RESUMO
BACKGROUND: Pancreatic cancer has a poor prognosis because of its high refractoriness to chemotherapy and tumour recurrence, and these properties have been attributed to cancer stem cells (CSCs). MicroRNA (miRNA) regulates various molecular mechanisms of cancer progression associated with CSCs. This study aimed to identify the candidate miRNA and to characterise the clinical significance. METHODS: We established gemcitabine-resistant Panc1 cells, and induced CSC-like properties through sphere formation. Candidate miRNAs were selected through microarray analysis. The overexpression and knockdown experiments were performed by evaluating the in vitro cell growth and in vivo tumourigenicity. The expression was studied in 24 pancreatic cancer samples after laser captured microdissection and by immunohistochemical staining. RESULTS: The in vitro drug sensitivity of pancreatic cancer cells was altered according to the miR-1246 expression via CCNG2. In vivo, we found that miR-1246 could increase tumour-initiating potential and induced drug resistance. A high expression level of miR-1246 was correlated with a worse prognosis and CCNG2 expression was significantly lower in those patients. CONCLUSIONS: miR-1246 expression was associated with chemoresistance and CSC-like properties via CCNG2, and could predict worse prognosis in pancreatic cancer patients.
Assuntos
Ciclina G2/fisiologia , Desoxicitidina/análogos & derivados , MicroRNAs/metabolismo , Células-Tronco Neoplásicas/patologia , Neoplasias Pancreáticas/tratamento farmacológico , Animais , Antimetabólitos Antineoplásicos , Linhagem Celular Tumoral , Desoxicitidina/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Camundongos , Neoplasias Pancreáticas/patologia , GencitabinaRESUMO
A 55-year-old man underwent rectal amputation for rectal cancer in August 2005. A tiny thin-walled cavity lesion in his left S1+2 was found on computed tomography (CT) of the chest in November 2008. The cavity lesion in the left S1+2 gradually increased in size over 3 months and positron emission tomography (PET) with 18F-fluorodeoxyglucose (FDG) showed FDG accumulation at the lesion. Videoassisted thoracoscopic (VATS) wedge resection was performed to make a definite diagnosis in March 2009. The pathological findings revealed a metastatic lung tumor from the rectal cancer. It is necessary to consider the possibility of metastatic lung tumors in a case with the cavity lesions especially in patients with a history of colon cancer.
Assuntos
Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Neoplasias Retais/patologia , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
A 74-year-old man was admitted to our hospital in order to treat a mediastinal mass and 2 ground-glass attenuations in the right upper lobe detected by chest X-ray and computed tomography (CT). Partial resection of right lung and thymectomy were performed. The mediastinal mass and 2 ground-glass attenuations in the right upper lobe proved to be thymoma and bronchioloalveolar carcinomas, respectively by pathology.
Assuntos
Adenocarcinoma Bronquioloalveolar/complicações , Neoplasias Pulmonares/complicações , Neoplasias Primárias Múltiplas , Timoma/complicações , Neoplasias do Timo/complicações , Idoso , Humanos , MasculinoRESUMO
F10 and BL6 sublines of B16 mouse melanoma cells are metastatic after intravenous injection, but only BL6 cells are metastatic after subcutaneous injection. We found that connexin (Cx) 26 is upregulated in BL6 cells. To examine gap junction formation, we devised a coculture system, in which an opened vein segment was placed at the bottom of a culture dish and then dye-labeled melanoma cells were seeded onto it. Immunohistochemistry indicated that the vein segment preserved the integrity of the endothelial monolayer. In this system, BL6 cells could transfer dye into endothelial cells but F10 cells could not. Transfection with wild-type Cx26 rendered F10 cells competent for coupling with endothelial cells and as spontaneously metastatic as BL6 cells. Conversely, transfection with a dominant-negative form of Cx26 rendered BL6 cells deficient in coupling and less metastatic. In human melanoma lesions, the level of Cx26 expression was low in melanoma cells residing in the basal layer, but significantly upregulated in melanoma cells invading the dermis. The results suggested that Cx26 plays a role in intravasation and extravasation of tumor cells through heterologous gap junction formation with endothelial cells.
