RESUMO
Alterations in the tumor suppressor ATRX are recurrently observed in mesenchymal neoplasms. ATRX has multiple epigenetic functions including heterochromatin formation and maintenance and regulation of transcription through modulation of chromatin accessibility. Here, we show in murine mesenchymal progenitor cells (MPCs) that Atrx deficiency aberrantly activated mesenchymal differentiation programs. This includes adipogenic pathways where ATRX loss induced expression of adipogenic transcription factors and enhanced adipogenic differentiation in response to differentiation stimuli. These changes are linked to loss of heterochromatin near mesenchymal lineage genes together with increased chromatin accessibility and gains of active chromatin marks. We additionally observed depletion of H3K9me3 at transposable elements, which are derepressed including near mesenchymal genes where they could serve as regulatory elements. Finally, we demonstrated that loss of ATRX in a mesenchymal malignancy, undifferentiated pleomorphic sarcoma, results in similar epigenetic disruption and de-repression of transposable elements. Together, our results reveal a role for ATRX in maintaining epigenetic states and transcriptional repression in mesenchymal progenitors and tumor cells and in preventing aberrant differentiation in the progenitor context.
Assuntos
Diferenciação Celular , Heterocromatina , Células-Tronco Mesenquimais , Proteína Nuclear Ligada ao X , Animais , Humanos , Camundongos , Adipogenia , Elementos de DNA Transponíveis/genética , Epigênese Genética , Heterocromatina/metabolismo , Heterocromatina/genética , Histonas/metabolismo , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , Proteína Nuclear Ligada ao X/genética , Proteína Nuclear Ligada ao X/metabolismoRESUMO
OBJECTIVE: To characterize the results of surgery for gastrointestinal stromal tumor (GIST) in the pre and post-imatinib eras at a single institution and to identify current prognostic clinicopathologic factors. BACKGROUND: Imatinib has radically changed the management of GIST, yet the magnitude of impact on outcome across the spectrum of GIST presentation and relevance of historical prognostic factors are not well defined. METHODS: We retrospectively analyzed 1000 patients who underwent surgery for GIST at our institution from 1982 to 2016. Patients were stratified by presentation status as primary tumor only (PRIM), primary with synchronous metastasis (PRIMâ+âMET), or metachronous recurrence/metastases (MET), and also imatinib era (before and after it became available). Cox proportional-hazard models and Kaplan-Meier methods were used to model and estimate overall survival (OS) and recurrence-free survival (RFS). RESULTS: OS was longer in the imatinib era compared with the pre-imatinib era in each presentation group, including in Miettinen high-risk primary tumors. Among PRIM patients from the pre-imatinib era, tumor site, size, and mitotic rate were independently associated with OS and RFS on multivariate analysis. PRIM patients in the imatinib era who received imatinib (neoadjuvant and/or adjuvant) had higher risk tumors, but after adjusting for treatment, only size >10âcm remained independently prognostic of RFS [hazard ratio (HR) 3.85, 95% confidence interval (CI) 2.00-7.40, P < 0.0001) and OS (HR 3.37, 95% CI 1.60-7.13, P = 0.001)]. CONCLUSIONS: Patients treated in the imatinib era had prolonged OS across all presentations. In the imatinib era, among site, size, and mitotic rate, high-risk features were associated with treatment with the drug, but only size >10âcm correlated with outcome. Imatinib should still be prescribed for patients with high-risk features.
Assuntos
Antineoplásicos/uso terapêutico , Tumores do Estroma Gastrointestinal/tratamento farmacológico , Tumores do Estroma Gastrointestinal/cirurgia , Mesilato de Imatinib/uso terapêutico , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Feminino , Tumores do Estroma Gastrointestinal/mortalidade , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Taxa de Sobrevida , Resultado do Tratamento , Adulto JovemRESUMO
Cellular mosaicism due to monoallelic autosomal expression (MAE), with cell selection during development, is becoming increasingly recognized as prevalent in mammals, leading to interest in understanding its extent and mechanism(s). We report here use of clonal cell lines derived from the CNS of adult female [Formula: see text] hybrid (C57BL/6 X JF1) mice to characterize MAE as neural stem cells (nscs) differentiate to astrocyte-like cells (asls). We found that different subsets of genes show MAE in the two populations of cells; in each case, there is strong enrichment for genes specific to the respective developmental state. Genes that exhibit MAE are 22% of nsc-specific genes and 26% of asl-specific genes. Moreover, the promoters of genes with MAE have reduced CpG dinucleotides but increased CpG differences between the two parental mouse strains. Extending the study of variability to wild populations of mice, we found evidence for balancing selection as a contributing force in evolution of those genes showing developmental specificity (i.e., expressed in either nsc or asl), not just for genes showing MAE. Furthermore, we found that genes showing skewed allelic expression (SKE) were similarly enriched among cell type-specific genes and also showed a heightened probability of balancing selection. Thus, developmental stage-specific genes and genes with MAE or SKE seem to make up overlapping classes subject to selection for increased diversity. The implications of these results for development and evolution are discussed in the context of a model with stochastic epigenetic modifications taking place only during a relatively brief developmental window.
