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1.
Clin Genet ; 87(5): 448-54, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-24814349

RESUMO

ABCC8 encodes a subunit of the ß-cell potassium channel (KATP ) whose loss of function is responsible for congenital hyperinsulinism (CHI). Patients with two recessive mutations of ABCC8 typically have severe diffuse forms of CHI unresponsive to diazoxide. Some dominant ABCC8 mutations are responsible for a subset of diffuse diazoxide-unresponsive forms of CHI. We report the analysis of 21 different ABCC8 mutations identified in 25 probands with diazoxide-unresponsive diffuse CHI and carrying a single mutation in ABCC8. Nine missense ABCC8 mutations were subjected to in vitro expression studies testing traffic efficiency and responses of mutant channels to activation by MgADP and diazoxide. Eight of the 9 missense mutations exhibited normal trafficking. Seven of the 8 mutants reaching the plasma membrane had dramatically reduced response to MgADP or to diazoxide (<10% of wild-type response). In our cohort, dominant KATP mutations account for 22% of the children with diffuse unresponsive-diazoxide CHI. Their clinical phenotype being indistinguishable from that of children with focal CHI and diffuse CHI forms due to two recessive KATP mutations, we show that functional testing is essential to make the most reliable diagnosis and offer appropriate genetic counseling.


Assuntos
Alelos , Hiperinsulinismo Congênito/tratamento farmacológico , Hiperinsulinismo Congênito/genética , Diazóxido/uso terapêutico , Resistência a Medicamentos/genética , Mutação , Receptores de Sulfonilureias/genética , Substituição de Aminoácidos , Hiperinsulinismo Congênito/diagnóstico , Análise Mutacional de DNA , Feminino , Genes Recessivos , Humanos , Lactente , Recém-Nascido , Masculino , Fenótipo , Receptores de Sulfonilureias/metabolismo , Resultado do Tratamento
2.
DNA Res ; 18(6): 451-61, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21908513

RESUMO

Somatic cells are equipped with different silencing mechanisms that protect the genome against retrotransposons. In Drosophila melanogaster, a silencing pathway implicating the argonaute protein PIWI represses retrotransposons in cells surrounding the oocyte, whereas a PIWI-independent pathway is involved in other somatic tissues. Here, we show that these two silencing mechanisms result in distinct chromatin structures. Using sensor transgenes, we found that, in somatic tissues outside of the ovaries, these transgenes adopt a heterochromatic configuration implicating hypermethylation of H3K9 and K27. We identified the Polycomb repressive complexes (PRC1 and 2), but not heterochromatin protein 1 to be necessary factors for silencing. Once established, the compact structure is stably maintained through cell divisions. By contrast, in cells where the silencing is PIWI-dependent, the transgenes display an open and labile chromatin structure. Our data suggest that a post-transcriptional gene silencing (PTGS) mechanism is responsible for the repression in the ovarian somatic cells, whereas a mechanism that couples PTGS to transcriptional gene silencing operates to silence retrotransposons in the other somatic tissues.


Assuntos
Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Drosophila melanogaster/genética , Interferência de RNA , Proteínas Repressoras/metabolismo , Retroelementos/genética , Animais , Homólogo 5 da Proteína Cromobox , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Feminino , Histonas/metabolismo , Metilação , Folículo Ovariano/citologia , Folículo Ovariano/metabolismo , Proteínas do Grupo Polycomb , Ligação Proteica , Transgenes
3.
J Med Genet ; 47(11): 752-9, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20685672