Assuntos
Conexinas/metabolismo , Endotélio Vascular/metabolismo , Junções Comunicantes/metabolismo , Melanoma Experimental/metabolismo , Melanoma Experimental/secundário , Animais , Técnicas de Cocultura , Conexina 26 , Conexinas/genética , Endotélio Vascular/patologia , Fluoresceínas/metabolismo , Corantes Fluorescentes/metabolismo , Humanos , Injeções Intravenosas , Injeções Subcutâneas , Melanoma Experimental/patologia , Camundongos , Metástase Neoplásica , Técnicas de Cultura de Órgãos , Veia Cava Inferior/metabolismo , Veia Cava Inferior/patologiaRESUMO
We have observed three calmodulin mRNA species in rat tissues. In order to know from how many expressed genes they are derived, we have investigated the genomic organization of calmodulin genes in the rat genome. From a rat brain cDNA library, we obtained two kinds of cDNAs (pRCM1 and pRCM3) encoding authentic calmodulin. DNA sequence analysis of these cDNA clones revealed substitutions of nucleotides at 73 positions of 450 nucleotides in the coding region, although the amino acid sequences of these calmodulins are exactly the same. DNA sequences in the 5' and 3' noncoding regions are quite different between these two cDNAs. From these results, we conclude that they are derived from two distinct bona fide calmodulin genes, CaMI (pRCM1) and CaMII (pRCM3). Total genomic Southern hybridization suggested four distinct calmodulin-related genes in the rat genome. By cloning and sequencing the calmodulin-related genes from rat genomic libraries, we demonstrated that the other two genes are processed pseudogenes generated from the CaMI (lambda SC9) and CaMII (lambda SC8) genes, respectively, through an mRNA-mediated process of insertions. Northern blotting showed that the CaMI gene is transcribed in liver, muscle, and brain in similar amounts, whereas the CaMII gene is transcribed mainly in brain. S1 nuclease mapping indicated that the CaMI gene produced two mRNA species (1.7 and 4 kilobases), whereas the CaMII gene expressed a single mRNA species (1.4 kilobases).
Assuntos
Calmodulina/genética , Genes , RNA Mensageiro/genética , Animais , Sequência de Bases , Encéfalo/fisiologia , Mapeamento Cromossômico , DNA/genética , Enzimas de Restrição do DNA , Endonucleases , Regulação da Expressão Gênica , Fígado/fisiologia , Músculos/fisiologia , Ratos , Homologia de Sequência do Ácido Nucleico , Endonucleases Específicas para DNA e RNA de Cadeia Simples , Distribuição TecidualRESUMO
Our recent studies with cell mutants indicate that a cascade shared by the epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) signals exists in NRK cells and mediates oncogenic signals induced by many oncogenes (A. Masuda, S. Kizaka-Kondoh, H. Miwatani, Y. Terada, H. Nojima, and H. Okayama, New Biol. 4:489-503, 1992). We have employed the antisense RNA technique to investigate possible involvement of Raf-1 kinase in this signal transduction cascade. NRK cell clones highly reduced in the Raf-1 production are generated by the expression of a c-raf-1 antisense RNA. They have no apparent growth defects and retain proper mitotic responses to growth factors but are refractory to transformation by EGF or PDGF plus transforming growth factor beta, v-erbB, v-fms, v-K-ras, v-mos, v-fos, v-src, simian virus 40 large T, and polyomavirus middle T but not by v-raf or adenovirus E1A. These results not only support our model for the oncogenic signal cascade but also lead to the conclusion that Raf-1 protein kinase is a downstream component of this oncogenic signal cascade shared by EGF and PDGF.
