RESUMO
Predicting the function of noncoding variation is a major challenge in modern genetics. In this study, we used massively parallel reporter assays to screen 5706 variants identified from genome-wide association studies for both Alzheimer's disease (AD) and progressive supranuclear palsy (PSP), identifying 320 functional regulatory variants (frVars) across 27 loci, including the complex 17q21.31 region. We identified and validated multiple risk loci using CRISPR interference or excision, including complement 4 (C4A) and APOC1 in AD and PLEKHM1 and KANSL1 in PSP. Functional variants disrupt transcription factor binding sites converging on enhancers with cell type-specific activity in PSP and AD, implicating a neuronal SP1-driven regulatory network in PSP pathogenesis. These analyses suggest that noncoding genetic risk is driven by common genetic variants through their aggregate activity on specific transcriptional programs.
Assuntos
Doença de Alzheimer , Cromossomos Humanos Par 17 , Redes Reguladoras de Genes , Variação Genética , Regiões não Traduzidas , Doença de Alzheimer/genética , Cromossomos Humanos Par 17/genética , Genes Reporter , Loci Gênicos , Estudo de Associação Genômica Ampla , Humanos , Fatores de Risco , Paralisia Supranuclear Progressiva/genética , Regiões não Traduzidas/genéticaRESUMO
CSF1R-related leukoencephalopathy is an autosomal dominant neurodegenerative disease caused by mutations in the tyrosine kinase domain of the colony stimulating factor 1 receptor (CSF1R). Several studies have found that hematogenic stem cell transplantation is an effective disease modifying therapy however the literature regarding prodromal and early symptoms CSF1R-related leukoencephalopathy is limited. We describe a 63-year-old patient with 4 years of repetitive scratching and skin picking behavior followed by 10 years of progressive behavioral, cognitive, and motor decline in a pattern suggesting behavioral variant of frontotemporal dementia. Brain MRI demonstrated prominent frontal and parietal atrophy accompanied by underlying bilateral patchy white matter hyperintensities sparing the U fibers and cavum septum pellucidum. Whole-exome sequencing revealed a novel, predicted deleterious missense variant in a highly conserved amino acid in the tyrosine kinase domain of CSF1R (p.Gly872Arg). Given this evidence and the characteristic clinical and radiological findings this novel variant was classified as likely pathogenic according to the American College of Medical Genetics standard guidelines. Detailed description of the prodromal scratching and skin picking behavior and possible underlying mechanisms in this case furthers knowledge about early manifestations of CSF1R-related leukoencephalopathy with the hope that early detection and timely administration of disease modifying therapies becomes possible.
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Transcriptional changes in Friedreich's ataxia (FRDA), a rare and debilitating recessive Mendelian neurodegenerative disorder, have been studied in affected but inaccessible tissues-such as dorsal root ganglia, sensory neurons and cerebellum-in animal models or small patient series. However, transcriptional changes induced by FRDA in peripheral blood, a readily accessible tissue, have not been characterized in a large sample. We used differential expression, association with disability stage, network analysis and enrichment analysis to characterize the peripheral blood transcriptome and identify genes that were differentially expressed in FRDA patients (n = 418) compared with both heterozygous expansion carriers (n = 228) and controls (n = 93 739 individuals in total), or were associated with disease progression, resulting in a disease signature for FRDA. We identified a transcriptional signature strongly enriched for an inflammatory innate immune response. Future studies should seek to further characterize the role of peripheral inflammation in FRDA pathology and determine its relevance to overall disease progression.