RESUMO

BACKGROUND: Congenital hyperinsulinism (CHI) is characterised by an over secretion of insulin by the pancreatic ß-cells. This condition is mostly caused by mutations in ABCC8 or KCNJ11 genes encoding the SUR1 and KIR6.2 subunits of the ATP-sensitive potassium (K(ATP)) channel. CHI patients are classified according to their responsiveness to diazoxide and to their histopathological diagnosis (either focal, diffuse or atypical forms). Here, we raise the benefits/limits of the genetic diagnosis in the clinical management of CHI patients. METHODS: ABCC8/KCNJ11 mutational spectrum was established in 109 diazoxide-unresponsive CHI patients for whom an appropriate clinical management is essential to prevent brain damage. Relationships between genotype and radiopathological diagnosis were analysed. RESULTS: ABCC8 or KCNJ11 defects were found in 82% of the CHI cases. All patients with a focal form were associated with a single K(ATP) channel molecular event. In contrast, patients with diffuse forms were genetically more heterogeneous: 47% were associated with recessively inherited mutations, 34% carried a single heterozygous mutation and 19% had no mutation. There appeared to be a predominance of paternally inherited mutations in patients diagnosed with a diffuse form and carrying a sole K(ATP) channel mutation. CONCLUSIONS: The identification of recessively inherited mutations related to severe and diffuse forms of CHI provides an informative genetic diagnosis and allows prenatal diagnosis. In contrast, in patients carrying a single K(ATP) channel mutation, genetic analysis should be confronted with the PET imaging to categorise patients as focal or diffuse forms in order to get the appropriate therapeutic management.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Hiperinsulinismo Congênito/genética , Mutação , Canais de Potássio Corretores do Fluxo de Internalização/genética , Receptores de Droga/genética , Hiperinsulinismo Congênito/diagnóstico , Hiperinsulinismo Congênito/tratamento farmacológico , Análise Mutacional de DNA , Diazóxido/uso terapêutico , Resistência a Medicamentos , Feminino , Genótipo , Humanos , Lactente , Recém-Nascido , Masculino , Receptores de Sulfonilureias , Vasodilatadores/uso terapêutico
4.
Gene ; 450(1-2): 25-31, 2010 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-19819312

RESUMO

Chromatin insulators are cis-regulatory sequences participating in the regulation of gene expression. Their presence within the genome is associated with two main functions. One of them is an enhancer-blocking function that blocks enhancer-promoter communication when the insulator is located in between. The second is a boundary or barrier function that insulates independent units of transcription. This latter is observed when two insulators flanking a gene and its regulatory sequences block the regulatory influences of surrounding chromatin. Some years ago, we reported the presence of an insulator within the retrotransposon Idefix from Drosophila melanogaster. This insulator displays an enhancer-blocking activity toward an enhancer located within a second retrotransposon called ZAM. Here, we show that this insulator is not specific to the ZAM enhancer but has the capacity to interfere in the communication established between a broad range of cis-regulatory enhancer and a promoter. Furthermore, we show that, if it is placed on both sides of a transgene, this insulator acts as a barrier able to isolate the transgene from its repressive or enhancing environment. Thus, the Idefix insulator carries both an enhancer-blocking and a barrier activity. According to these properties, the Idefix insulator might prove to be a useful tool to isolate artificial transgenes from positive or negative influences from their integration sites.


Assuntos
Drosophila melanogaster/genética , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica , Elementos Isolantes/fisiologia , Retroelementos/fisiologia , Animais , Elementos Isolantes/genética , Regiões Promotoras Genéticas , Retroelementos/genética
5.
Nucleic Acids Res ; 35(8): 2661-70, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17426135

RESUMO

Insulators play important roles in controlling gene activity and maintaining regulatory independence between neighbouring genes. In this article, we show that the enhancer-blocking activity of the insulator present within the LTR retrotransposon Idefix can be abolished if two copies of the region containing the insulator--specifically, the long terminal repeat (LTR)--are fused to the retrotransposon's 5' untranslated region (5' UTR). The presence of this combination of two [LTR-5' UTR] modules is a prerequisite for the loss of enhancer-blocking activity. We further show that the 5' UTR causes flanking genomic sequences to be displaced to the nuclear periphery, which is not observed when two insulators are present by themselves. This study thus provides a functional link between insulators and independent genomic modules, which may cooperate to allow the specific regulation of defined genomic loci via nuclear repositioning. It further illustrates the complexity of genomic regulation within a chromatic environment with multiple functional elements.