Assuntos
Transformação Celular Neoplásica , Fator de Crescimento Epidérmico/metabolismo , Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Western Blotting , Divisão Celular , Linhagem Celular , Clonagem Molecular , Oncogenes , Proteínas Proto-Oncogênicas c-raf , Fator de Crescimento Transformador beta/fisiologiaRESUMO
Multiple species of G1 cyclins and cyclin-dependent kinases are induced sequentially during G1 phase, and the expression of cyclin A and cdc2 genes is subsequently induced at the G1/S boundary. To analyze the mechanism of cdc2 promoter activation, the 5'-flanking region of the rat cdc2 gene was isolated and its structural features were characterized. The highly conserved sequence between human and rat cdc2 genes is present in the basal promoter region from positions -183 to -122, which contains the E box, SpI, and E2F motifs. The expression of 5' sequential deletion derivatives of the promoter fused to luciferase cDNA in rat 3Y1 cells revealed the presence of the enhancer element. The presumed enhancer region was further analyzed by the introduction of base substitutions and by the formation of DNA-protein complexes with cell extracts prepared at various times during the G1-to-S-phase progression. These analyses revealed that the enhancer sequence, AAGTTACAAATA, located from -276 to -265, confers strong inducibility on the basal promoter at the G1/S boundary. The base substitutions introduced into the motifs of transcription factors indicated that the E2F motif is essential for the enhancer-dependent activation of the cdc2 promoter at the G1/S boundary. Electrophoretic mobility shift assays and DNase I footprinting showed that a factor which interacts with the enhancer element is induced late in G1 phase.
Assuntos
Proteína Quinase CDC2/genética , Ciclo Celular/genética , Elementos Facilitadores Genéticos , Regiões Promotoras Genéticas , Animais , Sequência de Bases , Linhagem Celular , DNA/genética , Fase G1/genética , Regulação da Expressão Gênica , Genes , Dados de Sequência Molecular , Mutação , Ratos , Fase S/genéticaRESUMO
Although many reports have described laparoscopic minor liver resections, major hepatic resection, including right or left lobectomy, has not been widely developed because of technical difficulties. This article describes a new technique for performing laparoscopy-assisted right or left hepatic lobectomy using hilar Glissonean pedicle transection. Laparoscopic mobilization of the right or left hepatic lobe is performed, including dissection of the round, faliciform, triangular, and coronary ligaments. The right or left Glissonean pedicle is encircled and divided laparoscopically. A parenchymal dissection is then performed though the upper median or right subcostal incision, through which the resected liver is removed. We successfully performed this procedure in 6 patients without blood transfusion or serious complications. Laparoscopy-assisted hepatic lobectomy using hilar Glissonean pedicle transection can be feasible and safe in highly selected patients.
Assuntos
Hepatectomia/métodos , Laparoscopia/métodos , HumanosRESUMO
We report here the isolation and functional analysis of the rfc3(+) gene of Schizosaccharomyces pombe, which encodes the third subunit of replication factor C (RFC3). Because the rfc3(+) gene was essential for growth, we isolated temperature-sensitive mutants. One of the mutants, rfc3-1, showed aberrant mitosis with fragmented or unevenly separated chromosomes at the restrictive temperature. In this mutant protein, arginine 216 was replaced by tryptophan. Pulsed-field gel electrophoresis suggested that rfc3-1 cells had defects in DNA replication. rfc3-1 cells were sensitive to hydroxyurea, methanesulfonate (MMS), and gamma and UV irradiation even at the permissive temperature, and the viabilities after these treatments were decreased. Using cells synchronized in early G2 by centrifugal elutriation, we found that the replication checkpoint triggered by hydroxyurea and the DNA damage checkpoint caused by MMS and gamma irradiation were impaired in rfc3-1 cells. Association of Rfc3 and Rad17 in vivo and a significant reduction of the phosphorylated form of Chk1 in rfc3-1 cells after treatments with MMS and gamma or UV irradiation suggested that the checkpoint signal emitted by Rfc3 is linked to the downstream checkpoint machinery via Rad17 and Chk1. From these results, we conclude that rfc3(+) is required not only for DNA replication but also for replication and damage checkpoint controls, probably functioning as a checkpoint sensor.