Assuntos
Biomarcadores/sangue , Ataxia de Friedreich/sangue , Ataxia de Friedreich/genética , Redes Reguladoras de Genes , Mediadores da Inflamação/sangue , Inflamação/genética , Transcriptoma , Adulto , Estudos de Casos e Controles , Feminino , Ataxia de Friedreich/patologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
OBJECTIVE: Changes in progranulin (GRN) expression have been hypothesized to alter risk for Alzheimer's disease (AD). We investigated the relationship between GRN expression in peripheral blood and clinical diagnosis of AD and mild cognitive impairment (MCI). METHODS: Peripheral blood progranulin gene expression was measured, using microarrays from Alzheimer's (n = 186), MCI (n = 118), and control (n = 204) subjects from the University of California San Francisco Memory and Aging Center (UCSF-MAC) and two independent published series (AddNeuroMed and ADNI). GRN gene expression was correlated with clinical, demographic, and genetic data, including APOE haplotype and the GRN rs5848 single-nucleotide polymorphism. Finally, we assessed progranulin protein levels, using enzyme-linked immunosorbent assay, and methylation status using methylation microarrays. RESULTS: We observed an increase in blood progranulin gene expression and a decrease in GRN promoter methylation in males (P = 0.007). Progranulin expression was 13% higher in AD and MCI patients compared with controls in the UCSF-MAC cohort (F2,505 = 10.41, P = 3.72*10-5). This finding was replicated in the AddNeuroMed (F2,271 = 17.9, P = 4.83*10-8) but not the ADNI series. The rs5848 SNP (T-allele) predicted decreased blood progranulin gene expression (P = 0.03). The APOE4 haplotype was positively associated with progranulin expression independent of diagnosis (P = 0.04). Finally, we did not identify differences in plasma progranulin protein levels or gene methylation between diagnostic categories. INTERPRETATION: Progranulin mRNA is elevated in peripheral blood of patients with AD and MCI and its expression is associated with numerous genetic and demographic factors. These data suggest a role in the pathogenesis of neurodegenerative dementias besides frontotemporal dementia.
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Patients with frontotemporal lobar degeneration (FTLD) can show superimposed amyloid pathology, though the impact of amyloid on the clinical presentation of FTLD is not well characterized. This cross-sectional case-control study compared clinical features, fluorodeoxyglucose-positron emission tomography metabolism and gray matter volume loss in 30 patients with familial FTLD in whom amyloid status was confirmed with autopsy or Pittsburgh compound B-PET. Compared to the amyloid-negative patients, the amyloid-positive patients performed significantly worse on several cognitive tests and showed hypometabolism and volume loss in more temporoparietal regions. Our results suggest that in FTLD amyloid positivity is associated with a more Alzheimer's disease-like pattern of neurodegeneration.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Amiloide/metabolismo , Encéfalo/patologia , Demência Frontotemporal/patologia , Degeneração Lobar Frontotemporal/patologia , Substância Cinzenta/patologia , Idoso , Encéfalo/metabolismo , Estudos de Casos e Controles , Estudos Transversais , Feminino , Demência Frontotemporal/metabolismo , Degeneração Lobar Frontotemporal/metabolismo , Substância Cinzenta/metabolismo , Humanos , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-IdadeRESUMO
With the completion of the zebrafish genome sequencing project, it becomes possible to analyze the function of zebrafish genes in a systematic way. The first step in such an analysis is to inactivate each protein-coding gene by targeted or random mutation. Here we describe a streamlined pipeline using proviral insertions coupled with high-throughput sequencing and mapping technologies to widely mutagenize genes in the zebrafish genome. We also report the first 6144 mutagenized and archived F1's predicted to carry up to 3776 mutations in annotated genes. Using in vitro fertilization, we have rescued and characterized ~0.5% of the predicted mutations, showing mutation efficacy and a variety of phenotypes relevant to both developmental processes and human genetic diseases. Mutagenized fish lines are being made freely available to the public through the Zebrafish International Resource Center. These fish lines establish an important milestone for zebrafish genetics research and should greatly facilitate systematic functional studies of the vertebrate genome.
Assuntos
Técnicas de Inativação de Genes , Estudo de Associação Genômica Ampla , Genômica , Peixe-Zebra/genética , Alelos , Animais , Mapeamento Cromossômico/métodos , Biologia Computacional/métodos , Gammaretrovirus/fisiologia , Anotação de Sequência Molecular , Mutagênese Insercional , Mutação , Fenótipo , Integração ViralRESUMO
ZInC (Zebrafish Insertional Collection, http://research.nhgri.nih.gov/ZInC/) is a web-searchable interface of insertional mutants in zebrafish. Over the last two decades, the zebrafish has become a popular model organism for studying vertebrate development as well as for modeling human diseases. To facilitate such studies, we are generating a genome-wide knockout resource that targets every zebrafish protein-coding gene. All mutant fish are freely available to the scientific community through the Zebrafish International Resource Center (ZIRC). To assist researchers in finding mutant and insertion information, we developed a comprehensive database with a web front-end, the ZInC. It can be queried using multiple types of input such as ZFIN (Zebrafish Information Network) IDs, UniGene accession numbers and gene symbols from zebrafish, human and mouse. In the future, ZInC may include data from other insertional mutation projects as well. ZInC cross-references all integration data with the ZFIN (http://zfin.org/).