Assuntos
Regiões 5' não Traduzidas/química , Regulação da Expressão Gênica , Elementos Isolantes , Elementos Reguladores de Transcrição , Retroelementos , Animais , Núcleo Celular/genética , Drosophila/genética , Genoma , Sequências Repetidas Terminais , Transgenes
6.
Retrovirology ; 3: 25, 2006 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-16684341

RESUMO

BACKGROUND: Retroviruses have evolved various mechanisms to optimize their transfer to new target cells via late endosomes. Here, we analyzed the transfer of ZAM, a retroelement from Drosophila melanogaster, from ovarian follicle cells to the oocyte at stage 9-10 of oogenesis, when an active yolk transfer is occurring between these two cell types. RESULTS: Combining genetic and microscopic approaches, we show that a functional secretory apparatus is required to tether ZAM to endosomal vesicles and to direct its transport to the apical side of follicle cells. There, ZAM egress requires an intact follicular epithelium communicating with the oocyte. When gap junctions are inhibited or yolk receptors mutated, ZAM particles fail to sort out the follicle cells. CONCLUSION: Overall, our results indicate that retrotransposons do not exclusively perform intracellular replication cycles but may usurp exosomal/endosomal traffic to be routed from one cell to another.


Assuntos
Drosophila melanogaster/virologia , Retrovirus Endógenos/fisiologia , Endossomos/virologia , Provírus/fisiologia , Vírion/metabolismo , Animais , Transporte Biológico/fisiologia , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Retrovirus Endógenos/metabolismo , Endossomos/metabolismo , Feminino , Oócitos/citologia , Oócitos/virologia , Folículo Ovariano/citologia , Provírus/metabolismo , Proteínas Virais/análise , Membrana Vitelina/metabolismo , Vitelogeninas/metabolismo
7.
Heredity (Edinb) ; 96(2): 195-202, 2006 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16369574

RESUMO

RNA silencing is a form of nucleic-acid-based immunity, targeting viruses and genomic repeated sequences. First documented in plants and invertebrate animals, this host defence has recently been identified in mammals. RNAi is viewed as a conserved ancient mechanism protecting genomes from nucleic acid invaders. However, these tamed sequences are known to occasionally escape this host surveillance and invade the genome of their host. This response is consistent with the overall idea that parasitic sequences compete with cells to systematically counter host defences. Using examples taken from the current literature, we illustrate the dynamic move-countermove game played between these two protagonists, the host cell and its parasitic sequences, and discuss the consequences of this game on genome stability.


Assuntos
Elementos de DNA Transponíveis , Interferência de RNA , Vírus/genética , Animais , Interações Hospedeiro-Parasita/genética , Vírus/patogenicidade
8.
Heredity (Edinb) ; 94(6): 571-6, 2005 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15815711

RESUMO

The properties of cis-regulatory elements able to influence gene transcription over large distances have led to the hypothesis that elements called insulators should exist to limit the action of enhancers and silencers. During the last decades, insulators have been identified in many eukaryotes from yeast to human. Insulators possess two main properties: (i) they can block enhancer-promoter communication ('enhancer blocker activity'), and (ii) they can prevent the spread of repressive chromatin ('barrier activity'). This review focuses on recent studies designed to elucidate the molecular mechanisms of the insulator function, and gives an overview of the critical role of insulators in nuclear organization and functional identity of chromatin.


Assuntos
Genoma , Elementos Isolantes/genética , Alelos , Animais , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromatina/genética , Cromatina/metabolismo , Mapeamento Cromossômico , Drosophila/genética , Drosophila/metabolismo , Regulação da Expressão Gênica , Modelos Biológicos
9.
Insect Biochem Mol Biol ; 35(4): 323-31, 2005 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15763468

RESUMO

ZAM is an LTR-retrotransposon from Drosophila melanogaster that belongs to the genus errantivirus, viruses similar in structure and replication cycle to vertebrate retroviruses. A key component to its lifecycle is its reverse transcriptase which copies single-stranded genomic RNA into DNA. Here, we provide a detailed characterization of the enzymatic activities of the reverse transcriptase encoded by ZAM. When expressed in vitro, the reverse transcriptase domain associated with the RNase H domain encoded by the ZAM pol gene forms homodimers and displays an efficient RNA-dependent DNA-polymerase activity. It requires either Mg2+ or Mn2+ divalent cations, and works in basic pH, with a peak at around pH9. The so-called [RT-RH] polypeptide displays an optimal activity at 22 degrees C, a property that makes it well-adapted to the temperature of its host. This study contributes to our understanding of the general structures and functions of retroviral reverse transcriptases, a necessary process in the search for novel inhibitors.


