RESUMEN
Highly charged iron (Fe(16+), here referred to as Fe XVII) produces some of the brightest X-ray emission lines from hot astrophysical objects, including galaxy clusters and stellar coronae, and it dominates the emission of the Sun at wavelengths near 15 ångströms. The Fe XVII spectrum is, however, poorly fitted by even the best astrophysical models. A particular problem has been that the intensity of the strongest Fe XVII line is generally weaker than predicted. This has affected the interpretation of observations by the Chandra and XMM-Newton orbiting X-ray missions, fuelling a continuing controversy over whether this discrepancy is caused by incomplete modelling of the plasma environment in these objects or by shortcomings in the treatment of the underlying atomic physics. Here we report the results of an experiment in which a target of iron ions was induced to fluoresce by subjecting it to femtosecond X-ray pulses from a free-electron laser; our aim was to isolate a key aspect of the quantum mechanical description of the line emission. Surprisingly, we find a relative oscillator strength that is unexpectedly low, differing by 3.6σ from the best quantum mechanical calculations. Our measurements suggest that the poor agreement is rooted in the quality of the underlying atomic wavefunctions rather than in insufficient modelling of collisional processes.
RESUMEN
By implementing a large-area, gain-stabilized microcalorimeter array on an electron beam ion trap, the electron-impact excitation cross sections for the dominant x-ray lines in the Fe XVII spectrum have been measured as a function of electron energy establishing a benchmark for atomic calculations. The results show that the calculations consistently predict the cross section of the resonance line to be significantly larger than measured. The lower cross section accounts for several problems found when modeling solar and astrophysical Fe XVII spectra.
RESUMEN
In laboratory experiments using the engineering spare microcalorimeter detector from the ASTRO-E satellite mission, we recorded the x-ray emission of highly charged ions of carbon, nitrogen, and oxygen, which simulates charge exchange reactions between heavy ions in the solar wind and neutral gases in cometary comae. The spectra are complex and do not readily match predictions. We developed a charge exchange emission model that successfully reproduces the soft x-ray spectrum of comet Linear C/1999 S4, observed with the Chandra X-ray Observatory.
RESUMEN
Sex hormone-binding globulin (SHBG) is a multifunctional protein that acts in humans to regulate the response to steroids at several junctures. It was originally described as a hepatically secreted protein that is the major binding protein for sex steroids in plasma, thereby regulating the availability of free steroids to hormone-responsive tissues. SHBG also functions as part of a novel steroid-signaling system that is independent of the classical intracellular steroid receptors. Unlike the intracellular steroid receptors that are ligand-activated transcription factors, SHBG mediates androgen and estrogen signaling at the cell membrane by way of cAMP. We have reviewed the current state of knowledge on the SHBG gene and the role of SHBG in steroid signaling (we shall not address its function as a plasma-binding protein).
Asunto(s)
Hígado/metabolismo , Globulina de Unión a Hormona Sexual/biosíntesis , Transducción de Señal/fisiología , Andrógenos/metabolismo , Estrógenos/metabolismo , Trompas Uterinas/metabolismo , Femenino , Humanos , Masculino , Próstata/metabolismo , Receptores de Superficie Celular/metabolismoRESUMEN
Activating mutations in the c-K-ras gene occur in about 40% of human colorectal carcinomas, yet the role of this oncogene in tumorigenesis is not known. We have developed a model cell culture system to study this problem, utilizing the immortalized but non-tumorigenic epithelial cell line IEC18, originally derived from normal rat intestine epithelium. These cells were cotransfected with the drug resistance selectable marker tk-neo and the plasmid pMIKcys, which encodes a mini human c-K-ras gene (15 kb) containing a cysteine mutation at codon 12. Drug resistant clones were isolated. Clones which also expressed the activated c-K-ras gene displayed a transformed morphology, decreased doubling time, increased level of diacylglycerol, anchorage independent growth in soft agar and an aneuploid karyotype and they were also tumorigenic when injected into nude mice. These clones also displayed increased expression, at both the mRNA and protein levels, of cyclin D1 and Rb. These findings may be of clinical relevance since human colorectal tumors also frequently display increased expression of both cyclin D1 and Rb. This model system may be useful for understanding the role and interrelationship between activation of the c-K-ras oncogene and increased expression of cyclin D1 and Rb in colorectal tumorigenesis.
