RESUMO
The far-upstream element-binding protein (FBP) is one of several recently described factors which bind to a single strand of DNA in the 5' region of the c-myc gene. Although cotransfection of FBP increases expression from a far-upstream element-bearing c-myc promoter reporter, the mechanism of this stimulation is heretofore unknown. Can a single-strand-binding protein function as a classical transactivator, or are these proteins restricted to stabilizing or altering the conformation of DNA in an architectural role? Using chimeric GAL4-FBP fusion proteins we have shown that the carboxyl-terminal region (residues 448 to 644) is a potent transcriptional activation domain. This region contains three copies of a unique amino acid sequence motif containing tyrosine diads. Analysis of deletion mutants demonstrated that a single tyrosine motif alone (residues 609 to 644) was capable of activating transcription. The activation property of the C-terminal domain is repressed by the N-terminal 107 amino acids of FBP. These results show that FBP contains a transactivation domain which can function alone, suggesting that FBP contributes directly to c-myc transcription while bound to a single-strand site. Furthermore, activation is mediated by a new motif which can be negatively regulated by a repression domain of FBP.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Saccharomyces cerevisiae , Fatores de Transcrição , Ativação Transcricional , Tirosina , Sequência de Aminoácidos , Animais , Sítios de Ligação , Linhagem Celular , Cloranfenicol O-Acetiltransferase/biossíntese , Chlorocebus aethiops , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/química , Proteínas Fúngicas/biossíntese , Células HeLa , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Plasmídeos , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/biossíntese , TATA Box , TransfecçãoRESUMO
5-fluorouracil (5-FU) is a basic agent used in chemotherapy. The aim of this study is to investigate the gene expression of 5-FU anabolic and catabolic enzymes in hepatocellular carcinoma (HCC) and non-tumor tissue, respectively to increase our knowledge of resistant mechanisms to 5-FU in HCC. The relative mRNA level of orotate phosphoribosyltransferase (OPRT), ribonucleotide reductase (RNR), dihydropyrimidine dehydrogenase (DPD) and target enzyme thymidylate synthase (TS), were analyzed in 30 matched samples of HCC (T) and non-tumor tissue (NT) using quantitative RT-PCR. The expression of OPRT, RNR-M1, RNR-M2 and TS is significantly higher in T compared with in NT (1.3-fold increase, 1.6-fold, 7.1-fold, 1.9-fold, respectively), but that of DPD showed no difference between T and NT. Our results show that HCC should not be treated with 5-FU alone because of its instability in liver.
Assuntos
Carcinoma Hepatocelular/enzimologia , Fluoruracila/metabolismo , Neoplasias Hepáticas/enzimologia , Fígado/enzimologia , Di-Hidrouracila Desidrogenase (NADP)/metabolismo , Feminino , Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Orotato Fosforribosiltransferase/metabolismo , Ribonucleotídeo Redutases/metabolismo , Timidilato Sintase/metabolismoRESUMO
Most human cancers show chromosomal instability (CIN), but the precise mechanisms remain uncertain. Annexin A2 is frequently overexpressed in human cancers, and its relationship to tumorigenesis is poorly understood. We found that annexin A2 is overexpressed in the nuclei of CIN cells compared with cells with microsatellite instability (MIN). Ectopic annexin A2 expression in MIN cells results in a high level of aneuploidy and induces lagging chromosomes; suppression of annexin A2 in CIN cells reduces such CIN signatures with apoptosis of highly aneuploid cells. Ectopic expression of annexin A2 in MIN cells reduces the expression of centromere proteins. Conversely, annexin A2-knockdown in CIN cells increases the expression of centromere proteins. Moreover, the endogenous expression levels of centromere proteins in CIN cells were greatly reduced compared with MIN cell lines. The reduced expression of centromere proteins likely occurred due to aberrant centromere localization of coilin, a major component of the Cajal bodies. These results suggest that nuclear accumulation of annexin A2 has a crucial role in CIN by disrupting centromere function.