Assuntos
Drosophila melanogaster/virologia , DNA Polimerase Dirigida por RNA/genética , Retroviridae/genética , Animais , Sequência de Bases , Clonagem Molecular , DNA/genética , Primers do DNA , Dimerização , Cinética , RNA/genética , DNA Polimerase Dirigida por RNA/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Retroviridae/enzimologia , Retroviridae/isolamento & purificação , Termodinâmica
11.
Mol Genet Genomics ; 267(2): 133-41, 2002 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-11976956

RESUMO

Retrotransposons are transcriptionally activated in different tissues and cell types by a variety of genomic and environmental factors. Transcription of LTR retrotransposons is controlled by cis-acting regulatory sequences in the 5' LTR. Mobilization of two LTR retroelements, Idefix and ZAM, occurs in the unstable RevI line of Drosophila melanogaster, in which their copy numbers are high, while they are low in all other stocks tested. Here we show that both a full-length and a subgenomic Idefix transcript that are necessary for its mobilization are present in the Rev1 line, but not in the other lines. Studies on transgenic strains demonstrate that the 5' LTR of Idefix contains sequences that direct the tissue-specific expression of the retroelement in testes and ovaries of adult flies. In ovaries, expression occurs in the early follicle and in other somatic cells of the germarium, and is strictly associated with the unstable genetic context conferred by the RevI line. Control of tissue-specific Idefix expression by interactions between cis-acting sequences of its LTR and trans-acting genomic factors provides an opportunity to use this retroelement as a tool for the study of the early follicle cell lineage in the germarium.


Assuntos
Drosophila melanogaster/genética , Retroelementos , Processamento Alternativo , Animais , Animais Geneticamente Modificados , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Genes de Insetos , Genoma , Óperon Lac , Masculino , Folículo Ovariano/citologia , Folículo Ovariano/crescimento & desenvolvimento , Folículo Ovariano/metabolismo , Ovário/citologia , Ovário/crescimento & desenvolvimento , Ovário/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sequências Repetidas Terminais , Testículo/citologia , Testículo/crescimento & desenvolvimento , Testículo/metabolismo , Distribuição Tecidual
12.
Oncogene ; 20(37): 5232-8, 2001 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-11526514

RESUMO

Loss of heterozygosity (LOH) represents the most frequent genetic alteration observed in hepatocellular carcinoma (HCC). Chromosome 16q is of particular interest as it exhibits LOH in 29% of HCC tumors and is frequently lost in breast, prostate, ovarian and gastric carcinomas. We genotyped 157 HCC tumors for 17 microsatellite markers distributed on chromosome 16q and determined a common region of LOH localized between the markers D16S518 and D16S504. By refining the boundaries of two interstitial LOH and two homozygous deletions, the critical region was delimited to 180 kb between D16S3096 and D16S3029. This region is located in intron 8 of the WWOX/FOR gene, but a search for mutations in all coding exons of this gene in 27 HCC tumors and cell lines did not reveal any tumor somatic alterations. Furthermore, by RT-PCR, no abnormal transcripts of this WWOX/FOR gene was detected in nine HCC cell lines. Finally, analysis of the p53 gene mutations with the clinical parameters of all tumors revealed that the two homozygous deletions have occurred in tumors presenting a R249S mutation. Our data revealed a relationship between chromosome 16q homozygous deletions and R249S p53 mutations in tumors where the patient had been exposed to aflatoxin B1 (P=0.002). These results are consistent with a role of aflatoxin B1 in the instability of chromosome 16q at the fragile site FRA16D. However, the nature of the specific gene that is altered during hepatocarcinogenesis remains to be elucidated.


Assuntos
Aflatoxina B1 , Carcinógenos , Carcinoma Hepatocelular/induzido quimicamente , Carcinoma Hepatocelular/genética , Deleção Cromossômica , Cromossomos Humanos Par 16 , Homozigoto , Neoplasias Hepáticas/induzido quimicamente , Neoplasias Hepáticas/genética , Alelos , Mapeamento Cromossômico , Éxons , Citometria de Fluxo , Genes p53/genética , Genótipo , Humanos , Perda de Heterozigosidade , Repetições de Microssatélites , Modelos Genéticos , Mutação , RNA Mensageiro/metabolismo , Células Tumorais Cultivadas
13.
J Virol ; 74(22): 10658-69, 2000 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-11044110