Assuntos
Sistema Nervoso Central/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Genes Controladores do Desenvolvimento/genética , Seleção Genética/genética , Alelos , Animais , Astrócitos/fisiologia , Diferenciação Celular/genética , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Neurais/fisiologia , Regiões Promotoras Genéticas/genéticaRESUMO
LESSONS LEARNED: The combination of pexidartinib and binimetinib was safe and tolerable and demonstrated encouraging signs of efficacy in two patients with advanced gastrointestinal stromal tumor (GIST) refractory to tyrosine kinase inhibitors (TKIs).Molecular profiling of GISTs at diagnosis and upon progression may provide insight into the mechanisms of response or resistance to targeted therapies.Additional trials are needed to further explore combined KIT and MEK inhibition in treatment-naïve and TKI-refractory patients with advanced GIST. BACKGROUND: Nearly all patients with advanced gastrointestinal stromal tumor (GIST) develop resistance to imatinib, and subsequent treatments have limited efficacy. Dual inhibition of KIT and MAPK pathways has synergistic antitumor activity in preclinical GIST models. METHODS: This was an investigator-initiated, phase I, dose escalation study of the MEK inhibitor binimetinib combined with pexidartinib, a potent inhibitor of CSF1R, KIT, and FLT3, in patients with advanced or metastatic GIST who progressed on imatinib. The primary endpoint was phase II dose determination; secondary endpoints included safety, tolerability, and efficacy. An expansion cohort to further evaluate safety and efficacy was planned. RESULTS: Two patients were treated at dose level one (binimetinib 30 mg b.i.d. and pexidartinib 400 mg every morning and 200 mg every evening), after which the study was terminated by the manufacturer. No dose-limiting toxicities (DLTs) were reported, and treatment was well tolerated. The only grade ≥3 treatment-emergent adverse event (TEAE) was asymptomatic elevated creatine phosphokinase (CPK). Both patients had a best response of stable disease (SD) by RECIST. Progression-free survival (PFS) and overall survival (OS) were 6.1 and 14.6 months, respectively, in one patient with five prior lines of therapy. The second patient with NF1-mutant GIST had a 27% decrease in tumor burden by RECIST and remains on study after 19 months of treatment. CONCLUSION: Pexidartinib combined with binimetinib was tolerable, and meaningful clinical activity was observed in two imatinib-refractory patients.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Gastrointestinais/tratamento farmacológico , Tumores do Estroma Gastrointestinal/tratamento farmacológico , Idoso , Aminopiridinas/administração & dosagem , Aminopiridinas/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/farmacocinética , Benzimidazóis/administração & dosagem , Benzimidazóis/efeitos adversos , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Feminino , Neoplasias Gastrointestinais/metabolismo , Neoplasias Gastrointestinais/patologia , Tumores do Estroma Gastrointestinal/metabolismo , Tumores do Estroma Gastrointestinal/patologia , Humanos , Masculino , Dose Máxima Tolerável , Pessoa de Meia-Idade , Pirróis/administração & dosagem , Pirróis/efeitos adversos , Distribuição TecidualRESUMO
BACKGROUND: Approximately 5% of gastrointestinal stromal tumors (GISTs) originate in the rectum, and historically, radical resection was commonly performed. Little is known about the outcome for rectal GIST in the era of imatinib. METHODS: Using a prospectively maintained database, this study retrospectively analyzed 47 localized primary rectal GISTs treated at our center from 1982 to 2016, stratified by when imatinib became available in 2000. Overall survival (OS), disease-specific survival (DSS), and recurrence-free survival (RFS) were analyzed by the Kaplan-Meier method. RESULTS: Rectal GISTs represented 7.1% of 663 primary GISTs. The findings showed 17 patients in the pre-imatinib era and 30 patients in the imatinib era. The two groups had similar follow-up evaluation, age, gender, Miettinen risk, and distance to the anal verge. In the imatinib era, tumors were smaller at diagnosis (median 4 vs. 5 cm; p = 0.029), and 24 of the 30 patients received perioperative imatinib. In the high-risk patients, organ preservation and negative margins were more common among the 13 patients treated with neoadjuvant imatinib than among the 21 patients treated directly with surgery. High-risk patients who received perioperative imatinib (n = 15) had greater (or nearly significantly greater) 5-year OS, DSS, local RFS, and distant RFS than those who did not (n = 19) (91, 100, 100, and 71% vs. 47, 65, 74, and 41%; p = 0.049, 0.052, 0.077, 0.051, respectively). In the imatinib era, no patient has had a local recurrence or death due to GIST. CONCLUSIONS: The use of imatinib is associated with organ preservation and improved oncologic outcome for patients with rectal GIST.