Assuntos
Anexina A2/genética , Centrômero/genética , Instabilidade Cromossômica , Proteínas Nucleares/genética , Aneuploidia , Anexina A2/metabolismo , Apoptose/genética , Autoantígenos/genética , Autoantígenos/metabolismo , Western Blotting , Células CACO-2 , Linhagem Celular Tumoral , Núcleo Celular/genética , Núcleo Celular/metabolismo , Centrômero/metabolismo , Proteína Centromérica A , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Eletroforese em Gel Bidimensional , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Células HT29 , Humanos , Instabilidade de Microssatélites , Proteínas Nucleares/metabolismo , Proteoma/genética , Proteoma/metabolismo , Proteômica/métodos , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: Although morphologic, radiographic, and manometric features of achalasia have been well defined, it has not been established by careful retrospective analysis whether achalasia is a progressive disorder resulting in complete decompensation. STUDY DESIGN: To verify the hypothesis that achalasia is a progressive disease, we retrospectively investigated manometric, radiographic, and symptomatic data in patients with achalasia. Sixty-three patients (36 women and 27 men) with a median age of 44 years (range 11 to 79 years) were evaluated. The duration of symptoms ranged from 1 to 442 months, with a median of 48 months. Patients were divided into four groups according to the duration of symptoms: 36 patients with less than 5 years, 11 with 5 to 10 years, 9 with 10 to 15 years, and 7 with 15 years or more. RESULTS: Contraction pressures of the esophageal body decreased significantly at every level when the duration of symptoms increased (p < 0.04). The percentage of simultaneous waves in the esophageal body rose as the duration of symptoms increased. All waves were synchronous in every patient who had had symptoms for more than 15 years. The maximal width of the esophageal body measured on esophagram became greater with an increase in the duration of symptoms, but this measurement did not reach statistical significance (p = 0.063). The tortuosity of the esophagus, measured by the maximal angle of the esophageal axis, was significantly greater in patients with a longer duration of symptoms (p < 0.02). The type of symptoms was not associated with the duration of symptoms. CONCLUSIONS: Achalasia is a progressive disease, as verified by manometric and radiographic findings. The classification of esophageal motor function expressed by amplitude of contraction pressure and angle of tortuosity is objective and useful. Classification of achalasia by duration of symptoms may be important in treatment selection and effectiveness.
Assuntos
Acalasia Esofágica/diagnóstico , Manometria , Radiografia , Adolescente , Adulto , Idoso , Sulfato de Bário , Criança , Meios de Contraste , Transtornos de Deglutição/etiologia , Acalasia Esofágica/fisiopatologia , Acalasia Esofágica/cirurgia , Esôfago/fisiopatologia , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Resultado do TratamentoRESUMO
Technical controversies abound regarding the surgical treatment of achalasia. To determine the value of a concomitant antireflux procedure, the best antireflux procedure, the correct length for gastric myotomy, the optimal surgical approach (thoracic or abdominal), and the equivalency of minimally invasive surgery, a literature review was carried out. The review is based on 23 articles on open transabdominal or transthoracic myotomy, 14 articles on laparoscopic myotomy, and four articles on thoracoscopic myotomy. Postoperative results of traditional open thoracic or transabdominal myotomy as determined by symptomatology were better with fundoplication than without fundoplication. The incidence of postoperative reflux as proved by pH monitoring was high in patients who had an open transabdominal myotomy without fundoplication. The type of antireflux procedure used and the length of gastric myotomy had little effect on results. The results of transthoracic Heller myotomy do not require a concomitant fundoplication. Laparoscopic and thoracoscopic myotomy had excellent results at short-term follow-up. A fundoplication must be added if the myotomy is performed transabdominally. A randomized prospective study is required to determine the best fundoplication and the extent of gastric myotomy. Although minimally invasive surgery for achalasia has excellent initial results, longer follow-up in a larger population of patients is needed.
