RESUMO
BACKGROUND: Sequential treatment of Panitumumab (Pb) plus Paclitaxel (Px) as induction treatment (IT) followed by concurrent bioradiotherapy (Bio-RT) with Pb may be an alternative for locally advanced squamous cell carcinoma of the head and neck (LA-SCCHN) in patients ineligible for high-dose cisplatin therapy. METHODS: Phase II, single-arm, multicentre study, with two-stage design, in patients ≥ 18 years with stage III-IVa-b LA-SCCHN unfit for platinum. Patients received Px + Pb (9 weeks) as IT followed by Bio-RT + Pb. Primary endpoint: overall response rate (ORR) after IT, defined as: more than 70% of patients achieving complete response (CR) or partial response (PR) to IT. Secondary end-points: progression-free survival, organ preservation rate, safety profile. RESULTS: Study ended prematurely (51 patients) due to slow recruitment. ORR: 66.7% (95% CI: 53.7-79.6), 8 (15.7%) CR and 26 (51.0%) PR. 39 patients (76%) completed radiotherapy (RT). Pb and/or Px-related adverse events (AEs) grade 3-4: 56.9% during IT and 63.4% during the concomitant phase, of which most common were skin toxicity (33.3%). Five deaths occurred during treatment, two of them (3.9%) were Pb and/or Px-related. CONCLUSIONS: Although underpowered, ORR was higher than the pre-specified boundary for considering the treatment active. Although Px + Pb as IT provides some benefit, the safety profile is worse than expected. To consider Pb + Px as IT as an alternative for platinum-unsuitable LA-SCCHN, further research/investigation would be needed.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/radioterapia , Paclitaxel/uso terapêutico , Panitumumabe/uso terapêutico , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Carcinoma de Células Escamosas de Cabeça e Pescoço/radioterapia , Idoso , Idoso de 80 Anos ou mais , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Causas de Morte , Término Precoce de Ensaios Clínicos , Feminino , Neoplasias de Cabeça e Pescoço/mortalidade , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Quimioterapia de Indução/métodos , Masculino , Pessoa de Meia-Idade , Tratamentos com Preservação do Órgão , Paclitaxel/efeitos adversos , Panitumumabe/efeitos adversos , Intervalo Livre de Progressão , Espanha , Carcinoma de Células Escamosas de Cabeça e Pescoço/mortalidade , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologiaRESUMO
BACKGROUND: Besides being building blocks for proteins, amino acids are also key metabolic intermediates in living cells. Surprisingly a variety of organisms are incapable of synthesizing some of them, thus named Essential Amino Acids (EAAs). How certain ancestral organisms successfully competed for survival after losing key genes involved in amino acids anabolism remains an open question. Comparative genomics searches on current protein databases including sequences from both complete and incomplete genomes among diverse taxonomic groups help us to understand amino acids auxotrophy distribution. RESULTS: Here, we applied a methodology based on clustering of homologous genes to seed sequences from autotrophic organisms Saccharomyces cerevisiae (yeast) and Arabidopsis thaliana (plant). Thus we depict evidences of presence/absence of EAA biosynthetic and nitrogen assimilation enzymes at phyla level. Results show broad loss of the phenotype of EAAs biosynthesis in several groups of eukaryotes, followed by multiple secondary gene losses. A subsequent inability for nitrogen assimilation is observed in derived metazoans. CONCLUSIONS: A Great Deletion model is proposed here as a broad phenomenon generating the phenotype of amino acids essentiality followed, in metazoans, by organic nitrogen dependency. This phenomenon is probably associated to a relaxed selective pressure conferred by heterotrophy and, taking advantage of available homologous clustering tools, a complete and updated picture of it is provided.