Assuntos
Neoplasias Gastrointestinais/mortalidade , Tumores do Estroma Gastrointestinal/mortalidade , Mesilato de Imatinib/uso terapêutico , Terapia Neoadjuvante/mortalidade , Recidiva Local de Neoplasia/mortalidade , Neoplasias Retais/mortalidade , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/uso terapêutico , Feminino , Seguimentos , Neoplasias Gastrointestinais/tratamento farmacológico , Neoplasias Gastrointestinais/patologia , Tumores do Estroma Gastrointestinal/tratamento farmacológico , Tumores do Estroma Gastrointestinal/patologia , Humanos , Masculino , Oncologia , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/tratamento farmacológico , Recidiva Local de Neoplasia/patologia , Preservação de Órgãos , Prognóstico , Estudos Prospectivos , Neoplasias Retais/tratamento farmacológico , Neoplasias Retais/patologia , Estudos Retrospectivos , Taxa de SobrevidaRESUMO
Synovial sarcoma (SyS) is an aggressive soft-tissue malignancy characterized by a pathognomonic chromosomal translocation leading to the formation of the SS18::SSX fusion oncoprotein. SS18::SSX associates with mammalian BAF complexes suggesting deregulation of chromatin architecture as the oncogenic driver in this tumour type. To examine the epigenomic state of SyS we performed comprehensive multi-omics analysis on 52 primary pre-treatment human SyS tumours. Our analysis revealed a continuum of epigenomic states across the cohort at fusion target genes independent of rare somatic genetic lesions. We identify cell-of-origin signatures defined by enhancer states and reveal unexpected relationships between H2AK119Ub1 and active marks. The number of bivalent promoters, dually marked by the repressive H3K27me3 and activating H3K4me3 marks, has strong prognostic value and outperforms tumor grade in predicting patient outcome. Finally, we identify SyS defining epigenomic features including H3K4me3 expansion associated with striking promoter DNA hypomethylation in which SyS displays the lowest mean methylation level of any sarcoma subtype. We explore these distinctive features as potential vulnerabilities in SyS and identify H3K4me3 inhibition as a promising therapeutic strategy.
RESUMO
Immune checkpoint blockade (ICB) has demonstrated clinical success in "inflamed" tumors with substantial T cell infiltrates, but tumors with an immune-desert tumor microenvironment (TME) fail to benefit. The tumor cell-intrinsic molecular mechanisms of the immune-desert phenotype remain poorly understood. Here, we demonstrated that inactivation of the polycomb-repressive complex 2 (PRC2) core components embryonic ectoderm development (EED) or suppressor of zeste 12 homolog (SUZ12), a prevalent genetic event in malignant peripheral nerve sheath tumors (MPNSTs) and sporadically in other cancers, drove a context-dependent immune-desert TME. PRC2 inactivation reprogramed the chromatin landscape that led to a cell-autonomous shift from primed baseline signaling-dependent cellular responses (e.g., IFN-γ signaling) to PRC2-regulated developmental and cellular differentiation transcriptional programs. Further, PRC2 inactivation led to diminished tumor immune infiltrates through reduced chemokine production and impaired antigen presentation and T cell priming, resulting in primary resistance to ICB. Intratumoral delivery of inactivated modified vaccinia virus Ankara (MVA) enhanced tumor immune infiltrates and sensitized PRC2-loss tumors to ICB. Our results identify molecular mechanisms of PRC2 inactivation-mediated, context-dependent epigenetic reprogramming that underline the immune-desert phenotype in cancer. Our studies also point to intratumoral delivery of immunogenic viruses as an initial therapeutic strategy to modulate the immune-desert TME and capitalize on the clinical benefit of ICB.
