Molecular structure and alternative splicing of the human carboxypeptidase M gene

Usando a tecnologia RACE os terminais 5' e 3' do RNAm da carboxipeptidase M ,PM) humana foram determinados e observamos ser divergente da seqüência publicada. Com esses resultados a estrutura completa do gene da CPM humana foi estabelecido baseado na seqüência genômica humana no GenBank. O gene apresentado contem 9 exons compreendendo pelo menos 75 kb da seqüência genômica. Um novo exon 1 de 30 pb foi identificado e uma seqüência promotora ipstream contendo vários sítios ligados a fatores de transcriçao foi encontrada pela análise no computador. Além disso, o códon de iniciaçao ATG foi detectado definindo uma regiao codificadora de 1329pb que codifica para uma proteína de 443 resíduos. adicionalmente, o sítio de poliadenilaçao foi descoberto determinado a regiao nao lodificadora de 2000 nucleotídeos. As fronteiras exon-intron divergem substancialmente comparado as outras carboxipeptidases básicas, CPD, CPE, CPN, AEBP1. Clonagem e sequenciamento dos produtos de RT-PCR de diferentes :tecidos revelaram um splicing alternativo dos exons 3 e 5 responsáveis pela geraçao de quatro diferentes isoformas do RNAm. RNA extraído de tecidos com tumor ,ontem mais RNAm de CPM de todas as isoformas dos o tecido controle sugerindo uma super regulaçao da expressao de CPM em tumores e originando uma questao do papel dessa atividade enzimática em câncer.BV UNIFESP: Teses e dissertaçõe

Kinin B1 and B2 receptors are overexpressed in the hippocampus of humans with temporal lobe epilepsy

Molecular biology tools have been employed to investigate the participation of peptides in human temporal lobe epilepsy ME). Active polypeptides and their receptors have been related to several brain processes, such as inflammation, apoptosis, brain development, K+ and Ca2+ channels' activation, cellular growth, and induction of neuronal differentiation. Previous works have shown a neuroprotector effect for kinin B2 receptor and a deleterious, pro-epileptogenic action for kinin B1 receptor in animal models of TLE. the present work was delineated to analyze the kinin B1 and B2 receptors expression in the hippocampus of patients presenting refractory mesial TLE. the hippocampi were removed during the patients surgery in a procedure used for seizure control and compared with tissues obtained after autopsy. Nissl staining was performed to study the tissue morphology and immunohistochemistry, and Western blot was used to compare the distribution and levels of both receptors in the hippocampus. in addition, real time PCR was employed to analyze the gene expression of these receptors. Nissl staining showed sclerotic hippocampi with hilar, granular, and pyramidal cell loss in TLE patients. Immunohistochemistry and Western blot analyses showed increased expression of kinin B1 and B2 receptors but the real-time PCR data demonstrated increased mRNA level only for kinin B2 receptors, when compared with controls. These data show for the first time a relationship between human TLE and the kallikrein-kinin system, confirming ours previous results, obtained from experimental models of epilepsy. (c) 2006 Wiley-Liss, Inc.Universidade Federal de São Paulo, Dept Expt Neurol, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Patol, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biofis, São Paulo, BrazilFCMUSP, Dept Anat Patol Incor, São Paulo, BrazilUniversidade Federal de São Paulo, Disciplina Neurol Clin, Dept Neurol Neurocirurgia, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Diagnost Imagem, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Bioquim, São Paulo, BrazilCtr Univ Nove de Julho, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Expt Neurol, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Patol, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biofis, São Paulo, BrazilUniversidade Federal de São Paulo, Disciplina Neurol Clin, Dept Neurol Neurocirurgia, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Diagnost Imagem, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Bioquim, São Paulo, BrazilWeb of Scienc

Identification of human chromosome 22 transcribed sequences with ORF expressed sequence tags

Transcribed sequences in the human genome can be identified with confidence only by alignment with sequences derived from cDNAs synthesized from naturally occurring mRNAs. We constructed a set of 250,000 cDNAs that represent partial expressed gene sequences and that are biased toward the central coding regions of the resulting transcripts. They are termed ORF expressed sequence tags (ORESTES). The 250,000 ORESTES were assembled into 81,429 contigs. Of these, 1,181 (1.45%) were found to match sequences in chromosome 22 with at least one ORESTES contig for 162 (65.6%) of the 247 known genes, for 67 (44.6%) of the 150 related genes, and for 45 of the 148 (30.4%) EST-predicted genes on this chromosome. Using a set of stringent criteria to validate our sequences, we identified a further 219 previously unannotated transcribed sequences on chromosome 22. Of these, 171 were in fact also defined by EST or full length cDNA sequences available in GenBank but not utilized in the initial annotation of the first human chromosome sequence. Thus despite representing less than 15% of all expressed human sequences in the public databases at the time of the present analysis, ORESTES sequences defined 48 transcribed sequences on chromosome 22 not defined by other sequences. All of the transcribed sequences defined by ORESTES coincided with DNA regions predicted as encoding exons by genscan. (http://genes.mit.edu/GENSCAN.html)

A Transcript Finishing Initiative for Closing Gaps in the Human Transcriptome

We report the results of a transcript finishing initiative, undertaken for the purpose of identifying and characterizing novel human transcripts, in which RT-PCR was used to bridge gaps between paired EST clusters, mapped against the genomic sequence. Each pair of EST clusters selected for experimental validation was designated a transcript finishing unit (TFU). A total of 489 TFUs were selected for validation, and an overall efficiency of 43.1% was achieved. We generated a total of 59,975 bp of transcribed sequences organized into 432 exons, contributing to the definition of the structure of 211 human transcripts. The structure of several transcripts reported here was confirmed during the course of this project, through the generation of their corresponding full-length cDNA sequences. Nevertheless, for 21% of the validated TFUs, a full-length cDNA sequence is not yet available in public databases, and the structure of 69.2% of these TFUs was not correctly predicted by computer programs. The TF strategy provides a significant contribution to the definition of the complete catalog of human genes and transcripts, because it appears to be particularly useful for identification of low abundance transcripts expressed in a restricted set of tissues as well as for the delineation of gene boundaries and alternatively spliced isoforms