Assuntos
Bases de Dados Genéticas , Mutagênese Insercional , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Técnicas de Inativação de Genes , Internet , MutaçãoRESUMO
As a vertebrate genetic model, the zebrafish has been well recognized for its strength in studying a variety of biological processes and human diseases. Traditional forward genetic screens in zebrafish have generated a large pool of mutants with interesting phenotypes resembling human diseases but the underlying mechanisms are not well understood. A powerful approach to elucidate the mechanisms of these mutants is the modifier screen, which identifies 2(nd)-site mutations that specifically enhance or block the phenotype of a given mutant. Here we described the first genetic suppressor screen in zebrafish, which identifies a novel transcriptional mechanism regulating erythropoiesis. In combination with the haploid genetics in zebrafish, we have shown the feasibility and strength of a modifier screen in zebrafish. This strategy will greatly broaden the utility of the zebrafish as a model for making original discoveries and establishing novel paradigms for understanding vertebrate biology.
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Genes Supressores , Testes Genéticos/métodos , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Cromossomos Artificiais Bacterianos/genética , Análise Mutacional de DNA , Etilnitrosoureia/farmacologia , Feminino , Genes Recessivos , Haploidia , Larva/genética , Masculino , Mutagênese , Mutagênicos/farmacologia , Mutação , Fatores de Transcrição/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
Recent genome-wide studies have demonstrated that pausing of RNA polymerase II (Pol II) occurred on many vertebrate genes. By genetic studies in the zebrafish tif1gamma mutant moonshine we found that loss of function of Pol II-associated factors PAF or DSIF rescued erythroid gene transcription in tif1gamma-deficient animals. Biochemical analysis established physical interactions among TIF1gamma, the blood-specific SCL transcription complex, and the positive elongation factors p-TEFb and FACT. Chromatin immunoprecipitation assays in human CD34(+) cells supported a TIF1gamma-dependent recruitment of positive elongation factors to erythroid genes to promote transcription elongation by counteracting Pol II pausing. Our study establishes a mechanism for regulating tissue cell fate and differentiation through transcription elongation.
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Eritropoese , Fatores de Transcrição/metabolismo , Transcrição Gênica , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Animais , Linhagem Celular Tumoral , Células Cultivadas , Células Eritroides/metabolismo , Humanos , RNA Polimerase II/metabolismo , Peixe-Zebra/metabolismoRESUMO
Transgenesis using bacterial artificial chromosomes (BAC) allows greater fidelity in directing desirable expression of transgenes. Application of this technology in the optically transparent zebrafish with fluorescent protein reporters enables unparalleled visual analysis of gene expression in a living organism. We have developed a streamlined procedure of directly selecting multiple BAC clones based on public sequence databases followed by rapid modification with green fluorescent protein or red fluorescent protein for transgenic analysis in zebrafish. In this chapter, experimental procedures for BAC DNA preparation and generation of transgenic zebrafish lines by microinjection are described.
Assuntos
Cromossomos Artificiais Bacterianos/metabolismo , Técnicas de Transferência de Genes , Peixe-Zebra/genética , Animais , DNA/genética , DNA/metabolismo , Bases de Dados de Ácidos Nucleicos , Escherichia coli/genética , Escherichia coli/metabolismo , Genes Reporter , Engenharia Genética/métodos , Proteínas de Fluorescência Verde , Proteínas Luminescentes , Recombinação Genética , Análise de Sequência de DNA , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Proteína Vermelha FluorescenteRESUMO
BACKGROUND: GATA-2 is a transcription factor required for hematopoietic stem cell survival as well as for neuronal development in vertebrates. It has been shown that specific expression of GATA-2 in blood progenitor cells requires distal cis-acting regulatory elements. Identification and characterization of these elements should help elucidating transcription regulatory mechanisms of GATA-2 expression in hematopoietic lineage. RESULTS: By pair-wise alignments of the zebrafish genomic sequences flanking GATA-2 to orthologous regions of fugu, mouse, rat and human genomes, we identified three highly conserved non-coding sequences in the genomic region flanking GATA-2, two upstream of GATA-2 and another downstream. Using both transposon and bacterial artificial chromosome mediated germline transgenic zebrafish analyses, one of the sequences was established as necessary and sufficient to direct hematopoietic GFP expression in a manner that recapitulates that of GATA-2. In addition, we demonstrated that this element has enhancer activity in mammalian myeloid leukemia cell lines, thus validating its functional conservation among vertebrate species. Further analysis of potential transcription factor binding sites suggested that integrity of the putative HOXA3 and LMO2 sites is required for regulating GATA-2/GFP hematopoietic expression. CONCLUSION: Regulation of GATA-2 expression in hematopoietic cells is likely conserved among vertebrate animals. The integrated approach described here, drawing on embryological, transgenesis and computational methods, should be generally applicable to analyze tissue-specific gene regulation involving distal DNA cis-acting elements.
