similar. Diferenças no comprimento entre os embriões gêmeos foram observadas aos 40 (2.0 e 2.2 cm 10%) e aos 60 (6,5 e 8,5 cm 24%) dias de gestação. Somente o plexo coróide do quarto ventrículo apresentou-se mais desenvolvido nos fetos com maior comprimento. Ao analisarmos fetos muares clonados em diferentes idades gestacionais e compará-los com muares, nos períodos embrionário, fetal e adulto, não foi observada diferença no padrão de metilação do gene SNRPN. No entanto, na décima passagem das células iPSC o padrão de metilação alterou, em relação aos muares estudados e ao padrão observado nos fibroblastos. Ao analisarmos os fetos clonados nas diferentes idades gestacionais observou-se no cérebro menor expressão dos gene H19 e UBE3A, e maior expressão do gene SNRPN. Contudo, a expressão do gene Necdin variou entre as estruturas estudadas. Em conclusão, apesar dos gêmeos eqüinos provenientes de TNCS diferirem quanto ao tamanho, morfologicamente são iguais. Dentre as estruturas cerebrais o plexo coróide se apresentou mais desenvolvido nos fetos de maior comprimento. Os fetos muares clonados não apresentaram diferença no padrão de metilação do gene SNRPN. No entanto, as iPSCs apresentaram alteração no padrão de metilação deste gene na décima passagem. Embora os genes SNRPN, Necdin e UBE3A sejam expressos no cérebro, o SNRPN apresentou-se prevalente nessa estrutura

Mutações inativadoras no gene MKRN3 são causa de puberdade precoce central familial

A maioria dos casos de puberdade precoce central (PPC) em meninas permanece idiopática. A hipótese de uma causa genética vem se fortalecendo após a descoberta de alguns genes associados a este fenótipo, sobretudo aqueles implicados com o sistema kisspeptina (KISS1 e KISS1R). Entretanto, apenas casos isolados de PPC foram relacionados à mutação na kisspeptina ou em seu receptor. Até recentemente, a maioria dos estudos genéticos em PPC buscava genes candidatos selecionados com base em modelos animais, análise genética de pacientes com hipogonadismo hipogonadotrófico, ou ainda, nos estudos de associação ampla do genoma. Neste trabalho, foi utilizado o sequenciamento exômico global, uma metodologia mais moderna de sequenciamento, para identificar variantes associadas ao fenótipo de PPC. Trinta e seis indivíduos com a forma de PPC familial (19 famílias) e 213 casos aparentemente esporádicos foram inicialmente selecionados. A forma familial foi definida pela presença de mais de um membro afetado na família. DNA genômico foi extraído dos leucócitos do sangue periférico de todos os pacientes. O estudo de sequenciamento exômico global realizado pela técnica ILLUMINA, em 40 membros de 15 famílias com PPC, identificou mutações inativadoras em um único gene, MKRN3, em cinco dessas famílias. Pesquisa de mutação no MKRN3 realizada por sequenciamento direto em duas famílias adicionais (quatro pacientes) identificou duas novas variantes nesse gene. O MKRN3 é um gene de um único éxon, localizado no cromossomo 15 em uma região crítica para a síndrome de Prader Willi. O gene MKRN3 sofre imprinting materno, sendo expresso apenas pelo alelo paterno. A descoberta de mutações em pacientes com PPC familial despertou o interesse para a pesquisa de mutações nesse gene em 213 pacientes com PPC aparentemente esporádica por meio de reação em cadeia de polimerase seguida de purificação enzimática e sequenciamento automático direto (Sanger). Três novas mutações e duas já anteriormente identificadas, incluindo quatro frameshifts e uma variante missense, foram encontradas, em heterozigose, em seis meninas não relacionadas. Todas as novas variantes identificadas estavam ausentes nos bancos de dados (1000 Genomes e Exome Variant Server). O estudo de segregação familial em três dessas meninas com PPC aparentemente esporádica e mutação no MKRN3 confirmou o padrão de herança autossômica dominante com penetrância completa e transmissão exclusiva pelo alelo paterno, demonstrando que esses casos eram, na verdade, também familiares. A maioria das mutações encontradas no MKRN3 era do tipo frameshift ou nonsense, levando a stop códons prematuros e proteínas truncadas e, portanto, confirmando a associação com o fenótipo. As duas mutações missenses (p.Arg365Ser e p.Phe417Ile) identificadas estavam localizadas em regiões de dedo ou anel de zinco, importantes para a função da proteína. Além disso, os estudos in silico dessas duas variantes demonstraram patogenicidade. Todos os pacientes com mutação no MKRN3 apresentavam características clínicas e hormonais típicas de ativação prematura do eixo reprodutivo. A mediana de idade de início da puberdade foi de 6 anos nas meninas (variando de 3 a 6,5) e 8 anos nos meninos (variando de 5,9 a 8,5). Tendo em vista o fenômeno de imprinting, análise de metilação foi também realizada em um subgrupo de 52 pacientes com PPC pela técnica de MS-MLPA, mas não foram encontradas alterações no padrão de metilação. Em conclusão, este trabalho identificou um novo gene associado ao fenótipo de PPC. Atualmente, mutações inativadoras no MKRN3 representam a causa genética mais comum de PPC familial (33%). O MKRN3 é o primeiro gene imprintado associado a distúrbios puberais em humanos. O mecanismo preciso de ação desse gene na regulação da secreção de GnRH necessita de estudos adicionais