Assuntos
Neoplasias , Vírus , Cromatina , Humanos , Complexo Repressor Polycomb 2/genética , Microambiente Tumoral , Vírus/genéticaRESUMO
PURPOSE: Because the Hedgehog and Notch pathways are often overexpressed in mesenchymal malignancies, we evaluated the efficacy of concurrent inhibition of Notch and Hedgehog signaling using the gamma-secretase inhibitor (GSI) RO4929097 and the smoothened antagonist vismodegib in unresectable or metastatic sarcoma. PATIENTS AND METHODS: In this investigator-initiated trial, phase Ib used standard 3+3 dose escalation in which patients first received vismodegib once daily for 21 days, followed by the combination of RO4929097 concurrently with vismodegib in 21-day cycles. In phase II, patients were randomized to RO4929097 alone or in combination with vismodegib. RESULTS: Nine patients were treated in phase Ib with no dose-limiting toxicities. RO4929097 at 15 mg daily in combination with 150 mg daily of vismodegib was declared the recommended phase II dose. Most adverse events were grade ≤ 2. In phase II (closed early due to discontinuation of RO4929097 evaluation), 34 patients were randomized to RO4929097 alone and 33 to RO4929097 plus vismodegib. RO4929097 did not interfere with the steady-state concentration of vismodegib, while vismodegib reduced the plasma concentration of RO492909. No patients had an objective response. Neither progression-free nor overall survival differed significantly between treatment arms. Paired tumor biopsies from a subset of patients demonstrated inhibition of cleaved Notch. CONCLUSIONS: The combination of RO4929097 plus vismodegib was generally well tolerated. Although accrual to this study was not completed, vismodegib did not meaningfully enhance the clinical efficacy of RO4929097 in an unplanned analysis. GSIs and GSIs plus vismodegib can inhibit intratumoral Notch and downstream phosphorylated Akt signaling.
Assuntos
Proteínas Hedgehog , Sarcoma , Secretases da Proteína Precursora do Amiloide , Anilidas/efeitos adversos , Benzazepinas , Fluorocarbonos , Humanos , PiridinasRESUMO
INTRODUCTION: Neoadjuvant imatinib (Neo-IM) therapy may facilitate R0 resection in primary gastrointestinal stromal tumors (GISTs) that are large or in difficult anatomic locations. While response to preoperative tyrosine kinase inhibitors is associated with better outcome in metastatic GIST, little is known about prognostic factors after Neo-IM in primary GIST. STUDY DESIGN: Patients with primary GIST with or without synchronous metastases who underwent Neo-IM were retrospectively analyzed from a prospective maintained institutional database for Response Evaluation Criteria in Solid Tumors (RECIST), tumor viability, and mitotic rate. Overall survival (OS) was estimated by Kaplan-Meier and compared by log-rank test. Cox proportionate hazard models were used for univariate and multivariate analysis. RESULTS: One hundred and fifty patients were treated for a median of 7.1 months (range 0.2-160). By RECIST, partial response, stable disease, and progressive disease were seen in 40%, 51%, and 9%, respectively. By pathologic analysis, ≤ 50% of the tumor was viable in 72%, and the mitotic rate was ≤ 5/50HPF in 74%. On multivariate analysis, RECIST response and tumor viability were not associated with OS, while post-treatment high mitotic rate (hazard ratio (HR) for death 5.3, CI 2.3-12.4), R2 margins (HR 6.0, CI 2.3-15.5), and adjuvant imatinib (HR 0.4, CI 0.2-0.9) were (p < 0.05). Five-year OS was 81 vs. 38% for low vs. high mitotic rate; 81, 59, and 39% for R0, R1, and R2 margins; and 75 vs 61% for adjuvant vs. no adjuvant imatinib therapy (p < 0.05). CONCLUSIONS: In primary GIST undergoing Neo-IM therapy, progression was uncommon, but substantial down-sizing occurred in the minority. High tumor mitotic rate and incomplete resection following Neo-IM were associated with poor outcome, while adjuvant imatinib was associated with prolonged survival.