Assuntos
Aminoácidos/biossíntese , Genoma , Nitrogênio/metabolismo , Deleção de Sequência , Animais , Arabidopsis/genética , Arabidopsis/metabolismo , Evolução Biológica , Análise por Conglomerados , Enzimas/classificação , Filogenia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
Nutrigenomics studies the effects of nutrients on the genome, transcriptome and proteome of organisms, and here an evolutionary standpoint on this new discipline is presented. It is well known that metazoan organisms are unable to synthesize all amino acids necessary to produce their proteins and that these essential amino acids (EAA) must be acquired from the diet. Here, we tested the hypothesis that conserved regions such as protein domains (DM) have different essentiality indexes and use different sets of amino acids when compared to extra-domains (ED) and proteins without mapped domains (WD). We found that auxotrophic organisms have a tendency to use less EAAs in DM than do prototrophic ones. Looking into the amino acid usage of eukaryotic proteins downloaded from KEGG and COG, we showed that WD have a usage of amino acids closer to DM, which suggests that proteins without mapped domains behave as large domains. Using an ED index that shows the proportion of prevalent amino acids in ED, a differential usage of amino acids in domains versus extra-domains was demonstrated. Protein domains were shown to be enriched with a higher number of EAA, and it may be related to the fact that these amino acids had lost their biosynthetic pathways in metazoans during a great amino acid pathway deletion, followed by a nutritional constraint that may have happened close to the conquest of the terrestrial environment. Thus, the proportion of EAA outside domains could have decreased during evolution due to nutritional constraints.
Assuntos
Aminoácidos Essenciais/metabolismo , Células Eucarióticas/metabolismo , Nutrigenômica/métodos , Proteínas/metabolismo , Aminoácidos Essenciais/genética , Animais , Evolução Molecular , Estrutura Terciária de Proteína , Proteínas/química , Proteínas/genéticaRESUMO
A procedure to recruit members to enlarge protein family databases is described here. The procedure makes use of UniRef50 clusters produced by UniProt. Current family entries are used to recruit additional members based on the UniRef50 clusters to which they belong. Only those additional UniRef50 members that are not fragments and whose length is within a restricted range relative to the original entry are recruited. The enriched dataset is then limited to contain only genomes from selected clades. We used the COG database - used for genome annotation and for studies of phylogenetics and gene evolution - as a model. To validate the method, a UniRef-Enriched COG0151 (UECOG) was tested with distinct procedures to compare recruited members with the recruiters: PSI-BLAST, secondary structure overlap (SOV), Seed Linkage, COGnitor, shared domain content, and neighbor-joining single-linkage, and observed that the former four agree in their validations. Presently, the UniRef50-based recruitment procedure enriches the COG database for Archaea, Bacteria and its subgroups Actinobacteria, Firmicutes, Proteobacteria, and other bacteria by 2.2-, 8.0-, 7.0-, 8.8-, 8.7-, and 4.2-fold, respectively, in terms of sequences, and also considerably increased the number of species.
Assuntos
Biologia Computacional/métodos , Bases de Dados de Proteínas , Reprodutibilidade dos TestesRESUMO
Following sequence alignment, clustering algorithms are among the most utilized techniques in gene expression data analysis. Clustering gene expression patterns allows researchers to determine which gene expression patterns are alike and most likely to participate in the same biological process being investigated. Gene expression data also allow the clustering of whole samples of data, which makes it possible to find which samples are similar and, consequently, which sampled biological conditions are alike. Here, a novel similarity measure calculation and the resulting rank-based clustering algorithm are presented. The clustering was applied in 418 gene expression samples from 13 data series spanning three model organisms: Homo sapiens, Mus musculus, and Arabidopsis thaliana. The initial results are striking: more than 91% of the samples were clustered as expected. The MESs (most expressed sequences) approach outperformed some of the most used clustering algorithms applied to this kind of data such as hierarchical clustering and K-means. The clustering performance suggests that the new similarity measure is an alternative to the traditional correlation/distance measures typically used in clustering algorithms.