RESUMO

ZAM is an env-containing member of the gypsy family of retrotransposons that represents a possible retrovirus of invertebrates. In this paper, we traced ZAM mobilization to get information about a potential path a retroelement may take to reach the germ line of its host. In situ hybridization on whole-mount tissues and immunocytochemistry analyses with antibodies raised against ZAM Gag and Env proteins have shown that all components necessary to assemble ZAM viral particles, i.e., ZAM full-length RNAs and Gag and Env polypeptides, are coexpressed in a small set of follicle cells surrounding the oocyte. By electron microscopy, we have shown that ZAM viral particles are indeed detected in this somatic lineage of cells, which they leave and enter the closely apposed oocyte. Our data provide evidence that the vesicular traffic and yolk granules in the process of vitellogenesis play an important role in ZAM transfer to the oocyte. Our data support the possibility that vitellogenin transfer to the oocyte may help a retroelement pass to the germ line with no need of its envelope product.


Assuntos
Drosophila melanogaster/virologia , Retrovirus Endógenos/crescimento & desenvolvimento , Retrovirus Endógenos/genética , Retroelementos/fisiologia , Animais , Drosophila melanogaster/fisiologia , Drosophila melanogaster/ultraestrutura , Feminino , Produtos do Gene env/genética , Produtos do Gene env/metabolismo , Produtos do Gene gag/genética , Produtos do Gene gag/metabolismo , Células Germinativas , Imuno-Histoquímica , Estágios do Ciclo de Vida , Oócitos/fisiologia , Oogênese/genética , Oogênese/fisiologia , Ovário/citologia , Ovário/metabolismo , Biossíntese de Proteínas , RNA Viral/metabolismo , Transcrição Gênica , Vírion/metabolismo
14.
Int J Cancer ; 87(6): 794-7, 2000 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-10956387

RESUMO

The human homolog of the Drosophila seven in absentia gene (SIAH1) may play an important role in apoptosis and tumor suppression. Transcription of SIAH1 is up-regulated in non-tumorigenic clonal populations of cells derived from 2 different tumorigenic parental cell lines. Intracellular SIAH1 mRNA concentration increases in intestinal cells as they migrate from the bottom of the crypt to the lumen, where they undergo apoptosis. Finally, SIAH1 is located on chromosome 16q12-q13, a region that is frequently deleted in a large variety of human tumors. These observations suggest SIAH1 as a candidate tumor suppressor gene that may be inactivated during tumorigenesis. To test this hypothesis, a search for mutation in the coding sequence of this gene was initiated in tumors exhibiting loss of heterozygosity (LOH) at 16q12-q13. No difference was found in 12 hepatocellular carcinomas, 19 breast carcinomas, 9 prostate carcinomas, 7 colon carcinomas and 5 human cell lines derived from colon cancer. One silent sequence variant (C to T transition at amino acid 270) was observed in the FET colon carcinoma cell line. It was subsequently found once in a group of 100 unrelated individuals from the CEPH families. A rapid real-time quantitative RT-PCR fluorescent method shows that SIAH1 remained transcriptionally active in the 6 colon cancer-derived cell lines, and the expression is comparable to the normal colon tissue. Taken together, these observations suggest that although many tumors may have lost one SIAH1 allele, the second allele would not be the site of frequent somatic mutations and may even remain functional.


Assuntos
Cromossomos Humanos Par 16/genética , Genes Supressores de Tumor/genética , Perda de Heterozigosidade , Proteínas de Neoplasias/genética , Neoplasias/genética , Proteínas Nucleares/genética , Alelos , Sequência de Bases , Neoplasias da Mama/genética , Carcinoma Hepatocelular/genética , Neoplasias do Colo/genética , Análise Mutacional de DNA , Feminino , Humanos , Neoplasias Hepáticas/genética , Masculino , Dados de Sequência Molecular , Mutação Puntual/genética , Neoplasias da Próstata/genética , RNA Mensageiro/genética , Ubiquitina-Proteína Ligases
15.
Genetica ; 109(1-2): 53-9, 2000.
Artigo em Inglês | MEDLINE | ID: mdl-11293795