Assuntos
Antineoplásicos , Neoplasias Gastrointestinais , Tumores do Estroma Gastrointestinal , Antineoplásicos/uso terapêutico , Neoplasias Gastrointestinais/tratamento farmacológico , Tumores do Estroma Gastrointestinal/tratamento farmacológico , Humanos , Mesilato de Imatinib/uso terapêutico , Terapia Neoadjuvante , Prognóstico , Estudos Prospectivos , Estudos RetrospectivosRESUMO
Here, we report on a phase IIa study to determine the intubation rate, survival, viral clearance, and development of endogenous Abs in patients with COVID-19 pneumonia treated with convalescent plasma (CCP) containing high levels of neutralizing anti-SARS-CoV-2 Abs. Radiographic and laboratory evaluation confirmed all 51 treated patients had COVID-19 pneumonia. Fresh or frozen CCP from donors with high titers of neutralizing Abs was administered. The nonmechanically ventilated patients (n = 36) had an intubation rate of 13.9% and a 30-day survival rate of 88.9%, and the overall survival rate for a comparative group based on network data was 72.5% (1625/2241). Patients had negative nasopharyngeal swab rates of 43.8% and 73.0% on days 10 and 30, respectively. Patients mechanically ventilated had a day-30 mortality rate of 46.7%; the mortality rate for a comparative group based on network data was 71.0% (369/520). All evaluable patients were found to have neutralizing Abs on day 3 (n = 47), and all but 1 patient had Abs on days 30 and 60. The only adverse event was a mild rash. In this study on patients with COVID-19 disease, we show therapeutic use of CCP was safe and conferred transfer of Abs, while preserving endogenous immune response.
Assuntos
Anticorpos Neutralizantes/uso terapêutico , Anticorpos Antivirais/uso terapêutico , COVID-19/terapia , Imunoglobulina G/uso terapêutico , Plasma , SARS-CoV-2/imunologia , Índice de Gravidade de Doença , Idoso , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , COVID-19/imunologia , COVID-19/mortalidade , COVID-19/virologia , Convalescença , Feminino , Humanos , Imunização Passiva , Hospedeiro Imunocomprometido , Imunoglobulina G/sangue , Masculino , Pessoa de Meia-Idade , Pneumonia , Respiração Artificial , Soroterapia para COVID-19RESUMO
Importance: Patients with advanced sarcoma have limited treatment options. Talimogene laherparepvec (T-VEC) has been shown to increase tumor-specific immune activation via augmenting antigen presentation and T-cell priming. Objective: To examine whether T-VEC in combination with pembrolizumab is associated with increased tumor-infiltrating lymphocyte infiltration and programmed death-ligand 1 expression and thus with increased antitumor activity in patients with locally advanced or metastatic sarcoma. Design, Setting, and Participants: This open-label, single-institution phase 2 interventional trial of T-VEC plus pembrolizumab enrolled 20 patients with locally advanced or metastatic sarcoma between March 16 and December 4, 2017, for whom at least 1 standard systemic therapy had failed. The median duration of therapy was 16 weeks (range, 7-67 weeks). Reported analyses include data through December 14, 2018. Intervention: Patients received pembrolizumab (200-mg flat dose) intravenously and T-VEC (first dose, ≤4 mL × 106 plaque-forming units [PFU]/mL; second and subsequent doses, ≤4 mL × 108 PFU/mL) injected into palpable tumor site(s) on day 1 of each 21-day cycle. Main Outcomes and Measures: The primary end point was objective response rate (ORR; complete response and partial response) at 24 weeks determined by Response Evaluation Criteria In Solid Tumors (RECIST), version 1.1, criteria. Secondary end points included best ORR by immune-related RECIST criteria, progression-free survival rate at 24 weeks, overall survival, and safety. Results: All 20 patients (12 women [60%]; median age, 63.5 years [range, 24-90 years]) were evaluable for response. The study met its primary end point of evaluating the best ORR at 24 weeks determined by RECIST, version 1.1, criteria; the best ORR was 30% (95% CI, 12%-54%; n = 6). The ORR overall was 35% (95% CI, 15%-59%; n = 7). The incidence of grade 3 treatment-related adverse events was low (4 patients [20%]). There were no grade 4 treatment-related adverse events or treatment-related deaths. Conclusions and Relevance: In this phase 2 clinical trial, treatment with T-VEC plus pembrolizumab was associated with antitumor activity in advanced sarcoma across a range of sarcoma histologic subtypes, with a manageable safety profile. This combination therapy met its predefined primary study end point; further evaluation of T-VEC in combination with pembrolizumab for patients with select sarcoma subtypes is planned. Trial Registration: ClinicalTrials.gov identifier: NCT03069378.
Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Antineoplásicos Imunológicos/uso terapêutico , Produtos Biológicos/uso terapêutico , Terapia Viral Oncolítica , Sarcoma/terapia , Adulto , Idoso , Idoso de 80 Anos ou mais , Terapia Combinada , Herpesvirus Humano 1 , Humanos , Pessoa de Meia-Idade , Resultado do Tratamento , Adulto JovemRESUMO
The 5' control region and first exon for human X-chromosome-linked phosphoglycerate kinase is contained in a G + C-rich island. We measured methylation at all HpaII sites in this 5' region of leukocyte DNA. By use of a blotting procedure that allows analysis of small DNA fragments, we found that the HpaII sites are entirely methylated when from an inactive X chromosome and entirely unmethylated when from an active one. In contrast, methylation of HpaII sites in more downstream regions of the gene is essentially the same in active and inactive X chromosomes.
Assuntos
Citosina , DNA/genética , Genes , Guanina , Fosfoglicerato Quinase/genética , Cromossomo X , Sequência de Bases , DNA/isolamento & purificação , Enzimas de Restrição do DNA , Feminino , Ligação Genética , Humanos , Masculino , MetilaçãoRESUMO
A HpaII-PCR assay was used to study DNA methylation in individual mouse embryos. It was found that HpaII site H-7 in the CpG island of the X-chromosome-linked Pgk-1 gene is less than or equal to 10% methylated in oocytes and male embryos but becomes 40% methylated in female embryos at 6.5 days; about the time of X-chromosome inactivation of the inner cell mass.
Assuntos
DNA/genética , Fosfatos de Dinucleosídeos , Embrião de Mamíferos/fisiologia , Fosfoglicerato Quinase/genética , Cromossomo X , Animais , Sequência de Bases , Desoxirribonuclease HpaII , Desoxirribonucleases de Sítio Específico do Tipo II , Feminino , Masculino , Metilação , Camundongos , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Reação em Cadeia da Polimerase , Mapeamento por RestriçãoRESUMO
We describe here a very sensitive technique for RNA structure analysis and the determination of transcription start sites and demonstrate its use for mapping the start site of the imprinted Snrpn gene in individual hippocampal neurons. The method is adapted from reverse transcription-terminal transferase-dependent PCR (RT-TDPCR) to include amplification of the antisense sequence by in vitro transcription just prior to the final PCR step. The method should be useful for analysis of all genes for which variation in promoter usage and/or differences in RNA secondary structure may be specific to a given cell type or developmental stage.
Assuntos
Neurônios/metabolismo , RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Ribonucleoproteínas Nucleares Pequenas/genética , Animais , DNA , DNA Nucleotidilexotransferase/metabolismo , Hipocampo/química , Hipocampo/citologia , Camundongos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas , RNA/química , RNA/metabolismo , Transcrição GênicaRESUMO
The human genome contains several DNA sequences homologous to cDNA for X-linked phosphoglycerate kinase (PGK). In the course of screening a genomic library containing a mutant gene for X-linked PGK (PGK-Matsue), we obtained a recombinant clone that cross-hybridizes to PGK cDNA. Restriction analysis suggests that the clone overlaps with a clone previously found to originate from chromosome 6, and which has been suggested to code for testis-specific PGK [Szabo et al., Proc. Natl. Acad. Sci. USA 81 (1984) 3167-3169]. The DNA fragment we have cloned contains 1252 bp that are homologous to the coding part of human PGK cDNA, and has an initiation codon, an in-phase termination codon, direct flanking repeats, and no introns. The maximum homology between the DNA and PGK cDNA is 85% at the coding nucleotide level, and it is 86.2% at the translated protein level; homology is also found in their 3'-flanking sequences and adenine-rich tails. These results suggest that the cloned DNA is most likely a processed pseudogene for PGK located on chromosome 6.