Assuntos
Algoritmos , Análise por Conglomerados , Perfilação da Expressão Gênica/estatística & dados numéricos , Animais , Arabidopsis/citologia , Arabidopsis/genética , Perfilação da Expressão Gênica/métodos , Humanos , Camundongos , Especificidade da EspécieRESUMO
The KEGG Orthology (KO) database was tested as a source for automated annotation of expressed sequence tags (ESTs). We used a control experiment where every EST was assigned to its cognate protein, and an annotation experiment where the ESTs were annotated by proteins from other organisms. Analyzing the results, we could assign classes to the annotation: correct, changed and speculated. The correct annotation ranged from 57 (Caenorhabditis elegans) to 81% (Homo sapiens). In spite of the changed annotation being low (1 in H. sapiens to 9% in Arabidopsis thaliana), the speculation was very high (18 in H. sapiens to 38% in C. elegans). We propose eliminating part of the speculated annotation using the KEGG Genes database to enrich KO clusters, decreasing the speculation from 38 to 2% in C. elegans. Thus, the KO database still demands some effort for moving sequences from Kegg GENES to KO, to complement the annotation performance.
Assuntos
Análise por Conglomerados , Bases de Dados Genéticas , Etiquetas de Sequências Expressas , Animais , Arabidopsis/genética , Caenorhabditis elegans/genética , Biologia Computacional/métodos , Drosophila melanogaster/genética , Humanos , Análise de Sequência de DNA/métodosRESUMO
The production of nucleic acid sequences by automatic DNA sequencer machines is always associated with some base-calling errors. In order to produce a high-quality DNA sequence from a molecule of interest, researchers normally sequence the same sample many times. Considering base-calling errors as rare events, re-sequencing the same molecule and assembling the reads produced are frequently thought to be a good way to generate reliable sequences. However, a relevant question on this issue is: how many times the sample needs to be re-sequenced to minimize costs and achieve a high-fidelity sequence? We examined how both the number of re-sequenced reads and PHRED trimming parameters affect the accuracy and size of final consensus sequences. Hundreds of single-pool reaction pUC18 reads were generated and assembled into consensus sequences with CAP3 software. Using local alignment against the published pUC18 cloning vector sequence, the position and number of errors in the consensus were identified and stored in MySQL databases. Stringent PHRED trimming parameters proved to be efficient for the reduction of errors; however, this procedure also decreased consensus size. Moreover, re-sequencing did not have a clear effect on the removal of consensus errors, although it was able to slightly increase consensus.
Assuntos
Sequência Consenso , Análise de Sequência de DNA/métodos , Pareamento Incorreto de Bases , Sequência de Bases , Plasmídeos/genéticaRESUMO
We have previously shown evidence of strong sex-biased genetic blending in the founding and ongoing history of the Brazilian population, with the African and Amerindian contribution being highest from maternal lineages (as measured by mitochondrial DNA) and the European contribution foremost from paternal lineages (estimated from Y-chromosome haplogroups). The same phenomenon has been observed in several other Latin American countries, suggesting that it might constitute a universal characteristic of the Iberian colonization of the Americas. However, it has also recently been detected in the Black population of the United States. We thus wondered if the same could be observed in American Caucasians. To answer that question, we retrieved 1387 hypervariable I Caucasian mitochondrial DNA sequences from the FBI population database and established their haplogroups and continental geographical sources. In sharp contrast with the situation of the Caucasian population of Latin American countries, only 3.1% of the American Caucasian sequences had African and/or Amerindian origin. To explain this discrepancy we propose that the finding of elevated genomic contributions from European males and Amerindian or African females depends not only on the occurrence of directional mating, but also on the "racial" categorization of the children born from these relations. In this respect, social practices in Latin America and in the United States diverge considerably; in the former socially significant "races" are normally designated according to physical appearance, while in the latter descent appears to be the most important factor.
