RESUMEN
Cardiomyopathy and neuropathy are the two commonly observed complications in diphtheria patients and in, some instances, individuals vaccinated against diphtheria. The nature of these complications remains not well understood. It was suggested that autoimmunity may play a role in the development of these afflictions. Based on functional similarities between diphtheria toxin (DT) and epidermal growth factor receptor (EGFR), which both can bind to the heparin-binding EGF-like growth factor (HB-EGF) precursors, we suggested that antibodies developed against DT can cross react with EGFR. Here, using serum from healthy donors (n = 10) and diphtheria patients (n = 15), we demonstrated that B-subunit of DT has the antigenic epitopes similar to those of EGFR. Diphtheria toxin as well as EGFR could be recognized by antibodies raised against EGFR and by serum antibodies from diphtheria patients. Moreover serum of diphtheria patients competitively inhibits binding of anti-EGFR antibodies to the receptor. The truncated diphtheria toxin without B-subunit could be detected by serum antibodies of diphtheria patients, but not by anti-EGFR antibodies. Collectively, these studies demonstrate cross-reactivity of antibodies raised against B-subunit of DT and extracellular domain of EGFR and suggest that clinically observed post-diphtheria complications may result from autoimmune inhibition of EGFR function and possible destruction of receptor-positive tissues.
Asunto(s)
Antitoxina Diftérica/inmunología , Toxina Diftérica/inmunología , Difteria/inmunología , Receptores ErbB/inmunología , Autoinmunidad/inmunología , Línea Celular Tumoral , Reacciones Cruzadas , Difteria/sangre , Difteria/complicaciones , Epítopos/inmunología , Humanos , Sueros Inmunes/sangre , Sueros Inmunes/inmunología , Subunidades de Proteína/inmunología , Vacunación/efectos adversosRESUMEN
The unicellular green microalga Chlamydomonas reinhardtii is a perspective model object for basic and applied research. However, its homologous recombination (HR) system which lies in the basis of double-strand DNA break repair have still not been studied. Last years the program of C. reinhardtii nuclear genome sequence is realized and different nucleotide repeats in the genome structure have been revealed that can explain a low level of HR relative to nonhomologous recombination events. Analyses of the C. reinhardtii EST (Expressed Sequence Tag)--and genome libraries permitted us to reconstruct and clone cDNA of the RAD51 gene. In present work, the cDNA was expressed, its product was purified and some basal biochemical activities were studied. The results show that Rad51C protein from lower eukaryote C. reinhardtii is identified as typical representative of the Rad51C-like subfamily of higher eukaryotes.
Asunto(s)
Chlamydomonas reinhardtii/metabolismo , Proteínas de Unión al ADN/metabolismo , Recombinación Genética , Adenosina Trifosfato/química , Animales , Chlamydomonas reinhardtii/genética , Clonación Molecular , Reparación del ADN/genética , ADN de Algas/química , ADN de Algas/genética , ADN Complementario/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Etiquetas de Secuencia Expresada , Concentración de Iones de Hidrógeno , Hidrólisis , Recombinasa Rad51 , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoAsunto(s)
Adenosina Trifosfatasas/química , ADN Helicasas/química , ADN Polimerasa I/química , ADN/síntesis química , Escherichia coli/genética , Escherichia coli/metabolismo , Geobacillus stearothermophilus/enzimología , Secuencia de Bases , Cartilla de ADN , AdnB Helicasas , Humanos , Datos de Secuencia Molecular , Peso Molecular , Moldes GenéticosRESUMEN
A new technique of PCR hot start using oligonucleotide primers with a stem-loop structure is developed here. The molecular beacon oligonucleotide structure without any chromophore addition to the ends was used. The 3'-end sequence of the primers was complementary to the target and five or six nucleotides complementary to the 3'-end were added to the 5'-end. During preparation of the reaction mixture and initial heating, the oligonucleotide has a stem-loop structure and cannot serve as an effective primer for DNA polymerase. After heating to the annealing temperature it acquires a linear structure and primer extension can begin.
Asunto(s)
Técnicas Biosensibles , Cartilla de ADN/química , Cartilla de ADN/genética , Calor , Conformación de Ácido Nucleico , Reacción en Cadena de la Polimerasa/métodos , Emparejamiento Base , Colorantes , ADN Polimerasa Dirigida por ADN/metabolismo , Fluorescencia , Genes p53/genética , Humanos , Linfocitos/metabolismo , Mycobacterium tuberculosis/genética , Hibridación de Ácido Nucleico , Inhibidores de la Síntesis del Ácido Nucleico , Sensibilidad y EspecificidadRESUMEN
A novel method for the hot start of PCR using DNA helicases is developed. The addition of a DNA helicase prevents the random annealing of primers and synthesis of nonspecific products during the preparation of the reaction mixture and initial heating. The hot start of PCR occurs automatically after inactivation of the DNA helicase upon heating of the reaction mixture.
Asunto(s)
ADN Helicasas/metabolismo , Reacción en Cadena de la Polimerasa/métodos , ADN Bacteriano/genética , Escherichia coli/enzimología , Humanos , Mycobacterium tuberculosis/genética , Thermus thermophilus/enzimologíaRESUMEN
A new method was developed for fast DNA amplification by polymerase chain reaction in tiny ultrathin microplates formed directly on the thermocycler's thermoblock. The microplates are made from thin (40-60 microns) polypropylene film by the thermal vacuum-formation method. Due to the effective heat transfer to 10-15 microliters samples and a high velocity of heating and cooling of the thermoblock (up to 7 degrees C/s), the total duration of the DNA amplification (30 cycles) is only 15-30 min.