MicroRNAs in pathology and as therapeutic target in gene therapy?

Dissertação para obtenção do grau de Mestre no Instituto Superior de Ciências da Saúde Egas Moniz

Tissue-specific gene expression in soybean (Glycine max) detected by cDNA microarray analysis

We have constructed cDNA microarrays for soybean (Glycine max L. Merrill), containing approximately 4,100 Unigene ESTs derived from axenic roots, to evaluate their application and utility for functional genomics of organ differentiation in legumes. We assessed microarray technology by conducting studies to evaluate the accuracy of microarray data and have found them to be both reliable and reproducible in repeat hybridisations. Several ESTs showed high levels (>50 fold) of differential expression in either root or shoot tissue of soybean. A small number of physiologically interesting, and differentially expressed sequences found by microarray analysis were verified by both quantitative real-time RT-PCR and Northern blot analysis. There was a linear correlation (r(2) = 0.99, over 5 orders of magnitude) between microarray and quantitative real-time RT-PCR data. Microarray analysis of soybean has enormous potential not only for the discovery of new genes involved in tissue differentiation and function, but also to study the expression of previously characterised genes, gene networks and gene interactions in wild-type, mutant or transgenic; plants.

Continued discovery of transcriptional units expressed in cells of the mouse mononuclear phagocyte lineage

The current RIKEN transcript set represents a significant proportion of the mouse transcriptome but transcripts expressed in the innate and acquired immune systems are poorly represented. In the present study we have assessed the complexity of the transcriptome expressed in mouse macrophages before and after treatment with lipopolysaccharide, a global regulator of macrophage gene expression, using existing RIKEN 19K arrays. By comparison to array profiles of other cells and tissues, we identify a large set of macrophage-enriched genes, many of which have obvious functions in endocytosis and phagocytosis. In addition, a significant number of LPS-inducible genes were identified. The data suggest that macrophages are a complex source of mRNA for transcriptome studies. To assess complexity and identify additional macrophage expressed genes, cDNA libraries were created from purified populations of macrophage and dendritic cells, a functionally related cell type. Sequence analysis revealed a high incidence of novel mRNAs within these cDNA libraries. These studies provide insights into the depths of transcriptional complexity still untapped amongst products of inducible genes, and identify macrophage and dendritic cell populations as a starting point for sampling the inducible mammalian transcriptome.