Assuntos
Negro ou Afro-Americano/genética , Fluxo Gênico , Caracteres Sexuais , População Branca/genética , Algoritmos , Brasil , Cromossomos Humanos Y , DNA Mitocondrial/genética , Bases de Dados de Ácidos Nucleicos , Feminino , Humanos , Masculino , Estados UnidosRESUMO
Data integration has become an important task for biological database providers. The current model for data exchange among different sources simplifies the manner that distinct information is accessed by users. The evolution of data representation from HTML to XML enabled programs, instead of humans, to interact with biological databases. We present here SRS.php, a PHP library that can interact with the data integration Sequence Retrieval System (SRS). The library has been written using SOAP definitions, and permits the programmatic communication through webservices with the SRS. The interactions are possible by invoking the methods described in WSDL by exchanging XML messages. The current functions available in the library have been built to access specific data stored in any of the 90 different databases (such as UNIPROT, KEGG and GO) using the same query syntax format. The inclusion of the described functions in the source of scripts written in PHP enables them as webservice clients to the SRS server. The functions permit one to query the whole content of any SRS database, to list specific records in these databases, to get specific fields from the records, and to link any record among any pair of linked databases. The case study presented exemplifies the library usage to retrieve information regarding registries of a Plant Defense Mechanisms database. The Plant Defense Mechanisms database is currently being developed, and the proposal of SRS.php library usage is to enable the data acquisition for the further warehousing tasks related to its setup and maintenance.
Assuntos
Bases de Dados Factuais , Bases de Dados Genéticas , Biologia Computacional , Biblioteca Genômica , InternetRESUMO
Data integration has become an important task for biological database providers. The current model for data exchange among different sources simplifies the manner that distinct information is accessed by users. The evolution of data representation from HTML to XML enabled programs, instead of humans, to interact with biological databases. We present here SRS.php, a PHP library that can interact with the data integration Sequence Retrieval System (SRS). The library has been written using SOAP definitions, and permits the programmatic communication through webservices with the SRS. The interactions are possible by invoking the methods described in WSDL by exchanging XML messages. The current functions available in the library have been built to access specific data stored in any of the 90 different databases (such as UNIPROT, KEGG and GO) using the same query syntax format. The inclusion of the described functions in the source of scripts written in PHP enables them as webservice clients to the SRS server. The functions permit one to query the whole content of any SRS database, to list specific records in these databases, to get specific fields from the records, and to link any record among any pair of linked databases. The case study presented exemplifies the library usage to retrieve information regarding registries of a Plant Defense Mechanisms database. The Plant Defense Mechanisms database is currently being developed, and the proposal of SRS.php library usage is to enable the data acquisition for the further warehousing tasks related to its setup and maintenance.
Assuntos
Bases de Dados Factuais , Bases de Dados Genéticas , Biologia Computacional , Biblioteca Genômica , InternetRESUMO
The production of nucleic acid sequences by automatic DNA sequencer machines is always associated with some base-calling errors. In order to produce a high-quality DNA sequence from a molecule of interest, researchers normally sequence the same sample many times. Considering base-calling errors as rare events, re-sequencing the same molecule and assembling the reads produced are frequently thought to be a good way to generate reliable sequences. However, a relevant question on this issue is: how many times the sample needs to be re-sequenced to minimize costs and achieve a high-fidelity sequence? We examined how both the number of re-sequenced reads and PHRED trimming parameters affect the accuracy and size of final consensus sequences. Hundreds of single-pool reaction pUC18 reads were generated and assembled into consensus sequences with CAP3 software. Using local alignment against the published pUC18 cloning vector sequence, the position and number of errors in the consensus were identified and stored in MySQL databases. Stringent PHRED trimming parameters proved to be efficient for the reduction of errors; however, this procedure also decreased consensus size. Moreover, re-sequencing did not have a clear effect on the removal of consensus errors, although it was able to slightly increase consensus.
Assuntos
Análise de Sequência de DNA/métodos , Sequência Consenso , Pareamento Incorreto de Bases , Sequência de Bases , Plasmídeos/genéticaRESUMO
We have previously shown evidence of strong sex-biased genetic blending in the founding and ongoing history of the Brazilian population, with the African and Amerindian contribution being highest from maternal lineages (as measured by mitochondrial DNA) and the European contribution foremost from paternal lineages (estimated from Y-chromosome haplogroups). The same phenomenon has been observed in several other Latin American countries, suggesting that it might constitute a universal characteristic of the Iberian colonization of the Americas. However, it has also recently been detected in the Black population of the United States. We thus wondered if the same could be observed in American Caucasians. To answer that question, we retrieved 1387 hypervariable I Caucasian mitochondrial DNA sequences from the FBI population database and established their haplogroups and continental geographical sources. In sharp contrast with the situation of the Caucasian population of Latin American countries, only 3.1% of the American Caucasian sequences had African and/or Amerindian origin. To explain this discrepancy we propose that the finding of elevated genomic contributions from European males and Amerindian or African females depends not only on the occurrence of directional mating, but also on the [quot ]racial[quot ] categorization of the children born from these relations. In this respect, social practices in Latin America and in the United States diverge considerably; in the former socially significant [quot ]races[quot ] are normally designated according to physical appearance, while in the latter descent appears to be the most important factor.