Asunto(s)
Colon/metabolismo , Ciclinas/genética , Genes de Retinoblastoma , Proteínas Oncogénicas/genética , Animales , Línea Celular Transformada , Colon/patología , Neoplasias Colorrectales/genética , Ciclina D1 , Genes ras , Humanos , Cariotipificación , Ratones , Ratones Desnudos , RatasRESUMEN
The cyclin D1 gene is amplified and overexpressed in a significant fraction of human esophageal tumors, and several other types of human cancer, but the functional significance of this overexpression has not been established. To further address the roles of cyclin D1 in growth control and tumorigenesis, we have overexpressed an antisense cyclin D1 cDNA construct, either constitutively or inducibly, in the HCE7 human esophageal cancer cell line in which cyclin D1 is amplified and expressed at high levels. The expression of antisense cyclin D1 led to decreased expression of cyclin D1 at both mRNA and protein levels, and this was associated with a marked inhibition of cell proliferation. Antisense cyclin D1 expressing cells displayed a decreased plating efficiency, increased doubling time, decreased saturation density, increased cell size, decreased cyclin D1-associated in vitro kinase activity, decreased anchorage-independent growth, and a loss of tumorigenicity in nude mice. These findings provide direct evidence that the overexpression of cyclin D1 in certain tumor cells contributes to their abnormal growth and tumorigenicity. The ability to revert the transformed phenotype of these cells with antisense cyclin D1 suggests that cyclin D1 may be a useful target in cancer therapy.
Asunto(s)
Ciclo Celular , Transformación Celular Neoplásica/patología , Ciclinas/antagonistas & inhibidores , ADN sin Sentido/administración & dosificación , Neoplasias Esofágicas/genética , Proteínas Oncogénicas/antagonistas & inhibidores , Adhesión Celular , Ciclina D1 , Ciclinas/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Técnicas In Vitro , Proteínas Oncogénicas/metabolismo , Fosforilación , Proteínas Quinasas/metabolismo , ARN Mensajero/genética , ARN Neoplásico/genética , Transfección , Células Tumorales CultivadasRESUMEN
UNLABELLED: BACKGROUND/DESIGN: The clonal theory of cancer predicts that transformed cells within a given tumor are derived from a single initiated precursor. Advancement of this precursor through various stages of tumor development occurs with the further accumulation of selective genetic and epigenetic lesions. Mammalian ras genes are important constituents of mitogenic signaling pathways, and when activated, they contribute to deregulated cellular growth. Activated ras genes play important roles in the development of certain skin tumors. Studies on a number of animal tumor model systems have shown that ras gene activation can be an early and perhaps initial event in the development of skin tumors. Activated ras genes are also found in a significant percentage of somatic human squamous cell carcinomas. To gain retrospective insight into the stages at which activated ras genes contribute to squamous cell carcinoma development, we investigated their incidence in actinic keratoses, premalignant precursors to squamous cell carcinomas. Using a nonradioactive polymerase chain reaction-based method developed in our laboratory, we examined a panel of 19 actinic keratoses and 33 squamous cell carcinomas for activated ras genes. RESULTS: DNA analysis revealed ras gene mutations in three (16%) of 19 actinic keratoses and in four (12%) of 33 squamous cell carcinomas. Activating mutations occurred at codon 12 of the K-ras gene, and codons 12, 13, and 61 of the H-ras gene. All positive actinic keratoses and squamous cell carcinomas occurred in sun-exposed regions. CONCLUSIONS: Activated ras genes can play important roles during early stages of squamous cell carcinoma development. Aberrant repair of UV-induced pyrimidine dimers is a likely cause of this activation.