Assuntos
Acalasia Esofágica/cirurgia , Procedimentos Cirúrgicos do Sistema Digestório/métodos , Humanos , Procedimentos Cirúrgicos Minimamente Invasivos/métodosRESUMO
The purpose of this study was to measure the length of the esophagus and assess its relationship to sex, weight, age, height, and various esophageal disorders. A retrospective analysis was undertaken of 617 esophageal manometric studies, which included 51 normal control subjects (27 males and 24 females) and 566 patients (297 males and 269 females) with esophageal disorders (50 with achalasia, 6 with diffuse esophageal spasm, 64 with strictures, 38 with nutcracker esophagus, 398 with gastroesophageal reflux disease [GERD] with positive 24-hour pH monitoring, and 66 with possible GERD but negative 24-hour pH monitoring). Manometry was performed in all of them by the station pull-through technique. The length of the esophagus was defined as the distance between the proximal end of the upper esophageal sphincter and the distal end of the lower esophageal sphincter. In the control group the mean (+/- standard deviation) length of the esophagus was 28.3 +/- 2.41 cm. In patients with esophageal disorders the mean length of the esophagus was 28.0 +/- 2.87 cm. Length of the esophagus is related to height but not to weight, sex, age, diffuse esophageal spasm, or nutcracker esophagus. Achalasia is associated with a longer esophagus, and GERD is associated with a shorter esophagus. Stricture is associated with a shorter esophagus, but this is in part due to the association between stricture and GERD. Patients with possible GERD but negative 24-hour pH monitoring have an esophageal length similar to that of GERD patients with positive 24-hour pH monitoring. Patients with GERD and stricture formation showed esophageal shortening in shorter patients. Achalasia, GERD, and GERD with stricture formation influence esophageal length. GERD-related strictures shorten the esophagus more significantly in short patients.
Assuntos
Doenças do Esôfago/fisiopatologia , Esôfago/patologia , Adulto , Feminino , Humanos , Masculino , Manometria , Pessoa de Meia-Idade , Estudos RetrospectivosRESUMO
BACKGROUND: Laparoscopic vagotomy represents a new and less invasive treatment for peptic ulcer disease, but the problem of postvagotomy dysphagia has not been solved. The aim of this study was to determine the etiologic factors related to long-term laparoscopic postvagotomy dysphagia. METHODS: Two female and 11 male patients with a mean age of 48.5 years who underwent laparoscopic vagotomy were investigated retrospectively. Preoperative diagnosis included duodenal ulcer resistant to medical treatment, gastric hypersecretion, gastric outlet obstruction, cholelithiasis, and gastroesophageal reflux disease (GERD). Ten patients underwent laparoscopic highly selective vagotomy, and three patients had laparoscopic truncal vagotomy with gastrojejunostomy or pyloroplasty. Nine of these patients had a Nissen fundoplication in conjunction with the vagotomy. RESULTS: The median long-term follow-up period was 47 months. Two patients complained of severe dysphagia, one of moderate dysphagia, and two of mild dysphagia. Neither type of vagotomy nor an additional fundoplication was correlated with the severity of postoperative long-term dysphagia. Severity of postoperative dysphagia was associated with severity of preoperative dysphagia (r = 0.752, p = 0.003) but not with heartburn (r = 0.358, p = 0.531) or regurgitation (r = 0.024, p = 0.938). The cause of preoperative dysphagia varied; however, all of these patients had GERD and consequent esophageal lesions. CONCLUSION: Preexisting dysphagia appears to play an integral role in persistent postoperative dysphagia. Care must be taken to construct a loose fundoplication in patients with dysphagia.
Assuntos
Transtornos de Deglutição/etiologia , Laparoscopia/efeitos adversos , Vagotomia/efeitos adversos , Colelitíase/cirurgia , Transtornos de Deglutição/diagnóstico , Diagnóstico Diferencial , Úlcera Duodenal/cirurgia , Feminino , Obstrução da Saída Gástrica/cirurgia , Refluxo Gastroesofágico/cirurgia , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Índice de Gravidade de Doença , Inquéritos e QuestionáriosRESUMO
STUDY DESIGN: Charts and radiographs of all patients treated with this halovest at one university hospital were reviewed retrospectively. OBJECTIVES: To describe the outcomes from an adjustable four-pad halovest and to compare them with those from standard halovests, as previously published. SUMMARY OF BACKGROUND DATA: With standard halovests, there can be cervical motion up to 70% of normal values, substantial loads between the halo and vest, and complications of pin loosening, pin infections, and scapular pressure sores. The four-pad vest reduces halovest loads and vest-torso motions. METHODS: The four-pad vest has four independently adjustable pads that completely avoid contact with the scapula, clavicle, and abdomen. Clinical records were analyzed to determine the incidences of halo pin loosening, pressure sores, injury or surgical site nonunion, and loss of cervical alignment. Lateral radiographs were taken with the patient in the upright and supine positions at various times to determine intervertebral rotations (flexion-extension). RESULTS: The clinical results with the four-pad vest were at least as good as those for standard vests. Scapular pressure sores were prevented completely by the absence of vest-scapula contact. Kyphosis did not increase significantly with time. The mean segmental rotations were all 3 degrees or less and showed a smoothly decreasing pattern from C1-C2 to C6-C7. The value at Oc-C1 was opposite to that at C1-C2 and is the subject of further analysis. CONCLUSIONS: The rotations occurring with the four-pad vest are less than or equal to those occurring with standard vests, for overall cervical rotation and for individual motion segment rotations. This is consistent with the smaller halovest forces seen with this vest. Prospective, comparative testing will assess the clinical significance of these findings.