Assuntos
Dano ao DNA/genética , Replicação do DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas Fúngicas/genética , Proteínas de Homeodomínio , Proteínas Proto-Oncogênicas c-bcl-2 , Proteínas Repressoras , Proteínas de Saccharomyces cerevisiae , Schizosaccharomyces/genética , Sequência de Bases , Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinase 1 do Ponto de Checagem , Clonagem Molecular , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas de Ligação a DNA/metabolismo , Eletroforese em Gel de Campo Pulsado , Epitopos , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/metabolismo , Raios gama , Hidroxiureia/toxicidade , Mesilatos/toxicidade , Antígenos de Histocompatibilidade Menor , Dados de Sequência Molecular , Mutação , Proteínas Nucleares , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteína de Replicação C , Schizosaccharomyces/efeitos dos fármacos , Schizosaccharomyces/metabolismo , Schizosaccharomyces/efeitos da radiação , Proteínas de Schizosaccharomyces pombe , Homologia de Sequência de Aminoácidos , Raios UltravioletaRESUMO
We report here a Schizosaccharomyces pombe gene (dmc1(+)) that resembles budding yeast DMC1 in the region immediately upstream of the rad24(+) gene. We showed by northern and Southern blot analysis that dmc1(+) and rad24(+) are co-transcribed as a bicistronic mRNA of 2.8 kb with meiotic specificity, whereas rad24(+) itself is constitutively transcribed as a 1.0-kb mRNA species during meiosis. Induction of the bicistronic transcript is under the control of a meiosis-specific transcription factor, Ste11. Disruption of both dmc1(+) and rad24(+) had no effect on mitosis or spore formation, and dmc1Delta cells displayed no change in sensitivity to UV or gamma irradiation relative to the wild type. Tetrad analysis indicated that Dmc1 is involved in meiotic recombination. Analysis of gene conversion frequencies using single and double mutants of dmc1 and rhp51 indicated that both Dmc1 and Rhp51 function in meiotic gene conversion. These observations, together with a high level of sequence identity, indicate that the dmc1(+) gene of S. POMBE: is a structural homolog of budding yeast DMC1, sharing both similar and distinct functions in meiosis.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Meiose/genética , Recombinases/fisiologia , Recombinação Genética , Proteínas de Schizosaccharomyces pombe/fisiologia , Schizosaccharomyces/genética , Proteínas de Ciclo Celular/genética , Divisão Celular , Troca Genética/genética , DNA Complementar/genética , Proteínas de Ligação a DNA/genética , Proteínas Fúngicas/fisiologia , Conversão Gênica/genética , Regulação Fúngica da Expressão Gênica , Genes Fúngicos/genética , Peptídeos e Proteínas de Sinalização Intracelular , Dados de Sequência Molecular , Mutação , Fases de Leitura Aberta/genética , RNA Fúngico/genética , RNA Fúngico/isolamento & purificação , Recombinases/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae , Proteínas de Schizosaccharomyces pombe/genética , Fatores de Transcrição/fisiologia , Transcrição GênicaRESUMO
In order to isolate meiosis-specific genes in Schizosaccharomyces pombe, we have constructed a subtracted cDNA library enriched in clones whose expression is enhanced during meiosis induced by nitrogen starvation. Using northern blot analysis, we isolated 31 kinds of clones whose expression was induced in a meiosis/sporulation-specific manner. We comprehensively named them meu after meiotic expression upregulated. The transcription of 20 meu genes was found to be dependent on the mei4(+) gene, which encodes a transcription factor required for the progression of meiosis. DNA sequencing indicated that most of the meu genes encode novel proteins. Notably, five of the meu genes harbor no apparent protein coding sequences, and the transcripts form stable hairpin structures, suggesting that they may generate non-coding RNAs or antisense RNAS: The results presented here imply that RNAs are also important for the comprehensive characterization of genomic expression.