Asunto(s)
Carcinoma de Células Escamosas/genética , Regulación Neoplásica de la Expresión Génica/genética , Genes ras/genética , Queratosis/genética , Lesiones Precancerosas/genética , Neoplasias Cutáneas/genética , Secuencia de Bases , Humanos , Datos de Secuencia Molecular , Mutación , Lesiones Precancerosas/patología , Activación Transcripcional , Rayos Ultravioleta/efectos adversosRESUMEN
To study the mutator phenotype characteristic of tumors showing widespread replication errors at simple DNA repeat sequences (RER+), we designed a selectable reporter system for the detection of such mutations in mammalian cells. A hygromycin B phosphotransferase gene was rendered out-of-frame by the insertion of a (CA)13 dinucleotide repeat tract immediately following the ATG start codon, and subcloned into a retroviral expression vector containing a G418 (neo) selectable marker. Following transduction of this construct into cultured cells, clonal neo+ cell lines were established and then tested for their ability to form colonies in hygromycin B-containing medium. Using this system, we found that the HCT116, LS174T and LS180 human colon carcinoma cell lines acquire hygromycin resistance (hygr) at a 100-fold higher frequency than the HT29, SW480, DLD-1 and HCT15 human colon carcinoma and NIH3T3 fibroblast cell lines, and at a 25-fold higher rate than the Rat 6 embyro fibroblast cell line. DNA sequence analysis indicated that frameshift mutations had occurred within the CA dinucleotide repeat tract in HCT116 cells that became hygr. Thus, the mutation rates at simple repeated sequences in mammalian cell lines can be readily determined and studied using this system.
Asunto(s)
Replicación del ADN , Mutación del Sistema de Lectura , Secuencias Repetitivas de Ácidos Nucleicos , Células 3T3 , Animales , Secuencia de Bases , Línea Celular , Clonación Molecular , Codón , Neoplasias del Colon , Cartilla de ADN , Humanos , Mamíferos , Ratones , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fenotipo , Reacción en Cadena de la Polimerasa , Retroviridae , Transfección , Células Tumorales CultivadasRESUMEN
We previously isolated a partial cDNA sequence, termed TPAR1 (TPA repressed gene 1), from a cDNA library constructed from C3H10T1/2 mouse embryo fibroblasts treated with TPA, using a differential screening procedure. (M.D. Johnson et al. Mol. Cell. Biol. 7, 2821-2829, 1987). In the present study, we have cloned two corresponding full-length 1.9- and 3.4-kb cDNAs of TPAR1 from murine cDNA libraries. Sequence analysis of these TPAR1 cDNAs revealed that they encode 89 and 93 amino acid polypeptides, respectively, with a putative leader sequence and show significant homology with the human cytokine interleukin-8 (IL-8) and its superfamily. Genomic DNA isolation and structural characterization provide evidence that the TPAR1 mRNAs are transcribed from a single gene with alternative splicing. TPAR1 mRNAs are expressed ubiquitously among adult mouse tissues as three major transcripts, 1.9, 3.4, and 6.5 kb, whose expression depends on the tissue type. The levels of TPAR1 mRNAs were markedly decreased in fibroblasts following TPA treatment and also in serum-deprived quiescent fibroblasts stimulated by serum. The levels of TPAR1 mRNAs were dramatically down-regulated in regenerating rat liver when compared to normal adult liver. In addition, there was no detectable expression of TPAR1 in three rat hepatoma cell lines and several transformed fibroblast cell lines. Thus, the TPAR1 gene is a new member of the cytokine IL-8 superfamily, whose expression is down-regulated in rapidly dividing cells. Further studies are required to determine whether it plays a negative role in controlling cell proliferation and tumorigenesis.
Asunto(s)
Expresión Génica/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Línea Celular Transformada , Clonación Molecular , ADN Complementario/genética , Regulación hacia Abajo , Humanos , Interleucina-8/genética , Neoplasias Hepáticas Experimentales/genética , Regeneración Hepática/genética , Ratones , Datos de Secuencia Molecular , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Mapeo Restrictivo , Homología de Secuencia de AminoácidoRESUMEN
The amino-terminal regulatory domain portion of each protein kinase C (PKC) family member (which in the case of PKC beta 1 includes the pseudosubstrate, C1, V1 and C2 domains) plays an important role in regulating the kinase activity of the carboxyl-terminal catalytic domain. To examine the possibility that this regulatory domain region (designated 'PAT') might have biological functions independent of the catalytic domain, we have developed derivatives of R6 cells which stably express a truncated PKC beta 1 cDNA that encodes the amino-terminal 317 amino acids, including the entire regulatory domain. These R6-plPAT cells express abundant amounts of a 38 kDa protein which binds a labeled phorbol ester, but lacks protein kinase activity. In contrast to the 79 kDa PKC beta 1 holoenzyme which, when overexpressed in R6 cells, is found mostly in the cytosol, the 38 kDa PAT protein is predominantly associated with the particulate subcellular fraction. Furthermore, the PAT protein fails to show down-regulation following treatment of R6-plPAT cells with 12-O-tetradecanoylphorbol-13-acetate (TPA). Evidence is also presented that TPA-stimulated growth is suppressed in R6-plPAT cells. These findings suggest that the PKC beta 1 regulatory domain could be involved in the suppression of mitogenic signaling.