Assuntos
Humanos , Masculino , Feminino , Negro ou Afro-Americano/genética , Caracteres Sexuais , Fluxo Gênico , População Branca/genética , Bases de Dados de Ácidos Nucleicos , Algoritmos , Brasil , Cromossomos Humanos Y , DNA Mitocondrial/genética , Estados UnidosRESUMO
The expressed sequence tag (EST) is an instrument of gene discovery. When available in large numbers, ESTs may be used to estimate gene expression. We analyzed gene expression by EST sampling, using the KOG database, which includes 24,154 proteins from Arabidopsis thaliana (Ath), 17,101 from Caenorhabditis elegans (Cel), 10,517 from Drosophila melanogaster (Dme), and 26,324 from Homo sapiens (Hsa), and 178,538 ESTs for Ath, 215,200 for Cel, 261,404 for Dme, and 1,941,556 for Hsa. BLAST similarity searches were performed to assign KOG annotation to all ESTs. We determined the amount of gene sampling or expression dedicated to each KOG functional category by each model organism. We found that the 25% most-expressed genes are frequently shared among these organisms. The KOG protein classification allowed the EST sampling calculation throughout the glycolysis pathway. We calculated the KOG cluster coverage and inferred that 50 to 80 K ESTs would efficiently cover 80-85% of the KOG database clusters in a transcriptome project. Since KOG is a database biased towards housekeeping genes, this is probably the number of ESTs needed to include the more commonly expressed genes in these organisms. We also examined a still unaddressed question: what is the minimum number of ESTs that should be produced in a transcriptome project?
Assuntos
Humanos , Animais , Etiquetas de Sequências Expressas , Expressão Gênica/genética , Proteínas de Arabidopsis/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Drosophila/genética , Análise de Sequência de Proteína , Análise por Conglomerados , Bases de Dados Genéticas , Bases de Dados de Proteínas , Modelos Genéticos , Proteínas de Arabidopsis/química , Proteínas de Caenorhabditis elegans/química , Proteínas de Drosophila/química , Transcrição GênicaRESUMO
Microorganisms with large genomes are commonly the subjects of single-round partial sequencing of cDNA, generating expressed sequence tags (ESTs). Usually there is a great distance between gene discovery by EST projects and submission of amino acid sequences to public databases. We analyzed the relationship between available ESTs and protein sequences and used the sequences available in the secondary database, clusters of orthologous groups (COG), to investigate ESTs from eight microorganisms of medical and/or economic relevance, selecting for candidate ESTs that may be further pursued for protein characterization. The organisms chosen were Paracoccidioides brasiliensis, Dictyostelium discoideum, Fusarium graminearum, Plasmodium yoelii, Magnaporthe grisea, Emericella nidulans, Chlamydomonas reinhardtii and Eimeria tenella, which have more than 10,000 ESTs available in dbEST. A total of 77,114 protein sequences from COG were used, corresponding to 3,201 distinct genes. At least 212 of these were capable of identifying candidate ESTs for further studies (E. tenella). This number was extended to over 700 candidate ESTs (C. reinhardtii, F. graminearum). Remarkably, even the organism that presents the highest number of ESTs corresponding to known proteins, P. yoelii, showed a considerable number of candidate ESTs for protein characterization (477). For some organisms, such as P. brasiliensis, M. grisea and F. graminearum, bioinformatics has allowed for automatic annotation of up to about 20 of the ESTs that did not correspond to proteins already characterized in the organism. In conclusion, 4093 ESTs from these eight organisms that are homologous to COG genes were selected as candidates for protein characterization
Assuntos