RESUMO

Transposable elements represent a large fraction of eukaryotic genomes and they are thought to play an important role in chromatin structure. ZAM and Idefix are two LTR-retrotransposons from Drosophila melanogaster very similar in structure to vertebrate retroviruses. In all the strains where their distribution has been studied, ZAM appears to be present exclusively in the intercalary heterochromatin while Idefix copies are mainly found in the centromeric heterochromatin with very few copies in euchromatin. Their distribution varies in a specific strain called RevI in which the mobilization of ZAM and Idefix is highly induced. In this strain, 15 copies of ZAM and 30 copies of Idefix are found on the chromosomal arms in addition to their usual distribution. Amongst the loci where new copies are detected, a hotspot for their insertion has been detected at the white locus where up to four elements occurred within a 3-kb fragment at the 5' end of this gene. This property of ZAM and Idefix to accumulate at a defined site provides an interesting paradigm to bring insight into the effect exerted by multiple insertions of transposable elements at an euchromatic locus.


Assuntos
Drosophila melanogaster/genética , Retroelementos , Animais , Hibridização in Situ Fluorescente
16.
J Virol ; 73(8): 7061-4, 1999 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-10400810

RESUMO

Retroviruses and retrotransposons insert into the host genome with no obvious sequence specificity. We examined the target sites of the retroelement ZAM by sequencing each host-ZAM junction in the genome of Drosophila melanogaster. Our overall data provide compelling evidence that ZAM integration machinery recognizes and leads to ZAM insertion into the sequence 5'-GCGCGCg-3'. This unique property of ZAM will facilitate the development of new tools to study the integration process of retroelements.


Assuntos
Drosophila melanogaster/genética , Retrovirus Endógenos/genética , Endonucleases/metabolismo , Genes de Insetos , Retroelementos , Animais , Especificidade por Substrato
17.
Mol Biol Evol ; 16(1): 54-66, 1999 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-10331252

RESUMO

We describe a novel transposition system in a line of Drosophila melanogaster called RevI in which two retroelements are mobilized. These elements are the retroelement ZAM, recently described in the literature, and a novel element designated Idefix. Like ZAM, Idefix displays the structural features of a vertebrate retrovirus. Its three open reading frames encode predicted products resembling the products of the gag, pol, and env genes of retroviruses. In situ hybridization and Southern analyses performed on the RevI genome revealed the presence of some 20 copies of ZAM and Idefix, whereas ZAM is absent and Idefix is present in only four copies on the chromosomal arms of the original parental line. From RevI, a series of mutations affecting eye coloration has been recovered. The genetic and molecular analyses of these mutations have shown that most of them affected the white locus through three rounds of mutational events. The first mutational event was previously shown to be caused by a ZAM insertion 3 kb upstream of the transcription start site of white. It confers a red-brick phenotype to the orange eye coloration of the parental line. The second event results from the insertion of an Idefix copy 1.7 kb upstream of the transcription start site of the white gene, which modifies the red-brick phenotype to orange. This second mutational event was recovered as a recurrent specific mutation in 11 independent individuals. The third event results from an additional Idefix located 1.7 kb upstream of white that is responsible for the full reversion of the orange phenotype to red-brick. The fact that such mutations due to recurrent appearances of both ZAM and Idefix at the white locus result in such a variety of phenotypes brings to light a new molecular system in which the interference of mobile elements with the correct expression of the host gene can be tested.


Assuntos
Drosophila melanogaster/genética , Retroelementos , Alelos , Sequência de Aminoácidos , Animais , Sequência de Bases , Primers do DNA/genética , Cor de Olho/genética , Feminino , Variação Genética , Hibridização in Situ Fluorescente , Masculino , Mamíferos/virologia , Dados de Sequência Molecular , Mutação , Fenótipo , Retroviridae/genética , Homologia de Sequência de Aminoácidos
18.
Hum Mol Genet ; 6(9): 1427-34, 1997 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-9285778

