dbSNP: the NCBI database of genetic variation

In response to a need for a general catalog of genome variation to address the large-scale sampling designs required by association studies, gene mapping and evolutionary biology, the National Center for Biotechnology Information (NCBI) has established the dbSNP database [S.T.Sherry, M.Ward and K.Sirotkin (1999) Genome Res., 9, 677–679]. Submissions to dbSNP will be integrated with other sources of information at NCBI such as GenBank, PubMed, LocusLink and the Human Genome Project data. The complete contents of dbSNP are available to the public at website: http://www.ncbi.nlm.nih.gov/SNP. The complete contents of dbSNP can also be downloaded in multiple formats via anonymous FTP at ftp://ncbi.nlm.nih.gov/snp/.

Database resources of the National Center for Biotechnology Information

In addition to maintaining the GenBank® nucleic acid sequence database, the National Center for Biotechnology Information (NCBI) provides data analysis and retrieval resources that operate on the data in GenBank and a variety of other biological data made available through NCBI’s Web site. NCBI data retrieval resources include Entrez, PubMed, LocusLink and the Taxonomy Browser. Data analysis resources include BLAST, Electronic PCR, OrfFinder, RefSeq, UniGene, HomoloGene, Database of Single Nucleotide Polymorphisms (dbSNP), Human Genome Sequencing, Human MapViewer, GeneMap’99, Human–Mouse Homology Map, Cancer Chromosome Aberration Project (CCAP), Entrez Genomes, Clusters of Orthologous Groups (COGs) database, Retroviral Genotyping Tools, Cancer Genome Anatomy Project (CGAP), SAGEmap, Gene Expression Omnibus (GEO), Online Mendelian Inheri­tance in Man (OMIM), the Molecular Modeling Database (MMDB) and the Conserved Domain Database (CDD). Augmenting many of the Web applications are custom implementations of the BLAST program optimized to search specialized data sets. All of the resources can be accessed through the NCBI home page at: http://www.ncbi.nlm.nih.gov.

Proteome Analysis Database: online application of InterPro and CluSTr for the functional classification of proteins in whole genomes

The SWISS-PROT group at EBI has developed the Proteome Analysis Database utilising existing resources and providing comparative analysis of the predicted protein coding sequences of the complete genomes of bacteria, archaea and eukaryotes (http://www.ebi.ac.uk/proteome/). The two main projects used, InterPro and CluSTr, give a new perspective on families, domains and sites and cover 31–67% (InterPro statistics) of the proteins from each of the complete genomes. CluSTr covers the three complete eukaryotic genomes and the incomplete human genome data. The Proteome Analysis Database is accompanied by a program that has been designed to carry out InterPro proteome comparisons for any one proteome against any other one or more of the proteomes in the database.

A comprehensive BAC resource

The Human Genome Project has generated extensive map and sequence data for a large number of Bacterial Artificial Chromosome (BAC) clones. In order to maximize the efficient use of the data and to minimize the redundant work for the research community, The Institute for Genomic Research (TIGR) comprehensive BAC resource (cBACr) (http://www.tigr.org/tdb/BacResource/BAC_resource_intro.html) was built as an expansion of the TIGR human BAC ends database. This resource collects, integrates and reports the information on library, maps, sequence, annotation and functions for each human and mouse BAC. The current database contains 635 016 human BACs and 265 617 mouse BACs that were characterized by various approaches, among which 22 705 human clones and 1000 mouse clones have sequence and annotation data.

Urocortin II: A member of the corticotropin-releasing factor (CRF) neuropeptide family that is selectively bound by type 2 CRF receptors

Here we describe the cloning and initial characterization of a previously unidentified CRF-related neuropeptide, urocortin II (Ucn II). Searches of the public human genome database identified a region with significant sequence homology to the CRF neuropeptide family. By using homologous primers deduced from the human sequence, a mouse cDNA was isolated from whole brain poly(A)+ RNA that encodes a predicted 38-aa peptide, structurally related to the other known mammalian family members, CRF and Ucn. Ucn II binds selectively to the type 2 CRF receptor (CRF-R2), with no appreciable activity on CRF-R1. Transcripts encoding Ucn II are expressed in discrete regions of the rodent central nervous system, including stress-related cell groups in the hypothalamus (paraventricular and arcuate nuclei) and brainstem (locus coeruleus). Central administration of 1–10 μg of peptide elicits activational responses (Fos induction) preferentially within a core circuitry subserving autonomic and neuroendocrine regulation, but whose overall pattern does not broadly mimic the CRF-R2 distribution. Behaviorally, central Ucn II attenuates nighttime feeding, with a time course distinct from that seen in response to CRF. In contrast to CRF, however, central Ucn II failed to increase gross motor activity. These findings identify Ucn II as a new member of the CRF family of neuropeptides, which is expressed centrally and binds selectively to CRF-R2. Initial functional studies are consistent with Ucn II involvement in central autonomic and appetitive control, but not in generalized behavioral activation.