Animais , Bases de Dados de Proteínas , Etiquetas de Sequências Expressas , Análise de Sequência de Proteína , Chlamydomonas reinhardtii/genética , Dictyostelium/genética , Eimeria tenella/genética , Emericella/genética , Fusarium/genética , Genoma , Magnaporthe/genética , Paracoccidioides/genética , Plasmodium yoelii/genética , Proteínas/genética , Homologia de Sequência de AminoácidosRESUMO
The yeast two-hybrid system is a powerful tool for screening protein-protein interactions and has also been used for large-scale studies. We evaluated two protein-coding sequences as reporter genes for the yeast two-hybrid system, to determine if it was suitable as an alternative screening strategy. Aspergillus awamori glucoamylase activity results in clear haloes around colonies producing this enzyme after growth on starch plates and staining with iodine vapors. However, transcription activation by Gal4 on Gal-regulated promoters was insufficient for this type of phenotypic visualization. A modified gene of Aequoria victoria enhanced green fluorescent protein (EGFP) was tested to determine its suitability for interaction screenings with flow cytometry. When the EGFP reporter gene system was incorporated into the cells, Gal4 transcriptional activation produced sufficient fluorescence for detection with the flow cytometer, especially when there were strong interactions
Assuntos
Genes Reporter , Leveduras/genética , Técnicas do Sistema de Duplo-Híbrido , Sequência de Bases , Clonagem Molecular , Citometria de Fluxo , Dados de Sequência Molecular , Proteínas Luminescentes/análise , Proteínas Luminescentes/genética , Alinhamento de SequênciaRESUMO
Transcription enhancer factor (TEF/TEAD) is a family of four transcription factors that share a common TEA-DNA binding domain and are involved in similar cellular functions, such as cell differentiation and proliferation. All adult tissues express at least one of the four TEAD genes, so this family of transcription factors may be of widespread importance, yet little is known about their regulation. Here we examine the factors that regulate TEAD activity in CHO cells. RT-PCR indicated the presence of TEAD-1, TEAD-3, and both isoforms of TEAD-4, but not TEAD-2. Quantitative measurements showed that TEAD-4 is most abundant, followed by TEAD-3, then TEAD-1. We examined the relative effects of nuclear and cytosolic Ca(2+) on TEAD activity, since TEAD proteins are localized to the nucleus and since free Ca(2+) within the nucleus selectively regulates transcription in some systems. Chelation of nuclear but not cytosolic Ca(2+) increased TEAD activity two times above control. Inhibition of mitogen-activated protein kinase (MAPK) also increased TEAD activity, while cAMP decreased TEAD activity, and protein kinase C had no effect. Together, these results show that nuclear Ca(2+), MAPK, and cAMP each negatively regulate the activity of the TEAD transcription factor.
Assuntos
Cálcio/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares , Transdução de Sinais/fisiologia , Fatores de Transcrição/metabolismo , Células 3T3 , Angiotensinas/metabolismo , Animais , Células CHO , Cricetinae , AMP Cíclico/metabolismo , Proteínas de Ligação a DNA/genética , Inibidores Enzimáticos/metabolismo , Genes Reporter , Humanos , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores de Angiotensina/genética , Receptores de Angiotensina/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Transcrição de Domínio TEA , Fatores de Transcrição/genéticaRESUMO
A new species of myxomycete, Calonema foliicola Estrada, J. M. Ramírez & Lado, recorded in the Mexican states of Chihuahua, Hidalgo and Tlaxcala is described. The most relevant characters of this species are the scattered, minute and stalked sporocarps, the red color of the sporotheca and the capillitium, with a faint and irregular reticulum.