RESUMO

Bloom's syndrome (BS), a human recessive disorder associated with an increased risk of malignancy, arises through mutations in both alleles of the BLM gene, which was recently identified as a member of the RecQ helicase family. BS cells are characterized by an increased rate of sister chromatid exchange (SCE). However, a subpopulation of lymphocytes exhibiting a normal level of SCE is observed in some patients. It has been proposed that reversion to a low-SCE phenotype involves an intragenic crossing over between the paternal and maternal BLM alleles, generating a wild-type allele. In this study we characterize a new BLM mutation in a BS patient leading to the replacement, in the C-terminal region of Blm, of a highly conserved cysteine by a phenylalanine in codon 1036. Moreover, our data show that this patient also inherited a BLM allele carrying a mutation affecting its expression and that a somatic intragenic crossing over was involved in reversion to the low-SCE phenotype. Further, we show that both topoisomerase II alpha mRNA and protein levels are decreased in the high-SCE cells derived from this patient, whereas they are normal in the corresponding low-SCE cells. Altogether, our data led us to propose that besides its putative helicase activity, Blm could be involved in transcription regulation.


Assuntos
Adenosina Trifosfatases/genética , Síndrome de Bloom/genética , DNA Helicases/genética , DNA Topoisomerases Tipo II/genética , Isoenzimas/genética , Sequência de Aminoácidos , Antígenos de Neoplasias , Proteínas de Ligação a DNA , Humanos , Dados de Sequência Molecular , Mutação , RecQ Helicases , Alinhamento de Sequência
19.
Genetica ; 100(1-3): 131-40, 1997.
Artigo em Inglês | MEDLINE | ID: mdl-9440265

RESUMO

The mobile element ZAM, recently identified in Drosophila melanogaster, is similar in structure and coding potential to vertebrate retroviruses. In this paper, we analyze the insertional and structural polymorphism of this element and show that members of this family appear to have a long evolutionary history in the genome of Drosophila. It is present in all the species of the D. melanogaster subgroup and in more distantly related species like D. takahashii, D. ananassae, or D. virilis but in a lower copy number or with a lower homology. Two categories of strains have been previously identified in D. melanogaster: strains with a high copy number of ZAM and strains with a low copy number. Here, we show that ZAM is at least in a low copy number in each tested strain of the species analyzed. The study of ZAM's genomic distribution by FISH mapping analysis to salivary gland polytene chromosomes or on mitotic chromosomes indicates that most of the insertion sites of ZAM elements are associated with the constitutive heterochromatin regardless of the ZAM copy number. In addition, our results suggest that multiple ZAM elements are present at the insertion sites visualized by in situ experiments.


Assuntos
Drosophila melanogaster/genética , Evolução Molecular , Genoma , Retroelementos/genética , Animais , Southern Blotting , Heterocromatina/genética , Filogenia , Especificidade da Espécie
20.
EMBO J ; 16(24): 7521-31, 1997 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-9405380

RESUMO

ZAM, a new retroelement of Drosophila melanogaster, was identified as a mutational insertion at the white locus. It displays all the structural features of a vertebrate retrovirus. Its three open reading frames encode predicted products resembling the products of the gag, pol and env genes of retroviruses. Its transcription gives rise to an 8.6 kb full-length RNA and a 1.7 kb spliced message for the env gene. The latter encodes an envelope protein that is typical of elements having an extracellular phase of the life cycle. The identification of a ZAM envelope retrogene provides evidence that ZAM is mobilized through a reverse trancriptional process in the germ line of flies. We report that ZAM is distributed differently among D.melanogaster strains. Two stocks out of >15 tested display a ZAM high copy number, with numerous copies distributed on chromosomal arms. This high copy number is associated with a high transcriptional rate of ZAM. The existence of these two categories of strains offers a new genetic system in which the properties of a potential invertebrate retrovirus can be tested.


Assuntos
Drosophila melanogaster/virologia , Retroviridae/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Cromossomos/ultraestrutura , Cromossomos/virologia , Drosophila melanogaster/genética , Drosophila melanogaster/fisiologia , Produtos do Gene env/química , Produtos do Gene pol/química , Produtos do Gene pol/genética , Genes env , Genes gag , Genes pol , Invertebrados , Estágios do Ciclo de Vida , Dados de Sequência Molecular , Fases de Leitura Aberta , Reação em Cadeia da Polimerase , Pseudogenes , Splicing de RNA , RNA Mensageiro/biossíntese , Mapeamento por Restrição , Retroviridae/isolamento & purificação , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transcrição Gênica
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