Coiled Bodies Preferentially Associate with U4, U11, and U12 Small Nuclear RNA Genes in Interphase HeLa Cells but Not with U6 and U7 Genes

Coiled bodies (CBs) are nuclear organelles involved in the metabolism of small nuclear RNAs (snRNAs) and histone messages. Their structural morphology and molecular composition have been conserved from plants to animals. CBs preferentially and specifically associate with genes that encode U1, U2, and U3 snRNAs as well as the cell cycle–regulated histone loci. A common link among these previously identified CB-associated genes is that they are either clustered or tandemly repeated in the human genome. In an effort to identify additional loci that associate with CBs, we have isolated and mapped the chromosomal locations of genomic clones corresponding to bona fide U4, U6, U7, U11, and U12 snRNA loci. Unlike the clustered U1 and U2 genes, each of these loci encode a single gene, with the exception of the U4 clone, which contains two genes. We next examined the association of these snRNA genes with CBs and found that they colocalized less frequently than their multicopy counterparts. To differentiate a lower level of preferential association from random colocalization, we developed a theoretical model of random colocalization, which yielded expected values for χ2 tests against the experimental data. Certain single-copy snRNA genes (U4, U11, and U12) but not controls were found to significantly (p < 0.000001) associate with CBs. Recent evidence indicates that the interactions between CBs and genes are mediated by nascent transcripts. Taken together, these new results suggest that CB association may be substantially augmented by the increased transcriptional capacity of clustered genes. Possible functional roles for the observed interactions of CBs with snRNA genes are discussed.

Identification of multiple quantitative trait loci linked to prion disease incubation period in mice

Polymorphisms in the prion protein gene are known to affect prion disease incubation times and susceptibility in humans and mice. However, studies with inbred lines of mice show that large differences in incubation times occur even with the same amino acid sequence of the prion protein, suggesting that other genes may contribute to the observed variation. To identify these loci we analyzed 1,009 animals from an F2 intercross between two strains of mice, CAST/Ei and NZW/OlaHSd, with significantly different incubation periods when challenged with RML scrapie prions. Interval mapping identified three highly significantly linked regions on chromosomes 2, 11, and 12; composite interval mapping suggests that each of these regions includes multiple linked quantitative trait loci. Suggestive evidence for linkage was obtained on chromosomes 6 and 7. The sequence conservation between the mouse and human genome suggests that identification of mouse prion susceptibility alleles may have direct relevance to understanding human susceptibility to bovine spongiform encephalopathy (BSE) infection, as well as identifying key factors in the molecular pathways of prion pathogenesis. However, the demonstration of other major genetic effects on incubation period suggests the need for extreme caution in interpreting estimates of variant Creutzfeldt–Jakob disease epidemic size utilizing existing epidemiological models.

Identification of urocortin III, an additional member of the corticotropin-releasing factor (CRF) family with high affinity for the CRF2 receptor

The corticotropin-releasing factor (CRF) family of neuropeptides includes the mammalian peptides CRF, urocortin, and urocortin II, as well as piscine urotensin I and frog sauvagine. The mammalian peptides signal through two G protein-coupled receptor types to modulate endocrine, autonomic, and behavioral responses to stress, as well as a range of peripheral (cardiovascular, gastrointestinal, and immune) activities. The three previously known ligands are differentially distributed anatomically and have distinct specificities for the two major receptor types. Here we describe the characterization of an additional CRF-related peptide, urocortin III, in the human and mouse. In searching the public human genome databases we found a partial expressed sequence tagged (EST) clone with significant sequence identity to mammalian and fish urocortin-related peptides. By using primers based on the human EST sequence, a full-length human clone was isolated from genomic DNA that encodes a protein that includes a predicted putative 38-aa peptide structurally related to other known family members. With a human probe, we then cloned the mouse ortholog from a genomic library. Human and mouse urocortin III share 90% identity in the 38-aa putative mature peptide. In the peptide coding region, both human and mouse urocortin III are 76% identical to pufferfish urocortin-related peptide and more distantly related to urocortin II, CRF, and urocortin from other mammalian species. Mouse urocortin III mRNA expression is found in areas of the brain including the hypothalamus, amygdala, and brainstem, but is not evident in the cerebellum, pituitary, or cerebral cortex; it is also expressed peripherally in small intestine and skin. Urocortin III is selective for type 2 CRF receptors and thus represents another potential endogenous ligand for these receptors.

Cyclic polyamides for recognition in the minor groove of DNA.

Small molecules that specifically bind with high affinity to any designated DNA sequence in the human genome would be useful tools in molecular biology and potentially in human medicine. Simple rules have been developed to rationally alter the sequence specificity of minor groove-binding polyamides containing N-methylimidazole and N-methylpyrrole amino acids. Crescent-shaped polyamides bind as antiparallel dimers with each polyamide making specific contacts with each strand on the floor of the minor groove. Cyclic polyamides have now been synthesized that bind designated DNA sequences at subnanomolar concentrations.