RESUMO
ESTs constitute rapid and informative tools with which to study gene-expression profiles of the diverse stages of the schistosome life cycle. Following a comprehensive EST study of adult worms, analysis has now targeted the cercaria, the parasite larval form responsible for infection of the vertebrate host. Two Schistosoma mansoni cercarial cDNA libraries were examined and partial sequence obtained from 957 randomly selected clones. On the basis of database searches, 551 (57.6%) ESTs generated had no homologs in the public databases whilst 308 (32.2%) were putatively identified, totaling 859 informative ESTs. The remaining 98 (10.2%) were uninformative ESTs (ribosomal RNA and non-coding mitochondrial sequences). By clustering analysis we have identified 453 different genes. The most common sequences in both libraries represented Sm8 calcium binding protein (8% of ESTs), fructose-1,6-bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase, cytochrome oxidase subunit 1, ATP guanidine kinase and triose phosphate isomerase. One hundred and nineteen identified genes were sorted into 11 functional categories, with genes associated with energy metabolism being the most abundant (13%) and diverse. The diversity and abundance of genes associated with the transcription/translation machinery and with regulatory/signaling functions were also marked. A paramyosin transcript was identified, indicating that this gene is not exclusively expressed in adult worms and sporocysts (as had been suggested previously). The possible physiological relevance to cercariae of the presence of transcripts with homology to calcium binding proteins of the EF-hand superfamily, Gq-coupled rhodopsin photoreceptor, rod phosphodiesterase 8 subunit and peripheral-type benzodiazepine receptor is discussed.
Assuntos
Etiquetas de Sequências Expressas , Genes de Helmintos , Schistosoma mansoni/genética , Animais , DNA Complementar/genética , DNA de Helmintos/genética , Expressão Gênica , Biblioteca Gênica , Dados de Sequência Molecular , Schistosoma mansoni/crescimento & desenvolvimentoRESUMO
To determine whether or not initiation sites for DNA replication in mammalian cells are defined by association with nuclear structure, attachments between the nucleoskeleton and the hamster DHFR gene initiation zone were examined. Nucleoskeletons were prepared by encapsulating cells in agarose and then extracting them with a nonionic detergent in a physiological buffer. The fraction of DNA that remained following endonuclease digestion was resistant to salt, sensitive to Sarkosyl, and essentially unchanged by glutaraldehyde crosslinking. Although newly replicated DNA was preferentially attached to the nucleoskeleton, no specific sequence was preferentially attached within a 65 kb locus containing the DHFR gene, two origins of bi-directional replication and at least one nuclear matrix attachment region. Instead, the entire region went from preferentially unattached to preferentially attached as cells progressed from G1 to late S-phase. Thus, initiation sites in mammalian chromosomes are not defined by attachments to the nucleoskeleton. To further assess the relationship between the nucleoskeleton and DNA replication, plasmid DNA containing the DHFR initiation region was replicated in a Xenopus egg extract. All of the DNA associated with the nucleoskeleton prior to S-phase without preference for a particular sequence and was released upon mitosis. However, about half of this DNA was trapped rather than bound to the nucleoskeleton. Thus, attachments to the nucleoskeleton can form in the absence of either DNA replication or transcription, but if they are required for replication, they are not maintained once replication is completed.
Assuntos
Replicação do DNA , DNA/metabolismo , Matriz Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Origem de Replicação , Animais , Células CHO , Núcleo Celular/química , Núcleo Celular/metabolismo , Cricetinae , DNA/biossíntese , Proteínas de Ligação a DNA/metabolismo , Oócitos/química , Ligação Proteica , XenopusRESUMO
A comparative study of the gene expression profile in different developmental stages of Schistosoma mansoni has been initiated based on the expressed sequence tag (EST) approach. A total of 1401 ESTs were generated from seven different cDNA libraries constructed from four distinct stages of the parasite life cycle. The libraries were first evaluated for their quality for a large-scale cDNA sequencing program. Most of them were shown to have less than 20% useless clones and more than 50% new genes. The redundancy of each library was also analyzed, showing that one adult worm cDNA library was composed of a small number of highly frequent genes. When comparing ESTs from distinct libraries, we could detect that most genes were present only in a single library, but others were expressed in more than one developmental stage and may represent housekeeping genes in the parasite. When considering only once the genes present in more than one library, a total of 466 unique genes were obtained, corresponding to 427 new S. mansoni genes. From the total of unique genes, 20.2% were identified based on homology with genes from other organisms, 8.3% matched S. mansoni characterized genes and 71.5% represent unknown genes.