Discovery of a new source of rifamycin antibiotics in marine sponge actinobacteria by phylogenetic prediction

Phylogenetic analysis of the ketosynthase (KS) gene sequences of marine sponge-derived Salinispora strains of actinobacteria indicated that the polyketide synthase (PKS) gene sequence most closely related to that of Salinispora was the rifamycin B synthase of Amycolatopsis mediterranei. This result was not expected from taxonomic species tree phylogenetics using 16S rRNA sequences. From the PKS sequence data generated from our sponge-derived Salinispora strains, we predicted that such strains might synthesize rifamycin-like compounds. Liquid chromatography-tandem mass spectrometry (LC/MS/MS) analysis was applied to one sponge-derived Salinispora strain to test the hypothesis of rifamycin synthesis. The analysis reported here demonstrates that this Salinispora isolate does produce compounds of the rifamycin class, including rifamycin B and rifamycin SV. A rifamycin-specific KS primer set was designed, and that primer set increased the number of rifamycin-positive strains detected by PCR screening relative to the number detectable using a conserved KS-specific set. Thus, the Salinispora group of actinobacteria represents a potential new source of rifamycins outside the genus Amycolatopsis and the first recorded source of rifamycins from marine bacteria.

A simple implementation of a normal mixture approach to differential gene expression in multiclass microarrays

Motivation: An important problem in microarray experiments is the detection of genes that are differentially expressed in a given number of classes. We provide a straightforward and easily implemented method for estimating the posterior probability that an individual gene is null. The problem can be expressed in a two-component mixture framework, using an empirical Bayes approach. Current methods of implementing this approach either have some limitations due to the minimal assumptions made or with more specific assumptions are computationally intensive. Results: By converting to a z-score the value of the test statistic used to test the significance of each gene, we propose a simple two-component normal mixture that models adequately the distribution of this score. The usefulness of our approach is demonstrated on three real datasets.

Analysing diversity in sugarcane resistance gene analogues

As resistance genes have been shown to contain conserved motifs and cluster in many plant genomes, the identification of resistance gene analogues can be used as a strategy for both the discovery of DNA markers linked to disease resistance loci and the map-based cloning of disease resistance genes. Sugarcane suffers from many important diseases and an analysis of resistance gene analogues offers a means to identify DNA markers linked to resistance loci. However, sugarcane has the most complex genome of any crop plant and initially it is important to understand the extent of resistance gene analogue diversity in the sugarcane genome before genetic analysis. We review herein how more than 100 expressed sequence tags with homology to different resistance genes have been identified in sugarcane with many mapped as single-dose restriction fragment length polymorphism markers. Importantly, some of these resistance gene analogues have been shown to be linked to disease resistance genes or disease quantitative trait loci. In an attempt to more efficiently analyse additional resistance gene analogues in sugarcane, we report on experiments aimed at investigating the molecular diversity of several resistance gene analogue families using a modified form of a technique termed Ecotilling. Using Ecotilling, we were able to rapidly detect single nucleotide polymorphisms in fragments amplified by PCR from four different resistance gene analogue families, SoRP1D, SoPTO, SoXa21 and SoHs1pro-1. An analysis of a diverse set of sugarcane varieties, including modern sugarcane cultivars and several S. officinarum and S. spontaneum clones, indicated that all amplicons, apart from SoHs1pro-1, contained significant polymorphism within the gene region studied. However, a comparison among these sugarcane clones, including between the parents of two sugarcane mapping populations, indicated that most polymorphisms were multi-dose, not single-dose, preventing their genetic map location or association with disease susceptibility or resistance from being determined.

Discovery of cyclotide-like protein sequences in graminaceous crop plants: Ancestral precursors of circular proteins?