Asunto(s)
Isoenzimas/química , Mitosis/fisiología , Fragmentos de Péptidos/biosíntesis , Proteína Quinasa C/química , Estructura Terciaria de Proteína , Transducción de Señal/fisiología , Animales , Secuencia de Bases , División Celular , Línea Celular , Clonación Molecular , Inhibición de Contacto , Citosol/enzimología , Inducción Enzimática/efectos de los fármacos , Fibroblastos , Isoenzimas/biosíntesis , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteína Quinasa C/biosíntesis , Ratas , Proteínas Recombinantes de Fusión/biosíntesis , Fracciones Subcelulares/enzimología , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
We present the extreme ultraviolet (75-110 angstroms) spectrum of the BL Lac object PKS 2155-304, the first spectrum of an extragalactic source obtained with the Extreme Ultraviolet Explorer. The spectrum shows a generally smooth continuum, which can be modeled by a single power law plus interstellar absorption, and possibly an absorption feature at approximately 80 angstroms. The best fit to the data suggests that the EUV spectrum can be interpreted as a simple extrapolation of the X-ray continuum, with an energy index alpha approximately 1.6; however, shallower or steeper power laws with indices between -0.4 and 2.7 cannot be ruled out by the existing EUV data alone. The data provide strong constraints on the interstellar neutral H and He along the line of sight. Using a column density of 1.36 x 10(20) cm-2 for the Galactic neutral hydrogen along the PKS 2155-304 line of sight, the neutral helium column density is constrained to be 9%-10% of the hydrogen amount.
Asunto(s)
Astronomía/métodos , Medio Ambiente Extraterrestre , Vuelo Espacial/instrumentación , Nave Espacial/instrumentación , Análisis Espectral/métodos , Rayos Ultravioleta , Astronomía/instrumentación , Helio , Hidrógeno , Modelos Teóricos , Análisis Espectral/instrumentación , Análisis Espectral/estadística & datos numéricos , Rayos XRESUMEN
Cyclin D1, a putative G1 cyclin, has been implicated in cell cycle control. The human cyclin D1 gene is located on chromosome 11q13 where DNA rearrangement and amplification have been detected in several types of human cancer. Previous studies demonstrated that the cyclin D1 gene is not only rearranged or amplified but also overexpressed in some of these human tumors and tumor-derived cell lines. To further address the roles of cyclin D1 in cell cycle control and tumorigenesis, we have stably overexpressed the human cyclin D1 cDNA in Rat6 embryo fibroblasts by using retrovirus mediated transduction. The cyclin D1 protein was overproduced about 10-fold and was localized predominately in the nucleus. Cyclin D1 overexpressing cells displayed a decrease in the duration of the G1 phase, decreased cell size, and induced tumors when injected into athymic (nude) mice. In addition, overexpression of cyclin D1 in Rat6 cells perturbed the expression of several cellular growth-related genes including c-myc, c-jun, and cyclin A, but not cyclin D3. Taken together, these results indicate that deregulated expression of the cyclin D1 gene can cause disturbances in cell cycle control and gene expression and also enhance tumorigenesis.