Assuntos
Braquetes , Vértebras Cervicais/lesões , Fraturas da Coluna Vertebral/terapia , Adulto , Vértebras Cervicais/diagnóstico por imagem , Vértebras Cervicais/cirurgia , Desenho de Equipamento , Feminino , Humanos , Masculino , Radiografia , Amplitude de Movimento Articular , Estudos Retrospectivos , Fraturas da Coluna Vertebral/cirurgiaRESUMO
The screening for yeasts incorporating exogenous eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) into their cellular lipids was conducted. Two percent of oil from fish scraps was added to a yeast isolation medium as a sole carbon source. From 143 soil samples, we isolated 23 yeast strains, nine of them were found to be capable of quickly assimilating the scrap fish oil. These nine strains, in addition to four previously isolated triglyceride-assimilating and lipase-producing strains, Yarrowia lipolytica and three Geotrichum species (FO274A, FO347-2 and FO401B), were cultured in a growth medium at 30 degrees C. Strains FO726A, FO765A and FO347-2 were selected on the basis of dry cell weight production and ability to store EPA and DHA in their cells, and their performance was further compared by varying cultivation temperature and time. From 1 g of the scrap fish oil, FO726A yielded 620 mg of dry cells, containing 47.1% lipid, 38.1% triglyceride, 3.3% EPA, and 4.9% DHA, when cultured at 25 degrees C for 36 h. Strain FO726A apparently has the highest ability to incorporate EPA and DHA into its cellular lipids. Results from further experiments showed that the incorporated EPA and DHA mainly existed in the form of triglyceride in the FO726A cell. These results suggest that FO726A is suitable for the production of cell mass rich in EPA and DHA for feed.
RESUMO
AIM: To determine the usefulness of endoscopically-delivered small intestinal submucosa (SIS) as a scaffold in enhancing the lower oesophageal sphincter (LOS) pressures. METHODS: Six dogs were endoscopically injected--four with the SIS and two with its glycerin carrier. Manometry was performed prior to injection and every four weeks post-op. RESULTS: Adequate and site correct injections were made in four dogs. In one dog, significant augmentation of pressures were obtained at four weeks. None had significant changes in pressure at eight weeks, differences in length at either four or eight weeks or significant differences in the thickness of the examined layers. Four of the six had capillary cushions on pathological examination. The dog injected with the carrier had a loose and disorganise collection, while the others were well organised. CONCLUSION: SIS is a biologically compatible material. Lack of an animal model for gastro-oesophageal reflux disease (GORD) makes determining the ability of injections of SIS to combat reflux problematic.
Assuntos
Junção Esofagogástrica/cirurgia , Refluxo Gastroesofágico/terapia , Mucosa Intestinal/transplante , Animais , Cães , Endoscopia , ManometriaRESUMO
To evaluate the scavenging effect of mannitol, vitamin E and betamethasone in cerebral ischemia, spin trapping technique was applied to the detection of the free radicals generated in ischemic brain homogenate. Thirty Wistar rats were used for this study. In the control group, the brain homogenate prepared immediately after decapitation was preserved at 37 degrees C under N2 gas. Before the preservation and at 30 min, 60 min and 120 min from the start of the preservation, two reaction mixtures containing of spin trapping reagent phenyl-t-butyl nitrone (PBN), NADPH, Fe-EDTA and brain homogenate was prepared from each brain sample--one to be incubated for 20 min at 37 degrees C in air and one to be incubated in nitrogen gas under similar condition. Then the free radical adducts of PBN were measured by electron spin resonance (ESR). In pre-treated group, mannitol, vitamin E and betamethasone were administered intravenously 30 min prior to the decapitation and spin adducts of PBN were detected by same procedures as in control group. The ESR spectra, which hyperfine coupling constants were AN = 16.0-16.6 G and AH beta = 3.0-3.8 G, were obtained from the reaction mixtures in each group. Analyses of their relative intensity in control group revealed that the formation of free radical adducts of PBN was increased dependent on the preservation period under aerobic incubation, and increased gradually for 60 min of preservation time followed by a decrease under anaerobic incubation.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Betametasona/análise , Química Encefálica , Infarto Cerebral/metabolismo , Manitol/análise , Vitamina E/análise , Animais , Betametasona/administração & dosagem , Quimioterapia Combinada , Espectroscopia de Ressonância de Spin Eletrônica , Manitol/administração & dosagem , Ratos , Ratos Endogâmicos , Vitamina E/administração & dosagemRESUMO