Assuntos
Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Meiose/genética , Schizosaccharomyces/genética , Sequência de Bases , DNA Complementar/genética , DNA Complementar/isolamento & purificação , Biblioteca Gênica , Genes Fúngicos/genética , Dados de Sequência Molecular , RNA Fúngico/genética , Schizosaccharomyces/fisiologia , Homologia de Sequência do Ácido Nucleico , Esporos Fúngicos/genética , Transcrição GênicaRESUMO
Cyclin G1 is one of the target genes of the transcription factor p53, and is induced in a p53-dependent manner in response to DNA damage. Although cyclin G1 has been implicated in a range of biological phenomena, its precise function remains unclear. Here we present an analysis of the physiological role of cyclin G1 using mice homozygous for a targeted disruption of the cyclin G1 gene. In order to clarify the role of cyclin G1 in the p53 pathway, downstream events such as apoptosis, cell growth and cell cycle checkpoint control were analysed in thymocytes and embryonic fibroblasts derived from cyclin G1-disrupted mice. No difference was detected in induction of apoptosis between mouse embryo fibroblasts (MEFs) derived from cyclin G1+/+ and cyclin G1-/- mice. Following irradiation, cyclin G1-/- MEFs proliferated more slowly and reached lower cell densities in culture dishes than cyclin G1+/+ MEFs. Analysis of cell survival showed that cyclin G1-/- MEFs were about twice as sensitive as cyclin G1+/+ MEFs to gamma radiation or UV radiation. Cyclin G1-/- mice were more sensitive to gamma radiation than wild-type mice. Flow cytometeric analysis revealed that the number of cyclin G1-/- MEFs in G2/M phase after irradiation was reduced by 50% relative to cyclin G1+/+ MEFs. Our results demonstrate that cyclin G1 plays roles in G2/M arrest, damage recovery and growth promotion after cellular stress.
Assuntos
Ciclinas/metabolismo , Dano ao DNA , Fase G2 , Mitose , Tolerância a Radiação , Animais , Ciclina G , Ciclina G1 , Ciclinas/genética , Relação Dose-Resposta à Radiação , Raios gama , Camundongos , Camundongos Mutantes , Mutagênese Insercional , Proteína Supressora de Tumor p53/metabolismo , Raios Ultravioleta , Irradiação Corporal TotalRESUMO
Initiation of DNA replication requires the function of MCM gene products, which participate in ensuring that DNA replication occurs only once in the cell cycle. Expression of all mammalian genes of the MCM family is induced by growth stimulation, unlike yeast, and the mRNA levels peak at G1/S boundary. In this study, we examined the transcriptional activities of isolated human MCM gene promoters. Human MCM5 and MCM6 promoters with mutation in the E2F sites failed in promoter regulation following serum stimulation and exogenous E2F expression. In addition, we identified a novel E2F-like sequence in human MCM6 promoter which cooperates with the authentic E2F sites in E2F-dependent regulation. Forced expression of E2F1 could induce expression of all members of the endogenous MCM genes in rat embryonal fibroblast REF52 cells. Our results demonstrated that the growth-regulated expression of mammalian MCM5 and MCM6 genes, and presumably other MCM members, is primarily regulated by E2F through binding to multiple E2F sites in the promoters.