Assuntos
Fator de Transcrição GATA2/genética , Regulação da Expressão Gênica no Desenvolvimento , Elementos Reguladores de Transcrição/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Linhagem Celular Tumoral , Sequência Conservada , Proteínas de Fluorescência Verde , Células-Tronco Hematopoéticas , Humanos , TransfecçãoRESUMO
Transgenesis using bacterial artificial chromosomes (BAC) offers greater fidelity in directing desirable expression of foreign genes. Application of this technology in the optically transparent zebrafish with fluorescent protein reporters enables unparalleled visual analysis of regulation of gene expression in a living organism. Here we describe a streamlined procedure of direct selecting multiple BAC clones based on public sequence databases followed by rapid modification with GFP or RFP for transgenic analysis in zebrafish. Experimental procedures for BAC DNA preparation, microinjection of zebrafish embryos and screening of transgenic zebrafish carrying GFP/RFP modified BAC clones are detailed.
Assuntos
Animais Geneticamente Modificados/genética , Cromossomos Artificiais Bacterianos/genética , Engenharia Genética/métodos , Peixe-Zebra/genética , Animais , Embrião não Mamífero , Escherichia coli/genética , Regulação da Expressão Gênica , Genes Bacterianos , Genes Reporter , Proteínas de Fluorescência Verde/análise , Proteínas Luminescentes/análise , Plasmídeos/genética , Recombinases Rec A/genética , Proteínas Recombinantes de Fusão/análise , Recombinação Genética , Peixe-Zebra/embriologia , Proteína Vermelha FluorescenteRESUMO
The pituitary hormone prolactin (PRL) regulates salt and water homeostasis by altering ion retention and water uptake through peripheral osmoregulatory organs. To understand the role of osmotic homeostasis in the development of PRL-secreting lactotrophs, we generated germline transgenic zebrafish coexpressing red fluorescent protein directed by Prolactin regulatory elements (PRL-RFP) and green fluorescent protein by the Pro-opiomelanocortin promoter (POMC-GFP). Transparent embryos expressing fluorescent markers specifically targeted to lactotrophs and corticotrophs, the two pituitary lineages involved in teleost osmotic adaptation, allowed in vivo dynamic tracing of pituitary ontogeny during altered environmental salinity. Physiological osmotic changes selectively regulate lactotroph but not corticotroph proliferation during early ontogeny. These changes are not suppressed by pharmacological dopamine receptor blockade but are completely abrogated by morpholino knockdown of the PRL receptor. PRL receptor signaling exerts robust effects on lactotroph development and plays a permissive role in lactotroph osmo-responsiveness, reflecting the dual peripheral and central interactions required for early pituitary development and embryonic homeostasis.
Assuntos
Adeno-Hipófise/fisiologia , Receptores da Prolactina/fisiologia , Peixe-Zebra/fisiologia , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Sequência de Bases , DNA Antissenso/genética , Dados de Sequência Molecular , Filogenia , Adeno-Hipófise/embriologia , Interferência de RNA , Receptores Dopaminérgicos/deficiência , Receptores Dopaminérgicos/genética , Receptores Dopaminérgicos/fisiologia , Receptores da Prolactina/deficiência , Receptores da Prolactina/genética , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Equilíbrio Hidroeletrolítico , Peixe-Zebra/embriologia , Peixe-Zebra/genéticaRESUMO