Cyclotides are peptides from plants of the Rubiaceae and Violaceae families that have the unusual characteristic of a macrocylic backbone. They are further characterized by their incorporation of a cystine knot in which two disulfides, along with the intervening backbone residues, form a ring through which a third disulfide is threaded. The cyclotides have been found in every Violaceae species screened to date but are apparently present in only a few Rubiaceae species. The selective distribution reported so far raises questions about the evolution of the cyclotides within the plant kingdom. In this study, we use a combined bioinformatics and expression analysis approach to elucidate the evolution and distribution of the cyclotides in the plant kingdom and report the discovery of related sequences widespread in the Poaceae family, including crop plants such as rice ( Oryza sativa), maize ( Zea mays), and wheat ( Triticum aestivum), which carry considerable economic and social importance. The presence of cyclotide-like sequences within these plants suggests that the cyclotides may be derived from an ancestral gene of great antiquity. Quantitative RT-PCR was used to show that two of the discovered cyclotide-like genes from rice and barley ( Hordeum vulgare) have tissue-specific expression patterns.

The identification and characterisation of alleles of sucrose phosphate synthase gene family III in sugarcane

Little is known about the extent of allelic diversity of genes in the complex polyploid, sugarcane. Using sucrose phosphate synthase (SPS) Gene (SPS) Family III as an example, we have amplified and sequenced a 400 nt region from this gene from two sugarcane lines that are parents of a mapping population. Ten single nucleotide polymorphisms (SNPs) were identified within the 400 nt region of which seven were present in both lines. In the elite commercial cultivar Q165(A), 10 sequence haplotypes were identified, with four haplotypes recovered at 9% or greater frequency. Based on SNP presence, two clusters of haplotypes were observed. In IJ76-514, a Saccharum officinarum accession, 8 haplotypes were identified with 4 haplotypes recovered at 13% or greater frequency. Again, two clusters of haplotypes were observed. The results suggest that there may be two SPS Gene Family III genes per genome in sugarcane, each with different numbers of different alleles. This suggestion is supported by sequencing results in an elite parental sorghum line, 403463-2-1, in which 4 haplotypes, corresponding to two broad types, were also identified. Primers were designed to the sugarcane SNPs and screened over bulked DNA from high and low Sucrose-containing progeny from a cross between Q165(A) and IJ76-514. The SNP frequency did not vary in the two bulked DNA samples, suggesting that these SNPs from this SPS gene family are not associated with variation in sucrose content. Using an ecotilling approach, two of the SPS Gene Family III haplotypes were mapped to two different linkage groups in homology group 1 in Q165(A). Both haplotypes mapped near QTLs for increased sucrose content but were not themselves associated with any sugar-related trait.

Molecular classification of cutaneous malignant melanoma by gene expression profiling

The most common human cancers are malignant neoplasms of the skin(1,2). Incidence of cutaneous melanoma is rising especially steeply, with minimal progress in non-surgical treatment of advanced disease(3,4). Despite significant effort to identify independent predictors of melanoma outcome, no accepted histopathological, molecular or immunohistochemical marker defines subsets of this neoplasm(2,3). Accordingly, though melanoma is thought to present with different 'taxonomic' forms, these are considered part of a continuous spectrum rather than discrete entities(2). Here we report the discovery of a subset of melanomas identified by mathematical analysis of gene expression in a series of samples. Remarkably, many genes underlying the classification of this subset are differentially regulated in invasive melanomas that form primitive tubular networks in vitro, a feature of some highly aggressive metastatic melanomas(5). Global transcript analysis can identify unrecognized subtypes of cutaneous melanoma and predict experimentally verifiable phenotypic characteristics that may be of importance to disease progression.

Expression-based strategies for discovery of genes involved in testis and ovary development

In recent years, strategies for gene identification based on differential gene expression have become increasingly popular, due in part to the development of microarray technology. These strategies are particularly well suited to the identification of genes involved in sex determination and gonadal development, which unlike the development of other organ systems, proceeds along two very different alternative courses, depending on the sex of the embryo. We have used a high-throughput, array-based expression screen to identify several genes expressed sex-specifically in developing mouse gonads. One of these, vanin 1, appears to play a role in mediating migration of mesonephric cells into the male genital ridge. Progress in characterizing other genes arising from the screen is discussed.