Assuntos
Proteínas de Transporte , Proteínas de Ciclo Celular/biossíntese , Ciclo Celular/fisiologia , Proteínas de Ligação a DNA , Proteínas Fúngicas/biossíntese , Regulação da Expressão Gênica/fisiologia , Fatores de Transcrição/fisiologia , Animais , Sequência de Bases , Sítios de Ligação , Proteínas de Ciclo Celular/genética , Linhagem Celular , Meios de Cultura/farmacologia , Replicação do DNA/fisiologia , Fatores de Transcrição E2F , Fator de Transcrição E2F1 , Sangue Fetal/fisiologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Proteínas Fúngicas/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Componente 6 do Complexo de Manutenção de Minicromossomo , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Ratos , Proteína 1 de Ligação ao Retinoblastoma , Proteínas de Schizosaccharomyces pombe , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Teratocarcinoma/patologia , Fator de Transcrição DP1 , Transcrição Gênica , Transfecção , Células Tumorais CultivadasRESUMO
A new gene encoding a cyclin-like protein has been isolated from a rat fibroblast cDNA library by cross-hybridization with a mixture of c-src family proto-oncogene kinase domains as a probe. This putative cyclin, called cyclin G, contains a typical cyclin box at the N-terminus but no apparent 'destruction box' or 'PEST' sequence. Interestingly, in its C-terminus region, it has a sequence homologous with a tyrosine phosphorylation site of the epidermal growth factor receptor. Although this cyclin is phylogenetically related to HCS26 of Saccharomyces cerevisiae, it most resembles Cig1, a B-type cyclin, of Schizosaccharomyces pombe, which has been suggested to act at the G1/S phase of the cell cycle. Cyclin G mRNA is induced within 3 h after growth stimulation and remains elevated with no apparent cell cycle dependency, indicating its close association with growth stimuli but not with the cell cycle.
Assuntos
Ciclinas/química , Proteínas Fúngicas/química , Schizosaccharomyces/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Células Cultivadas , Ciclina G , Ciclina G1 , Ciclinas/genética , DNA/isolamento & purificação , Humanos , Dados de Sequência Molecular , Filogenia , Proto-Oncogene Mas , RNA Mensageiro/análise , Ratos , Homologia de Sequência de AminoácidosRESUMO
The human embryonal carcinoma cells NEC14 can be induced to differentiate morphologically by the addition of 10(-2) M N, N'-hexamethylene-bis-acetamide and cease to grow in several days. Transcription factors of the E2F/DP family have been shown to be closely related to the regulation of cell proliferation. To analyse cellular proteins which interact with E2F in NEC14 cells, cDNA clones encoding E2F binding proteins were isolated from a lambdaZAP II NEC14 cell library with the 32P-labeled GST (Glutathione S-transferase)-E2F-1 fusion protein as a probe. One of the clones encodes E2FBP1 which has the helix-loop-helix (HLH) motif, but lacks the basic domain and the zipper structure usually found at N- and C-terminal sides to the HLH motif, respectively. The arrangement of amino acids in the helix 1 and helix 2 regions is quite similar to those of Mxi and Mad, but different from those of E2F-1 and DP-1. Western blot analysis of the immunoprecipitates prepared with anti-E2FBP1 antibody showed that E2FBP1 associates with both E2F-1 and DP-1 in vivo. E2FBP1 alone has no DNA binding activity, but bind to the E2F site through heterodimerization with E2F-1 but not with DP-1. Although E2FBP1 lacks the transactivation domain, it stimulates E2F site-dependent transcription in cooperation with E2F-1.