Asunto(s)
Ciclinas/genética , Ciclinas/fisiología , Fibroblastos/citología , Expresión Génica/genética , Proteínas Oncogénicas/genética , Proteínas Oncogénicas/fisiología , Animales , Northern Blotting , Western Blotting , Ciclo Celular/fisiología , División Celular/fisiología , Núcleo Celular/química , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Células Cultivadas , Mapeo Cromosómico , Cromosomas Humanos Par 11 , Ciclina D1 , Ciclinas/análisis , ADN/análisis , ADN/genética , Embrión de Mamíferos/química , Embrión de Mamíferos/citología , Fibroblastos/química , Fibroblastos/metabolismo , Citometría de Flujo , Fase G1 , Amplificación de Genes , Expresión Génica/fisiología , Humanos , Inmunohistoquímica , Ratones , Ratones Desnudos , Proteínas Oncogénicas/análisis , Proteínas Proto-Oncogénicas c-jun/análisis , Proteínas Proto-Oncogénicas c-jun/genética , Proteínas Proto-Oncogénicas c-myc/análisis , Proteínas Proto-Oncogénicas c-myc/genética , RatasRESUMEN
Amplification of the chromosome 11q13 region occurs in several types of human cancer including esophageal, breast, lung, bladder and hepatocellular carcinoma (HCC). The gene cyclin D1 maps to this region in close proximity to two proto-oncogenes hst-1 and int-2. We previously demonstrated that cyclin D1 was not only amplified but also overexpressed in about 30% of human esophageal cancers. To investigate the role of cyclin D1 in human hepatocellular carcinoma (HCC), DNA from 30 HCC and 5 control liver tissues from Taiwan and also the HCC cells lines HepG2 and Hep3B, were examined for amplification of the cyclin D1 gene. A 3 to 20-fold amplification was found in 4 of the 30 (13%) HCC samples but not in any of the 5 control tissues or the 2 cell lines. Immunohistochemical analysis of cyclin D1 indicated overexpression of this protein in tumors that displayed gene amplification. Weak or negative staining was observed in the other HCC samples as well as in the control tissues and cell lines. These data suggest that increased expression of cyclin D1 may play an important role in the development of a subset of human HCC, perhaps by perturbing normal control of the cell cycle.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Cromosomas Humanos Par 11 , Ciclinas/biosíntesis , Ciclinas/genética , Amplificación de Genes , Neoplasias Hepáticas/metabolismo , Proteínas Oncogénicas/biosíntesis , Proteínas Oncogénicas/genética , Northern Blotting , Southern Blotting , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Ciclina D1 , Ciclinas/análisis , ADN/química , ADN de Neoplasias/química , ADN de Neoplasias/aislamiento & purificación , Expresión Génica , Humanos , Inmunohistoquímica , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Proteínas Oncogénicas/análisis , ARN Neoplásico/aislamiento & purificaciónRESUMEN
We have examined DNA from four human esophageal carcinoma cell lines and 50 primary esophageal carcinomas obtained from China, Italy, and France for amplification of the cyclin D1 gene. We also examined 36 of these 50 carcinomas for expression of the cyclin D1 and retinoblastoma (RB) proteins by immunohistochemistry. We found a 3- to 10-fold amplification of the cyclin D1 gene in 16 of the 50 (32%) tumors and in two of the four cell lines. Cyclin D1 protein was overexpressed in 12 of 13 tumors and the two cell lines that showed gene amplification when compared to normal controls. Studies on RB protein expression indicated that 6 of the 36 (17%) tumor samples examined and one cell line did not show detectable expression of this protein. The tumors and cell lines that had cyclin D1 gene amplification and overexpression exhibited normal levels of expression of RB protein. By contrast, the tumors and cell line that did not appear to express the RB protein did not show amplification of the cyclin D1 gene and expressed only low levels of the cyclin D1 protein (P = 0.03). These results suggest that the inhibitory effect of RB on cell cycle progression can be abrogated during tumor development either by loss of expression of the RB gene or by increased expression of the cyclin D1 gene.
Asunto(s)
Ciclinas/genética , Neoplasias Esofágicas/genética , Regulación Neoplásica de la Expresión Génica , Genes de Retinoblastoma , Proteínas Oncogénicas/genética , Southern Blotting , Western Blotting , Línea Celular , Ciclina D1 , Ciclinas/análisis , Ciclinas/aislamiento & purificación , ADN de Neoplasias/genética , ADN de Neoplasias/aislamiento & purificación , Neoplasias Esofágicas/química , Neoplasias Esofágicas/patología , Expresión Génica , Humanos , Inmunohistoquímica , Peso Molecular , Proteínas Oncogénicas/análisis , Proteínas Oncogénicas/aislamiento & purificación , Proteína de Retinoblastoma/análisis , Proteína de Retinoblastoma/genética , Proteína de Retinoblastoma/aislamiento & purificación , Células Tumorales CultivadasRESUMEN
We found that the human colon cancer cell line SW480 consists of two distinct subpopulations which we have designated E-type (epithelial) and R-type (round). Pure cultures of each type were obtained by subcloning, and both have maintained their characteristic phenotypes for at least 1 year (40 passages). E-type cells are the major (> 98%) type in the parental SW480 cell line. They form flat epithelial-like colonies. In contrast, R-type cells, which constitute a minor fraction (< 2%) of the parental cell line, have a rounded shape and grow in clusters of piled-up cells. Compared to E-type cells or the parental SW480 cells, isolated R-type cells display decreased doubling time, loss of contact inhibition, less adhesiveness to culture plates, higher anchorage-independent growth in soft agar, and a much more aneuploid karyotype. When injected s.c. into nude mice, R-type cells produce much larger tumors within the same period of time than E-type cells, and the tumors are less differentiated than those produced by the E-type cells. Cell fusion experiments between R-type and E-type cells revealed that the R-type phenotype is dominant, and the results suggest that this is due to one or a few genetic changes. Taken together, these findings suggest that the R-type cells represent a more malignant variant of the E-type cells. They may be useful, therefore, for studying mechanisms involved in tumor progression.
