RESUMO
PURPOSE OF INVESTIGATION: The fragile histidine triad (FHIT) gene is a tumor suppressor frequently inactivated in various types of tumors. The authors evaluated the occurrence of loss of heterozygosity (LOH) in the FHIT locus and FHIT protein changes in breast tissue. MATERIALS AND METHODS: Blood and breast tissue samples were obtained from 35 women with mammary disorders. The occurrence of LOH in FHIT locus was assayed by polymerase chain reaction (PCR), and the results obtained from blood and breast tissues from each patient were compared. FHIT protein expression was evaluated by immunohistochemistry. RESULTS: LOH in the FHIT gene occurred in 48.6% (17/35) of patients with mammary disorder. Among patients with malignant breast disorders, 59.1% (13/22) presented LOH in the FHIT gene in comparison with patients with benign breast lumps, in which the LOH was observed in 30.8% (4/13) of women, suggesting that changes in this gene occur prior to the process of mammary carcinogenesis. The changes in the locus of the FHIT gene occur with greater frequency in the coded region of the gene, principally near exons 5 and 8, where the FRA3B site and the histidine triad respectively are found. Changes in FHIT did not modify protein expression. The association between menopause and LOH in the FHIT gene was evident. CONCLUSIONS: LOH in the FHIT gene may be related to menopause in women with breast disorders.
Assuntos
Hidrolases Anidrido Ácido/genética , Neoplasias da Mama/genética , Proteínas de Neoplasias/genética , Hidrolases Anidrido Ácido/metabolismo , Neoplasias da Mama/metabolismo , Estudos Transversais , Feminino , Humanos , Perda de Heterozigosidade , Menopausa/genética , Pessoa de Meia-Idade , Proteínas de Neoplasias/metabolismo , Fatores de RiscoRESUMO
This study presents the first genetic characterization of five Trypanosoma rangeli isolates from Minas Gerais, in the southeast of Brazil and their comparison with Colombian populations by minicircle classification, RAPD-PCR and LSSP-PCR analyses. Our results demonstrated a homogenous T. rangeli population circulating among Didelphis albiventris as reservoir host in Brazil while heterogeneous populations were found in different regions of Colombia. KP1(+) minicircles were found in 100% isolates from Brazil and in 36.4% of the Colombian samples, whereas the KP2 and KP3 minicircles were detected in both groups. RAPD-PCR and LSSP-PCR profiles revealed a polymorphism within KP1(+) and KP1(-) T. rangeli populations and allowed the division of T. rangeli in two branches. The Brazilian KP1(+) isolates were more homogenous than the KP1(+) isolates from Colombia. The RAPD-PCR were entirely consistent with the distribution of KP1 minicircles while those obtained by LSSP-PCR were associated in 88.9% and 71.4% with KP1(+) and KP1(-) populations, respectively.
Assuntos
DNA de Cinetoplasto/isolamento & purificação , Trypanosoma/genética , Animais , Brasil , Análise por Conglomerados , Colômbia , DNA de Cinetoplasto/química , Didelphis , Reservatórios de Doenças , Variação Genética , Humanos , Reação em Cadeia da Polimerase/métodos , Técnica de Amplificação ao Acaso de DNA Polimórfico , Rhodnius , Trypanosoma/classificaçãoRESUMO
Leishmania mutants have contributed greatly to extend our knowledge of this parasite's biology. Here we report the use of the mariner in vitro transposition system as a source of reagents for shuttle mutagenesis and targeted disruption of Leishmania genes. The locus-specific integration was achieved by the disruption of the subtelomeric gene encoding a DNA-directed RNA polymerase III subunit (RPC2). Further inactivation of RPC2 alleles required the complementation of the intact gene, which was transfected in an episomal context. However, attempts to generate a RPC2 chromosomal null mutant resulted in genomic rearrangements that maintained copies of the intact locus in the genome. The maintenance of the RPC2 chromosomal locus in complemented mutants was not mediated by an increase in the number of copies and did not involve chromosomal translocations, which are the typical characteristics of the genomic plasticity of this parasite. Unlike the endogenous locus, the selectable marker used to disrupt RPC2 did not display a tendency to remain in its chromosomal location but was targeted into supernumerary episomal molecules.
Assuntos
Leishmania major/genética , Mutagênese/genética , Mutação/genética , RNA Polimerase III/genética , Telômero/genética , Animais , Linhagem Celular , Regulação da Expressão Gênica , Genes Essenciais/genética , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , RNA Polimerase III/metabolismoRESUMO
The 36 chromosomes of the parasite Leishmania major range in size from 200 kb to approximately 2.5 Mb and variation between homologues seems to be restricted to the telomeric and subtelomeric regions. We have isolated three cosmids carrying the telomere hexameric repeat and assigned them to the extreme location of chromosomes 3, 7 and 20. When considering the distribution of repetitive sequences, Southern analysis of the three chromosomal ends indicated the existence of at least two classes of chromosomal extremities: one of them is composed almost exclusively of unique sequences and the other is characterised by patches of both reiterated and unique sequences. We devised a transfection-based strategy that allowed the determination of a map of transcripts in each of the regions examined. Sequencing of the chromosome 20 cosmid revealed the existence of a novel class of reiterated sequence, LST-R378, and 10 ORFs drawing a map of putative genes compatible with the map of transcripts.