The majority of human cancer shows chromosomal instability (CIN). Although the precise mechanism remains largely uncertain, proper progression of mitosis is crucial. B-type lamins were suggested to be components of the spindle matrix of mitotic cells and to be involved in mitotic spindle assembly; thus, B-type lamins may contribute to the maintenance of chromosome integrity. Here, using a proteomic approach, we identified lamin B2 as a novel protein involved in CIN. Lamin B2 expression decreased in colorectal cancer cell lines exhibiting CIN, as compared with colorectal cancer cell lines exhibiting microsatellite instability (MIN), which is mutually exclusive to CIN. Importantly, lamin B2 knockdown in MIN-type colorectal cancer cells induced CIN phenotypes such as aneuploidy, chromosome mis-segregation and aberrant spindle assembly, whereas ectopic expression of lamin B2 in CIN-type colorectal cancer cells prevented their CIN phenotypes. Additionally, immunohistochemical analysis showed a lower expression of lamin B2 in cancer tissues extracted from patients with sporadic colorectal cancer (CIN-type) than that from patients with hereditary non-polyposis colorectal cancer (HNPCC; MIN type). Intriguingly, mitotic lamin B2 in MIN cancer cells was localized outside the spindle poles and mitotic lamin B2 localization was diminished in CIN cancer cells, suggesting an important role of lamin B2 in proper mitotic spindle formation. The obtained results suggest that lamin B2 maintains chromosome integrity by ensuring proper spindle assembly and that its downregulation causes CIN in colorectal cancer.
RESUMO
Pancreatic cancer still remains one of the most lethal diseases and establishment of new therapy is needed. The purpose of this study is to find novel factors involved in pancreatic cancer progression by proteomic approach. We compared pre- and postoperative serum protein profiling obtained from pancreatic cancer patients who had curative pancreatectomy using surface-enhanced laser desorption ionization time-of-flight mass spectrometry. The peak intensity levels of both 6630 and 6420 Da were significantly higher in the preoperative serum than in the postoperative serum (P<0.002). Sequential amino acid analysis identified these proteins to be apolipoprotein C-1 (ApoC-1). The high level of ApoC-1 in preoperative serum significantly correlated with poor prognosis. Furthermore, ApoC-1 was abundantly expressed in pancreas neoplastic epithelium, and was detected in the culture medium of the pancreatic cancer cell line in vitro, which suggests that cancer cells secrete ApoC-1. Inhibition of ApoC-1 expression by short interfering RNA suppressed cell proliferation and induced apoptosis of pancreatic cancer cells. The specific expression of ApoC-1 and its role in preventing from spontaneous apoptosis in pancreatic cancer cells suggest that ApoC-1 contributes to the aggressiveness of pancreatic cancer and will be useful as a new therapeutic target.
Assuntos
Apolipoproteína C-I/fisiologia , Apoptose/fisiologia , Sobrevivência Celular/fisiologia , Neoplasias Pancreáticas/patologia , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Apolipoproteína C-I/sangue , Apolipoproteína C-I/metabolismo , Biomarcadores Tumorais/química , Carcinoma Adenoescamoso/metabolismo , Carcinoma Adenoescamoso/patologia , Linhagem Celular Tumoral , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Pancreáticas/sangue , Neoplasias Pancreáticas/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Células Tumorais CultivadasRESUMO
Chromosomal instability (CIN) has been recognized as a hallmark of human cancer and is caused by continuous chromosome missegregation during mitosis. Proper chromosome segregation requires a physical connection between spindle microtubules and centromeric DNA and this attachment occurs at proteinaceous structures called kinetochore. Thus, defect in kinetochore function is a candidate source for CIN and the generation of aneuploidy. Recently, a number of kinetochore components have been shown to be mutated and/or aberrantly expressed in human cancers, which suggests an important role of kinetochore for CIN and carcinogenesis. In this article, we will discuss about how kinetochore dysfunction causes CIN and might lead to the development of cancer.