Real-time detection of DNA hybridization and melting on oligonucleotide arrays by using optical wave guides.

The challenge of the Human Genome Project is to increase the rate of DNA sequence acquisition by two orders of magnitude to complete sequencing of the human genome by the year 2000. The present work describes a rapid detection method using a two-dimensional optical wave guide that allows measurement of real-time binding or melting of a light-scattering label on a DNA array. A particulate label on the target DNA acts as a light-scattering source when illuminated by the evanescent wave of the wave guide and only the label bound to the surface generates a signal. Imaging/visual examination of the scattered light permits interrogation of the entire array simultaneously. Hybridization specificity is equivalent to that obtained with a conventional system using autoradiography. Wave guide melting curves are consistent with those obtained in the liquid phase and single-base discrimination is facile. Dilution experiments showed an apparent lower limit of detection at 0.4 nM oligonucleotide. This performance is comparable to the best currently known fluorescence-based systems. In addition, wave guide detection allows manipulation of hybridization stringency during detection and thereby reduces DNA chip complexity. It is anticipated that this methodology will provide a powerful tool for diagnostic applications that require rapid cost-effective detection of variations from known sequences.

Da patenteabilidade de genes humanos

São inegáveis o caráter universal e a importância dos avanços tecnológicos e científicos originados das pesquisas genéticas. O sequenciamento do genoma humano, a identificação das principais sequências de DNA contidas nos seus genes e suas respectivas funções biológicas, bem como suas possíveis aplicações biomédicas, são de incalculável importância. Os genes, muito embora possam ser biologicamente caracterizados como compostos químicos, possuem um conteúdo informacional que se revela indispensável ao desenvolvimento da engenharia genética, figurando como elemento básico e central de suporte às inovações biotecnológicas. Desta forma, importante analisar a relevância da aplicação de mecanismos jurídicos como forma de fomento à contínua evolução biotecnológica sob a ótica do desenvolvimento econômico e social do país, princípios constitucionais justificadores da proteção de referidos desenvolvimentos técnicos por meio do intelecto e intervenção humanos na natureza. Para tanto, deve-se levar em consideração que a inexistência de tutela jurídica específica pode gerar desincentivo aos investimentos capazes de possibilitar o desenvolvimento de tais tecnologias, ao passo que uma tutela jurídica muito ampla poderá ocasionar indevida restrição ao acesso a tais insumos biológicos, de modo a gerar um efeito adverso àquele buscado. Assim, deve-se compatibilizar a proteção dos resultados obtidos através do desenvolvimento biotecnológico em relação à potencial dificuldade originada de uma eventual restrição ao acesso a tais elementos fundamentais à pesquisa e desenvolvimento genéticos. É neste contexto que se procura um balizamento entre os diferentes interesses e posicionamentos a respeito da patenteabilidade dos genes humanos, visando solução jurídica que permita um ambiente seguro e propício ao desenvolvimento da engenharia genética, e dos inúmeros benefícios que poderão daí se originar. O presente estudo se voltará, portanto, à análise da necessidade, condições, suficiência e extensão da tutela jurídica a ser conferida pela outorga de direitos patentários aos genes humanos.

Surdez genética na idade pediátrica : O papel da genética na abordagem à doença

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Next-generation sequencing of iron-metabolism related genes in Portuguese patients with iron overload: novel pathogenic genetic variants

Objective: In Southern European countries up to one-third of the patients with hereditary hemochromatosis (HH) do not present the common HFE risk genotype. In order to investigate the molecular basis of these cases we have designed a gene panel for rapid and simultaneous analysis of 6 HH-related genes (HFE, TFR2, HJV, HAMP, SLC40A1 and FTL) by next-generation sequencing (NGS). Materials and Methods: Eighty-eight iron overload Portuguese patients, negative for the common HFE mutations, were analysed. A TruSeq Custom Amplicon kit (TSCA, by Illumina) was designed in order to generate 97 amplicons covering exons, intron/exon junctions and UTRs of the mentioned genes with a cumulative target sequence of 12115bp. Amplicons were sequenced in the MiSeq instrument (IIlumina) using 250bp paired-end reads. Sequences were aligned against human genome reference hg19 using alignment and variant caller algorithms in the MiSeq reporter software. Novel variants were validated by Sanger sequencing and their pathogenic significance were assessed by in silico studies. Results: We found a total of 55 different genetic variants. These include novel pathogenic missense and splicing variants (in HFE and TFR2), a very rare variant in IRE of FTL, a variant that originates a novel translation initiation codon in the HAMP gene, among others. Conclusion: The merging of TSCA methodology and NGS technology appears to be an appropriate tool for simultaneous and fast analysis of HH-related genes in a large number of samples. However, establishing the clinical relevance of NGS-detected variants for HH development remains a hard-working task, requiring further functional studies.

To know ourselves /

"July 1996."

The evolution of microRNA in primates

Thesis (Ph.D.)--University of Washington, 2016-06