RESUMO
BACKGROUND: The Fragile Histidine Triad gene (FHIT) is an oncosuppressor implicated in many human cancers, including vesical tumors. FHIT is frequently hit by deletions caused by fragility at FRA3B, the most active of human common fragile sites, where FHIT lays. Vesical tumors affect also cattle, including animals grazing in the wild on bracken fern; compounds released by the fern are known to induce chromosome fragility and may trigger cancer with the interplay of latent Papilloma virus. RESULTS: The bovine FHIT was characterized by assembling a contig of 78 BACs. Sequence tags were designed on human exons and introns and used directly to select bovine BACs, or compared with sequence data in the bovine genome database or in the trace archive of the bovine genome sequencing project, and adapted before use. FHIT is split in ten exons like in man, with exons 5 to 9 coding for a 149 amino acids protein. VISTA global alignments between bovine genomic contigs retrieved from the bovine genome database and the human FHIT region were performed. Conservation was extremely high over a 2 Mb region spanning the whole FHIT locus, including the size of introns. Thus, the bovine FHIT covers about 1.6 Mb compared to 1.5 Mb in man. Expression was analyzed by RT-PCR and Northern blot, and was found to be ubiquitous. Four cDNA isoforms were isolated and sequenced, that originate from an alternative usage of three variants of exon 4, revealing a size very close to the major human FHIT cDNAs. CONCLUSION: A comparative genomic approach allowed to assemble a contig of 78 BACs and to completely annotate a 1.6 Mb region spanning the bovine FHIT gene. The findings confirmed the very high level of conservation between human and bovine genomes and the importance of comparative mapping to speed the annotation process of the recently sequenced bovine genome. The detailed knowledge of the genomic FHIT region will allow to study the role of FHIT in bovine cancerogenesis, especially of vesical papillomavirus-associated cancers of the urinary bladder, and will be the basis to define the molecular structure of the bovine homologue of FRA3B, the major common fragile site of the human genome.
Assuntos
Hidrolases Anidrido Ácido/genética , Mapeamento Cromossômico/métodos , Cromossomos Artificiais Bacterianos , Perfilação da Expressão Gênica , Proteínas de Neoplasias/genética , Animais , Bovinos , Mapeamento de Sequências Contíguas , DNA Complementar/metabolismo , Éxons , Biblioteca Genômica , Genômica/métodos , Humanos , Isoformas de Proteínas , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The PRND gene encodes Doppel (Dpl), a protein that is strongly expressed in testis and at much lower levels in other tissues. Despite the recent discovery of Dpl involvement in spermiogenesis and in apoptotic death of cerebellar neurons, respectively in wild type and transgenic mice, the physiological role of this prion-like protein remains unknown. To better understand which factors may contribute to the modulation of PRND activity, a study of the bovine promoter region was performed. First, the transcription start site of PRND mRNA was identified using an innovative fluorescently labelled oligonucleotide extension (FLOE) method. The initiation site mapped 129 nt upstream of the protein coding sequence and represents a refinement of a previous assignment based on RACE. Second, deletion mutants of the 4530 nt encompassing 2704 nt 5' of the bovine PRND, exon 1, intron 1, and the first 6 nt of exon 2, have been investigated with CAT-reporter assays in order to identify critical elements for the activation of the gene. The results showed that the region -323/+32 (+1 is the transcription start site mapped by FLOE) represents the promoter region and contains positive cis-acting elements (CCAAT and E box) confirming previous reports with the mouse gene. Additional regulatory elements, including binding sites for repressor molecules, have been identified upstream of that region and in the first portion of intron 1, suggesting a complex tissue-specific regulation of Doppel gene expression.
Assuntos
Bovinos/genética , Regiões Promotoras Genéticas/genética , Proteínas/genética , Sequências Reguladoras de Ácido Nucleico/genética , Animais , Sequência de Bases , Sítios de Ligação/genética , Western Blotting , Linhagem Celular Tumoral , Cloranfenicol O-Acetiltransferase/genética , Cloranfenicol O-Acetiltransferase/metabolismo , Biologia Computacional/métodos , Regulação da Expressão Gênica , Humanos , Masculino , Dados de Sequência Molecular , Mutação , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Elementos de Resposta/genética , Análise de Sequência de DNA/métodos , Testículo/metabolismo , Sítio de Iniciação de Transcrição , TransfecçãoRESUMO
SPRN is a new prion-like gene coding for Sho, a protein with significant similarity to PrP. SPRN was initially described in zebrafish; however, the strong evolutionary conservation led to the hypothesis that SPRN might be the ancestral prion-like gene. We mapped SPRN in Bos taurus by comparative analysis of the locus and of the predicted flanking genes. BACs, spanning the whole SPRN genomic locus, were assigned to BTA26q23 by radiation hybrid mapping and fluorescent in situ hybridization (FISH). Sequencing of five genes flanking SPRN, namely, ECHS1, PAOX, MTG1, SPRN, and CYP2E1, high-resolution FISH on mechanically stretched chromosomes, and combed BAC DNA allowed us to establish their order and reciprocal orientation. The results confirmed that BTA26q23 corresponds to HSA10q24.3-26.3, which is the site where the human SPRN is located. The gene order in Bos taurus is the same as in man, cen-ECHS1-PAOX-MTG1-SPRN-CYP2E1-tel, but PAOX has a different orientation in the two species. SPRN has the typical two-exon PRNP arrangement, with the CDS fully contained within exon 2; furthermore, it codes for a 143-amino-acid protein with 74.8% identity and 84.7% similarity with the human PRNP. RT-PCR and Northern blot analysis showed that SPRN is expressed at high levels in brain and less in testis and lung.
Assuntos
Clonagem Molecular/métodos , Proteínas do Tecido Nervoso/genética , Príons/genética , Animais , Bovinos , Cromossomos Artificiais Bacterianos/genética , Citocromo P-450 CYP2E1/genética , Enoil-CoA Hidratase/genética , Etiquetas de Sequências Expressas , Feminino , GTP Fosfo-Hidrolases/genética , Humanos , Hibridização in Situ Fluorescente , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The genomic structure of the caprine Doppel gene (PRND) was determined using the ovine sequence as a scaffold to generate PCR fragments that were aligned with a cDNA sequence obtained from testicular mRNA. The caprine gene contains two exons, 89 and >2291 bp long, separated by a 1689-bp intron. Two mRNA isoforms of 3.2 and 4.8 kb were identified in the testis, as well as the exact transcription start site by fluorescently labeled oligonucleotide extension (FLOE). Like in sheep and cattle, the open reading frame (ORF) (537 bp) lies within exon 2 and is very much conserved in sheep (99.3%) and cattle (97%). The intronic sequence is also highly conserved (95.3%) compared with sheep, with the only exception of a 47-bp insertion. The PRND ORF was sequenced in 47 healthy and 17 TSE-affected goats of the Italian Ionica breed. Seven nucleotide positions showed variation: T28C, C65T, A151G, G286A, C385G, T451C, and T528C. Five were commonly represented polymorphisms: T28C, T451C, and T528C are silent mutations at codons L10, L151, and I176, respectively, while A151G and C385G determine a T51A and L129V amino acid change, respectively. The two remaining variants, C65T and G286A, were rare, leading to the amino acid substitutions S22F and E96K, respectively. None of the polymorphisms was significantly relatable to the TSE status, and the same result was obtained by the analysis of the combined haplotypes at the five major polymorphic sites, namely, T28C, C65T, A151G, G286A, and C385G.