Assuntos
Instabilidade Cromossômica , Cinetocoros/patologia , Mutação , Neoplasias/patologia , Humanos , Neoplasias/genéticaRESUMO
To quantitatively analyze the electromyographic activity of the shoulder girdle muscles during elevation of the arm, the action potentials of nine muscles were integrated over 10-degree intervals of corresponding elevation angle. The trapezius, supraspinatus and deltoideus (anterior and middle fibers) muscles showed high activity and their activity increased in accordance with the magnitude of the elevation angle. The integrated EMG activity in the trapezius, supraspinatus and deltoideus (middle and posterior fibers) muscles increased as the plane of elevation was changed from the sagittal plane to the frontal plane. The correlation between the calculated work and the integrated EMG activity during scapular abduction from 0 to 90 degrees was analyzed. The regression coefficients of the trapezius, supraspinatus and deltoideus (anterior and middle fibers) muscles were higher than those of other muscles. The correlation coefficient of the supraspinatus was significantly lower than those of the trapezius, deltoideus and pectoralis major muscles.
Assuntos
Articulação do Ombro/fisiologia , Adolescente , Adulto , Eletromiografia , Humanos , Masculino , MovimentoRESUMO
We have previously reported that heterogeneous nuclear ribonucleoprotein K (hnRNP K) binds to the pyrimidine-rich strand of the CT element found in the human c-myc gene and activates CT reporter-driven gene expression in vivo. We now characterize the DNA and protein requirements for the interaction of hnRNP K with the CT element. First, hnRNP K is shown to preferentially bind single-stranded DNA over RNA or native double-stranded DNA. Using specific oligoribonucleotide or deoxyribonucleotide probes with specific or nonspecific RNA or DNA competitors, electrophoretic mobility shift assay revealed hnRNP K to be a DNA-binding protein. Specific binding was not simply a reflection of binding to pyrimidine-rich sequences as the number and arrangement of individual CT elements governed interactions with hnRNP K; at least two CT repeats separated by at least three nucleotides are required for binding, indicating the existence of particular stereochemical constraints regulating CT-hnRNP K complex formation. Deletion analysis showed that hnRNP K possesses several nonoverlapping, DNA binding domains, each capable of specific binding with the CT element and preferring DNA over RNA. Each sequence recognition domain is composed of at least one K homology motif, while a larger portion of hnRNP K may be required for stable RNA binding. Additional experiments indicate that the N-terminal 35 residues of hnRNP K are necessary for transactivating the CT element. These results indicate that hnRNP K is a DNA-binding protein and transcriptional activator.
Assuntos
DNA/metabolismo , Ribonucleoproteínas/metabolismo , Transativadores/metabolismo , Animais , Sequência de Bases , Linhagem Celular , DNA de Cadeia Simples/metabolismo , Células HeLa , Ribonucleoproteínas Nucleares Heterogêneas Grupo K , Ribonucleoproteínas Nucleares Heterogêneas , Humanos , Dados de Sequência Molecular , Ligação Proteica , Ativação TranscricionalRESUMO
Sequence specific regulators of eukaryotic gene expression, axiomatically, act through double stranded DNA targets. Proteins that recognize DNA cis-elements as single strands but for which compelling evidence has been lacking to indicate in vivo involvement in transcription are orphaned in this scheme. We sought to determine whether sequence specific single strand binding proteins can find their cognate elements and modify transcription in vivo by studying heterogeneous nuclear ribonucleoprotein K (hnRNP K), which binds the single stranded sequence (CCCTCCCCA; CT-element) of the human c-myc gene in vitro. To monitor its DNA binding in vivo, the ability of hnRNP K to activate a reporter gene was amplified by fusion with the VP16 transactivation domain. This chimeric protein was found to transactivate circular but not linear CT-element driven reporters, suggesting that hnRNP K recognizes a single strand region generated by negative supercoiling in circular plasmid. When CT-elements were engineered to overlap with lexA operators, addition of lexA protein, either in vivo or in vitro, abrogated hnRNP K binding most likely by preventing single strand formation. These results not only reveal hnRNP K to be a single strand DNA binding protein in vivo, but demonstrate how a segment of DNA may modify the transcriptional activity of an adjacent gene through the interconversion of duplex and single strands.