Assuntos
Proteínas de Transporte , Proteínas de Ciclo Celular , Proteínas de Ligação a DNA/metabolismo , Oncogenes , Transativadores , Fatores de Transcrição/metabolismo , Transcrição Gênica , Acetamidas/farmacologia , Sequência de Aminoácidos , Sequência de Bases , Carcinoma Embrionário , Diferenciação Celular/efeitos dos fármacos , Clonagem de Organismos , DNA Complementar , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Dimerização , Fatores de Transcrição E2F , Fator de Transcrição E2F1 , Glutationa Transferase/genética , Sequências Hélice-Alça-Hélice , Humanos , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/metabolismo , Proteína 1 de Ligação ao Retinoblastoma , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Fator de Transcrição DP1 , Fatores de Transcrição/química , Fatores de Transcrição/genética , Células Tumorais CultivadasRESUMO
F1-type ATPase is the central enzyme for ATP synthesis in most organisms. Because of the extreme reconstitutability of thermophilic ATPase (TF1) and diversity of the minor subunits of F1 type ATPase, an operon coding for TF1 was isolated from DNA of thermophilic bacterium PS3, and its terminal region containing the epsilon subunit (TF1 epsilon) and terminator was sequenced. The primary structure of the epsilon subunit (Mr = 14 333) was deduced from the nucleotide sequence (396 base-pairs) and amino-acid sequence of its amino terminus. The conclusions drawn from the results are as follows. Homologies: TF1 epsilon shows only 6% homology with the epsilon subunits of eight species reported, but 50% homology with Escherichia coli epsilon and 41% with chloroplast. The residues having a tendency to form reverse turns (Gly, Pro and Tyr) and His are relatively well conserved. Unlike some F1 epsilon types TF1 epsilon has no ATPase inhibitor activity and is not homologous with ATPase inhibitor. TF1 epsilon is essential to connect F1 to F0, like the b subunit, and is weakly homologous with the b subunit of F0F1. The cause of 3 beta: 1 epsilon subunit stoichiometry: The ribosome binding sequence of TF1 epsilon is TAGGN7, which is incomplete compared with that of TF1 beta. The codon usage for TF1 epsilon is similar to that for TF1 epsilon. The cause of stability of TF1 epsilon and its gene: There are 18 ionic groups at the putative reverse turns and the N- and C-termini of TF1 epsilon, but only 10 ionic groups in the corresponding sites of E. coli epsilon subunit. These ionic groups enhance the external polarity of TF1 epsilon and may intensify subunit-subunit interaction. There is a terminator at the 3' end of the TF1 epsilon gene, which is stabilized by a long (13 base-pairs) stem.
Assuntos
Bactérias/enzimologia , Proteínas de Bactérias/genética , Genes Bacterianos , ATPases Translocadoras de Prótons/genética , Sítios de Ligação , Cloroplastos , Clonagem Molecular , Códon , Escherichia coli/genética , Genes , Conformação de Ácido Nucleico , Conformação Proteica , Homologia de Sequência do Ácido Nucleico , Regiões Terminadoras GenéticasRESUMO
The 3-phosphoglycerate kinase (EC 2.7.2.3) of yeast which contains two tryptophyl and eight tyrosyl residues per molecule, displayed an unusualy fluorescence emission spectrum with a maximum at 308 nm when excited at 280 nm. The emission peak shifted to 329 nm when excited at 295 nm. We could confirm that it was due to the efficient quenching of tryptophyl fluorescence as well as to the incomplete energy transfer from tyrosyl to tryptophyl residues. The average fluorescence quantum yield of this protein was 0.076 (excitation at 280 nm) and that of tryptophyl residues was 0.046 (excitation at 295 nm). As the pH of the solution was lowered, the fluorescence intensity of phosphoglycerate kinase at 329 nm dramatically increased between pH 5 and 4, while the position of the peak remained unchanged. When denatured in 4 M guanidine hydrochloride, the protein showed two emission peaks, one at 343 nm and the other at 303 nm.
Assuntos
Fosfoglicerato Quinase , Saccharomyces cerevisiae/enzimologia , Sítios de Ligação , Concentração de Íons de Hidrogênio , Iodetos , Cinética , Ligação Proteica , Teoria Quântica , Espectrometria de FluorescênciaRESUMO
Elsewhere, we have reported the structure of a rat calmodulin gene and two distinct rat calmodulin cDNAs, pRCM1 and pRCM3. Here, I report the cloning and sequencing of the third calmodulin cDNA (pRCM4) and two additional rat calmodulin genes. The original calmodulin gene is named CaM I (pRCM1) and the newly discovered calmodulin genes are named CaM II (pRCM3) and CaM III (pRCM4). CaM II spans about 10 x 10(3) base-pairs and consisted of five exons, while CaM III spans about 7.2 x 10(3) base-pairs and consisted of six exons. One of the introns (intron 3) observed in CaM I and CaM III is lost in CaM II. Otherwise, the intron/exon organization of these genes is exactly the same. In all calmodulin genes, the first intron separates the initiation codon (ATG) from the coding region of the protein. Northern blotting showed that CaM I is transcribed primarily into 1.7 x 10(3) base-pair mRNA in various tissues examined and 4.0 x 10(3) base-pair mRNA mainly in skeletal muscle, CaM II is transcribed into 1.4 x 10(3) base-pair mRNA almost exclusively in brain and CaM III is transcribed predominantly into 2.3 x 10(3) base-pair mRNA and faintly into 1.0 x 10(3) base-pair mRNA mainly in skeletal muscle and brain. DNA sequences in the promoter-regulator regions of these genes are partly homologous but essentially distinct and possess a number of direct repeats, palindromes and feasible stem-loop structures. Together with these, I report here the structures of the third and fourth calmodulin retropseudogenes.