Gata2 is an essential hematopoietic transcriptional factor that is also expressed prominently in the nervous system. The early lethality of knockout mice due to severe anemia has largely precluded studies of gata2 neural regulation and function. In this report, we describe the identification of zebrafish Pur alpha and Sp8 orthologs as two factors that function to regulate neuronal expression of gata2. During embryogenesis, Pur alpha is expressed widely, whereas Sp8 has an overlapping pattern of expression with gata2 in the nervous system. Knockdown and ectopic expressions of Pur alpha and Sp8 indicate that these factors function, respectively, as a repressor and an activator of gata2 gene expression in the nervous system. With consideration given to the previously established roles for these factors, we propose a model for how the transcriptional regulation of neural gata2 expression may be involved in controlling cellular proliferation in the nervous system.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Helminto/metabolismo , Neurônios/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Fator de Transcrição GATA2 , Gástrula/fisiologia , Regulação da Expressão Gênica/fisiologia , Genes Reporter , Proteínas de Helminto/genética , Ligação Proteica/fisiologia , Fatores de Transcrição/genética , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/genéticaRESUMO
Oocyte maturation and egg fertilization in both vertebrates and invertebrates are marked by orchestrated cytoplasmic translocation of secretory vesicles known as cortical granules. It is thought that such redistribution of cellular content is critical for asymmetrical cell division during early development, but the mechanism and regulation of the process is poorly understood. Here we report the identification, purification and cDNA cloning of a C-type lectin from oocytes of a freshwater fish species gibel carp (Carassius auratus gibelio). The purified protein has been demonstrated to have lectin activity and to be a Ca(2+)-dependent C-type lectin by hemagglutination activity assay. Immunocytochemistry revealed that the lectin is associated with cortical granules, gradually translocated to the cell surface during oocyte maturation, and discharged to the egg envelope upon fertilization. Interestingly, the lectin becomes phosphorylated on threonine residues upon induction of exocytosis by fertilization and returns to its original state after morula stage of embryonic development, suggesting that this posttranslational modification may represent a critical molecular switch for early embryonic development.
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Grânulos Citoplasmáticos/metabolismo , Fertilização/fisiologia , Lectinas Tipo C/metabolismo , Oócitos/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Eletroforese Capilar , Imunofluorescência , Carpa Dourada , Lectinas Tipo C/genética , Lectinas Tipo C/isolamento & purificação , Dados de Sequência Molecular , Óvulo/metabolismoRESUMO
The solitary LTRs of ERV-9 human endogenous retrovirus are middle repetitive DNAs associated with 3,000-4,000 human gene loci including the beta-globin gene locus where the ERV-9 LTR is juxtaposed to the locus control region (beta-LCR) far upstream of the globin genes. The ERV-9 LTRs are conserved during primate evolution, but their function in the primate genomes is unknown. Here, we show that in transgenic zebrafish harboring the beta-globin ERV-9 LTR coupled to the GFP gene, the LTR enhancer was active and initiated synthesis of GFP mRNA in oocytes but not in spermatozoa, and GFP expression in the embryos was maternally inherited. The LTR enhancer was active also in stem/progenitor cell regions of adult tissues of transgenic zebrafish. In human tissues, ERV-9 LTR enhancer was active also in oocytes and stem/progenitor cells but not in spermatozoa and a number of differentiated, adult somatic cells. Transcriptional analyses of the human beta-globin gene locus showed that the beta-globin ERV-9 LTR enhancer initiated RNA synthesis from the LTR in the direction of the downstream beta locus control region and globin genes in ovary and erythroid progenitor cells. The findings suggest that, during oogenesis, ERV-9 LTR enhancers in the human genome could activate the cis-linked gene loci to synthesize maternal mRNAs required for early embryogenesis. Alternatively, the ERV-9 LTR enhancers, in initiating RNA syntheses into the downstream genomic DNAs, could transcriptionally potentiate and preset chromatin structure of the cis-linked gene loci in oocytes and adult stem/progenitor cells.