Assuntos
Fosfoglicerato Quinase/genética , Sequência de Bases , Mapeamento Cromossômico , Cromossomos Humanos 6-12 e X , Clonagem Molecular , DNA/genética , Feminino , Genes , Ligação Genética , Humanos , Masculino , Testículo/enzimologia , Cromossomo XRESUMO
A clone containing cDNA for X chromosome-linked phosphoglycerate kinase (PGK-1) was isolated from a mouse myeloma cDNA library. The nucleotide (nt) sequence of the cDNA has been determined, and the amino acid (aa) sequence of the enzyme thereby deduced. At the nt level, the coding region of mouse PGK cDNA has 93% homology with human X-linked cDNA and 60% homology with the yeast gene. Mouse PGK-1 protein contains 416 aa and is 98%, 96% and 64% homologous with human, horse, and yeast enzyme sequences, respectively.
Assuntos
Clonagem Molecular , DNA/metabolismo , Fosfoglicerato Quinase/genética , Cromossomo X , Sequência de Aminoácidos , Animais , Sequência de Bases , Ligação Genética , Cavalos , Humanos , Camundongos , Plasmocitoma/enzimologia , Plasmocitoma/genética , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Homologia de Sequência do Ácido Nucleico , Especificidade da EspécieRESUMO
Genomic imprinting is a special form of epigenetic system that determines the parent-of-origin-specific, or monoallelic, expression of a small number of genes, termed "imprinted" genes. Considerable sequence and methylation analysis of imprinted genes has revealed a common theme: Regions of allele-specific methylation inherited from the gametes, or primary differentially methylated regions (DMRs), are associated with CpG islands and repeat elements, and this overall structure suggests functional significance. For at least three imprinted genes the sequence of the primary DMR constitutes an element able to regulate gene activity in cis--a chromatin insulator and a promoter of an antisense transcript. In these cases the unique feature of imprinting appears to be in the ability to switch the regulatory capacity of these elements on or off by the absence or presence of inherited methylation. Increasing evidence therefore suggests that genomic imprinting for at least some genes constitutes the regulation of gene regulatory elements by methylation. An important challenge now is to determine how the differential methylation of primary DMR sequences is established in the germ line. If methylation is the primary imprint, then the processes establishing it are the primary imprinting mechanisms. Trans-acting factors that are expressed in one sex of germ line and not the other are likely to be involved, and their ability to methylate may be mediated through repeat elements associated with the sequence of primary DMRs.
Assuntos
Metilação de DNA , Impressão Genômica , Alelos , Síndrome de Angelman/genética , Animais , Células Germinativas , Humanos , Fator de Crescimento Insulin-Like II/genética , Síndrome de Prader-Willi/genética , RNA Longo não Codificante , RNA não Traduzido/genéticaRESUMO
We have determined the sequence of an 812-bp BamHI-EcoRI restriction fragment containing the 5' region of the human gene for PGK (3-phosphoglycerate kinase or ATP:3-phospho-D-glycerate 1-phosphotransferase; EC 2.7.2.3). The fragment contains 450 bp 5' to three start points for transcription (located by primer extension and S1 nuclease mapping), a leader sequence 85-94 bp long, the first exon of gene (65 bp), and part of the first intron. The promoter region is extremely G + C-rich, lacks a TATA box, and has an 8-bp direct repeat. A comparison of the promoter region for PGK with other promoters on the X-chromosome reveals homology with the promoter for HPRT, but not with the operator for factor IX.
Assuntos
Fosfoglicerato Quinase/genética , Regiões Promotoras Genéticas , Cromossomo X , Sequência de Bases , Clonagem Molecular , Enzimas de Restrição do DNA , Fator IX/genética , Feminino , Regulação da Expressão Gênica , Humanos , Hipoxantina Fosforribosiltransferase/genética , Dados de Sequência Molecular , Transcrição GênicaRESUMO
The primary structures of six phosphoglycerate kinases (PGKs) are known: three from mammals, one from yeast, and two from trypanosomes. Comparison of the amino acid sequence of these enzymes reveals 154 invariant positions out of 392 positions in the aligned sequences. Most of the conserved positions fall into the twelve beta-sheets and adjacent peptide regions that form the inner loops surrounding the ATP and 3-phosphoglycerate-binding cleft. The homology between mammalian and yeast PGKs is greater than 94% for the inner-loop region, even though the overall homology is less than 65%. Trypanosome PGK has only 44% overall homology with the mammalian enzyme, but shows 74% homology in the inner-loop region. Trypanosome PGK contains a polypeptide segment in its N-terminal domain that is transposed in comparison with the other species.