Asunto(s)
Neoplasias del Colon/patología , Animales , División Celular , Fusión Celular , Aberraciones Cromosómicas , Células Clonales , Neoplasias del Colon/genética , Femenino , Citometría de Flujo , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Células Tumorales CultivadasRESUMEN
Amplification of the hst-1 and int-2 genes on chromosome 11q13 has previously been found in over 20% of human primary esophageal cancers. However, these two genes do not appear to be transcribed in appreciable amounts. Recently, the human cyclin D gene (also referred to as prad1) has been mapped to the 11q13 locus. Here, we report coamplification of the cyclin D and hst-1 genes in 5 of 20 (25%) human squamous esophageal tumors. We also detected significant levels of cyclin D transcription in two esophageal carcinoma cell lines, even though they did not express detectable amounts of hst-1 transcription. These findings provide the first evidence for the amplification of a cyclin gene in human esophageal cancer and suggest that an increase in cyclin D gene dosage could be an important factor in the pathogenesis of esophageal cancer. Additionally, because the 11q13 locus is found to be amplified in many types of human tumors, cyclin gene amplification could also play an important role in the development of other forms of human cancer.
Asunto(s)
Carcinoma de Células Escamosas/genética , Ciclinas/genética , Neoplasias Esofágicas/genética , Expresión Génica/genética , Proteínas Oncogénicas/genética , Secuencia de Bases , Ciclina D1 , Sondas de ADN , ADN de Neoplasias/genética , Amplificación de Genes/genética , Humanos , Datos de Secuencia Molecular , Transcripción Genética/genéticaRESUMEN
We have developed a rapid and highly sensitive nonradioactive method for the detection of a mutant codon 12 human c-K-ras allele in the presence of as many as 10(4) copies of the wild type codon 12 allele. This sensitivity is achieved by selective polymerase chain reaction (PCR) amplification of mutant K-ras gene sequences employing a two stage procedure. The first stage entails the amplification of both K-ras mutant and wild type codon 12 sequences, followed by a selective restriction enzyme digestion of only wild type sequences. The second stage involves a subsequent amplification of undigested amplified fragments, enriched in mutant codon 12 sequences. These products are subject to restriction length polymorphism analysis for the detection of point mutations at codon 12. This technique is rapid, nonradioactive, and eliminates the need for either oligonucleotide hybridization or DNA sequencing. Variations of this selective amplification procedure may prove promising for the detection of specific point mutations in heterogenous cell populations.
Asunto(s)
Amplificación de Genes/genética , Genes ras/genética , Mutación/genética , Adenocarcinoma/genética , Adenocarcinoma/patología , Alelos , Secuencia de Bases , Línea Celular , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Humanos , Queratinocitos/patología , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de RestricciónRESUMEN
Helicobacter pylori is an organism thought to play an important causative role in gastritis and peptic ulcer diseases. We have designed an RNA dot blot assay for the detection of H. pylori, using as probe a synthetic oligonucleotide complementary to its 16S rRNA. We have also used oligonucleotide primers, complementary to conserved sequences within bacterial ribosomal 16S genes, to amplify a H. pylori ribosomal 16S DNA fragment via the polymerase chain reaction (PCR). After determining the DNA sequence of this amplified H. pylori fragment, primers were designed for specific PCR amplification of H. pylori ribosomal 16S DNA sequences. Samples from clinical endoscopic biopsies were PCR amplified with universal 16S ribosomal primers to detect the presence of bacteria and with H. pylori-specific primers to uniquely detect H. pylori. Finally, by comparing the H. pylori-specific PCR assay to commonly used diagnostic tests, we demonstrate that the molecular technique of PCR amplification shows promising applications for the clinical detection of H. pylori.