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Retrovirus Endógenos/genética , Elementos Facilitadores Genéticos , Oócitos/virologia , Células-Tronco/virologia , Sequências Repetidas Terminais , Animais , Animais Geneticamente Modificados , Diferenciação Celular , Feminino , Globinas/genética , Proteínas de Fluorescência Verde , Humanos , Região de Controle de Locus Gênico , Proteínas Luminescentes/genética , Masculino , Oócitos/metabolismo , Oogênese/genética , Proteínas Recombinantes/genética , Espermatozoides/virologia , Células-Tronco/metabolismo , Peixe-ZebraRESUMO
We characterized zebrafish proopiomelanocortin (POMC) gene promoter, and sequence analysis revealed that the promoter contains regulatory elements conserved among vertebrate species. To monitor the ontogeny of the pituitary POMC lineage in living vertebrates, we generated transgenic zebrafish expressing green fluorescent protein (GFP) driven by the POMC promoter. Zebrafish POMC-GFP is first expressed asymmetrically as two bilateral groups of cells most anterior to the neural ridge midline at 18-20 h post fertilization (hpf). POMC-GFP-positive cells then fuse into a single-cell mass within the pituitary anlage after 24 hpf and subsequently organize as distinct anterior and posterior domains between 48 and 64 hpf. Immunohistochemical studies with ACTH and alphaMSH antisera showed that POMC-GFP was mainly targeted to both anterior and posterior pituitary corticotrophs, whereas posterior pituitary region melanotrophs did not express GFP. To determine in vivo zebrafish corticotroph responses, dexamethasone (10(-5) m) was added to live embryos, which selectively suppressed POMC-GFP expression in the anterior group of corticotrophs, suggesting a distinct domain that is responsive to glucocorticoid feedback. Transgenic zebrafish with specific POMC-GFP expression in pituitary corticotrophs offers a powerful genetic system for in vivo study of vertebrate corticotroph lineage development.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Hipófise/fisiologia , Pró-Opiomelanocortina/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Sequência de Bases , Linhagem da Célula , Sequência Conservada , Dexametasona/farmacologia , Regulação para Baixo/efeitos dos fármacos , Embrião não Mamífero/efeitos dos fármacos , Feminino , Proteínas de Fluorescência Verde , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Dados de Sequência Molecular , Hipófise/citologia , Hipófise/efeitos dos fármacos , Hipófise/embriologia , Pró-Opiomelanocortina/metabolismo , Regiões Promotoras Genéticas , Vertebrados/genética , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/metabolismoRESUMO
Silver crucian carp (Carassius auratus gibelio) is a unique gynogenetic fish. Because of its specific genetic background and reproduction mode, it is an intriguing model system for understanding regulatory mechanism of oocyte maturation division. It keeps its chromosomal integrity by inhibiting the first meiotic division (no extrusion of the first pole body). The spindle behavior during oocyte maturation is significantly different from that in gonochoristic fish. The chromosomes are first arranged in a tripolar spindle, and then they turn around and are reunited mutually to form a normal bipolar spindle. A new member of the fish A-type cyclin gene, cyclin A2, has been isolated by suppression of subtractive hybridization on the basis of its differential transcription in fully-grown oocytes between the gynogenetic silver crucian carp and gonochoristic color crucian carp. There are 18 differing amino acids in the total 428 residues of cyclin A2 between the two forms of crucian carps. In addition, cDNAs of cyclin Al and cyclin B have also been cloned from them. Thus two members of A-type cyclins, cyclin Al and cyclin A2, are demonstrated to exist in fish, just as in frog, humans, and mouse. Northern blotting reveals that cyclin A2 mRNA is more than 20-fold and cyclin A1 mRNA is about 2-fold in fully grown oocytes of gynogenetic silver crucian carp compared to gonochoristic color crucian carp. However, cyclin B does not show such a difference between them. Western blot analysis also shows that the cyclin A2 protein stockpiled in fully grown oocytes of gynogenetic crucian carp is much more abundant than in gonochoristic crucian carp. Moreover, two different cyclin A2 expression patterns during oocyte maturation have been revealed in the two closely related crucian carps. For color crucian carp, cyclin A2 protein is translated only after hormone stimulation. For silver crucian carp, cyclin A2 protein can be detected throughout the process of maturation division. The different expression of cyclin A2 may be a clue to understanding the special maturation division of gynogenetic silver crucian carp.
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Carpas/classificação , Carpas/genética , Ciclina A/genética , Regulação da Expressão Gênica , Oócitos/crescimento & desenvolvimento , Oócitos/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Clonagem Molecular , Ciclina A/análise , Ciclina A/química , Ciclina A/imunologia , DNA Complementar/genética , Feminino , Soros Imunes/imunologia , Fator Promotor de Maturação/metabolismo , Meiose , Dados de Sequência Molecular , Oócitos/citologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Although mammals have been cloned from genetically manipulated cultured cells, a comparable achievement has not been realized in lower vertebrates. Here we report that fertile transgenic zebrafish can be obtained by nuclear transfer using embryonic fibroblast cells from long-term cultures. The donor nuclei, modified by retroviral insertions expressing green fluorescent protein (GFP), were transplanted into manually enucleated eggs. Overall, a 2% success rate was achieved, resulting in 11 adult transgenic zebrafish expressing GFP. These nuclear transplants produced fertile, diploid offspring, and their F1/F2 progeny continued to express GFP in a pattern identical to that of the founder fish. This finding demonstrates that slowly dividing nuclei from cultured cells can be reprogrammed to support rapid embryonic development and sets up a foundation for targeted genetic manipulation in zebrafish.