Assuntos
Calmodulina/genética , Genes , Animais , Bacteriófago lambda , Sequência de Bases , DNA/genética , Éxons , Immunoblotting , Íntrons , Dados de Sequência Molecular , Plasmídeos , Pseudogenes , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Mapeamento por RestriçãoRESUMO
The structural organization of the entire rat calmodulin gene was determined by cloning and sequencing overlapping genomic and cDNA clones from rat genomic and brain cDNA libraries. The intron/exon organization was determined by direct comparison of these sequences. Rat calmodulin gene is 9000 bases long and consisted of six exons interrupted by introns of variable sizes. The first intron separates the initiation codon (ATG) from the coding region of the protein. Three out of four intron/exon junctions in the coding region reside in the middle of calcium binding subdomains and do not correlate with the quarterly divided intramolecular homology of the protein. Their positions exactly coincide with those of the corrected version of chicken calmodulin gene. The rat calmodulin gene harbors a stretch of sequences homologous to a rat middle repetitive "identifier sequence" in the middle of the third intron. Analysis of the immediate 5' upstream region detected a TATA box (TATATATAT) and three C-G boxes (CCGCCC) but not a CAT box (CCAAT). A conserved sequence (GCGCCGCGYCYYGGGGGC) was found at -125 for rat and at -204 for chicken calmodulin genes.
Assuntos
Calmodulina/genética , Genes , Ratos/genética , Animais , Sequência de Bases , Galinhas , Mapeamento Cromossômico , DNA , Endonucleases , Éxons , Íntrons , Endonucleases Específicas para DNA e RNA de Cadeia Simples , Transcrição GênicaRESUMO
Two distinct processed calmodulin genes of rat (lambda SC8 and lambda SC9) were identified, cloned and their DNA sequences determined. The existence of direct repeats of 19 base-pairs for lambda SC8 or 9 base-pairs for lambda SC9 at both ends of the coding plus non-coding regions suggested a possible involvement of a mRNA-mediated process of insertion. Total genomic Southern hybridization suggested the existence of at least three different calmodulin-related genes in the rat genome. The other gene was the bona fide calmodulin gene (lambda SC4) which was split into at least five exons. lambda SC9 contained insertions of one nucleotide and two 17 base-pair direct repeats in the coding region. These insertions cause frameshift mutations probably preventing it from encoding a functional calmodulin. It also carried an insertion of a rat middle repetitive sequence, identifier sequence (IDS: Sutcliffe et al., 1982) in the 3'-non-coding region. Otherwise, it consisted of an almost identical DNA sequence to that of the bona fide calmodulin gene (lambda SC4), including the 3'-non-coding region down to the poly(A) recognition signal, A-A-T-A-A-A. On the other hand, lambda SC8 did not possess frameshift mutations in the coding region, and hence was capable of encoding a functional protein. In fact, a probe specific to the lambda SC8 sequence identified a band in Northern blotting whose size was 300 nucleotides smaller than that of authentic calmodulin mRNA. Comparison of the nucleotide sequences showed that only the coding regions of these two processed genes were homologous, indicating that the divergence of these two processed genes from the common ancestor calmodulin was an ancient event.