Asunto(s)
ADN Viral , Amplificación de Genes , Reacción en Cadena de la Polimerasa/métodos , Genes Virales , VIH-1/genética , Humanos , Reacción en Cadena de la Polimerasa/instrumentaciónRESUMEN
Abnormalities of some oncogenes, antioncogenes and losses of heterozygosity (LOH) of chromosome 11p, 17p, and 17q in colorectal carcinomas (CC) was studied. Amplification of ERBB-1/HER-1 oncogene was detected in 2 of 56 cases; ERBB-2/HER-2- in 4 of 62. There was a lack of evidence for C-MYC oncogene amplification (67 cases). LOH of chromosome 11p (HRAS-1 probe) was found in 2 of 37 informative (heterozygous) cases; such events were not accompanied by point mutations in "hot" codons (12th or 61st) in the remaining allele. Prevalence of A3 and A4 alleles of HRAS-1 oncogene (68 cases) as compared to healthy donors was noted. RB-1 (41 cases) and p53 (62 cases) suppressor genes did not show any alterations in Southern-blot analysis. LOH of chromosome 17p (YNZ-22 probe) was found in 15 of 26 heterozygous CC; 17q (THH-59 probe)--in 4 of 16. Analysis of 175th codon of p53 gene revealed only one case of mutation in 35 CC studied. Finally, we were able to detect genetic alterations in 23 of 40 (58%) CC, that were studied on each parameter using Southern-blot. We failed to find any correlation between various molecular abnormalities or clinical characteristics. The data obtained are in disagreement with the view concerning frequent involvement of p53 antioncogene in chromosome 17p deletions.
Asunto(s)
Cromosomas Humanos Par 17 , Neoplasias del Colon/genética , Eliminación de Gen , Genes Supresores de Tumor , Oncogenes , Proteínas Proto-Oncogénicas/genética , Secuencia de Bases , Southern Blotting , Genes myc , Genes p53 , Genes ras , Heterocigoto , Humanos , Datos de Secuencia Molecular , Oligonucleótidos , Plásmidos , Reacción en Cadena de la Polimerasa , Células Tumorales CultivadasRESUMEN
The methods for enzymatic DNA amplification in vitro that allow to avoid the step of preliminary DNA extraction and purification are proposed. Lysates of blood cells in the solution or immobilized on the nylon membrane filters and dried blood spots on the filter paper blotters were used directly in amplification permitting one to solve the problems of adapting the method of polymerase chain reaction in clinical practice, for instance, in massive screening of genome mutations, viral infections etc.
Asunto(s)
ADN Polimerasa Dirigida por ADN/metabolismo , ADN/genética , Amplificación de Genes , Alelos , Electroforesis en Gel de Poliacrilamida , Humanos , Hibridación de Ácido Nucleico , Sondas de Oligonucleótidos , Reacción en Cadena de la PolimerasaRESUMEN
RELP analysis of DNA loci MET, D7S8 and D7S23 was carried out in Leningrad population and partially in populations of Moscow, Azerbaijan, Ukraine, Buryatia as well as in individuals from high risk families and in cystic fibrosis (CF) patients by means of blot hybridization and polymerase chain reaction. Allelic polymorphism of all loci studied in these three groups was found to be quite similar to that in the North-Western Europe and in whites of the North America. Linkage disequilibrium of the alleles studied with the CF gene was especially pronounced for alleles of the D7S23 locus and gradually decreases from KM-19 through CS-7 to XV-2c DNA probes. The data witness genetic homogeneity of the CF mutation in European populations of the USSR and its similarity to this mutation in Western Europe. The significance of these data for potential diagnosis of CF and for heterozygous carrier detection is discussed.
Asunto(s)
Alelos , Fibrosis Quística/genética , ADN/genética , Polimorfismo Genético , Sondas de ADN , Ligamiento Genético , Humanos , Mutación , Hibridación de Ácido Nucleico , Reacción en Cadena de la Polimerasa , Factores de Riesgo , U.R.S.S.RESUMEN
Beta zero-thalassaemia comprises a series of closely related haemoglobinopathies which are widely spread in some areas (the Mediterranean, Caucasus, Central Asia, and others). It is caused by a variety of mutations in the beta-globin gene which damage its expression, thus leading to severe illness, which is often lethal at an early age. By means of the polymerase chain reaction (PCR), restriction analysis, and sequencing by the Maxam-Gilbert method, we have identified a number of mutations in the beta-globin gene that cause beta zero-thalassaemia in the Azerbaijanian population, viz AA deletion in codon 8, C----T transition in codon 39, and a previously unknown G deletion in codons 82/83.
Asunto(s)
Talasemia/genética , Azerbaiyán , Secuencia de Bases , ADN/genética , Análisis Mutacional de ADN , Globinas/genética , Haplotipos/genética , Humanos , Lactante , Datos de Secuencia Molecular , Polimorfismo de Longitud del Fragmento de RestricciónRESUMEN
Upon amplification in vitro of the 12th exon area of the human phenylalanine hydroxylase gene followed by allele-specific hybridisation of the amplification product with synthetic probes and its sequencing by the Maxam-Gilbert method, a C----T transition causing phenylketonuria has been identified in Latvian patients.
Asunto(s)
Exones , Mutación , Fenilalanina Hidroxilasa/genética , Fenilcetonurias/genética , Alelos , Secuencia de Bases , ADN/genética , Humanos , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Fenilcetonurias/enzimologíaRESUMEN
Molecular nature of two beta 0-thalassaemia-causing mutations in beta-globin gene in Azerbaijanian population has been elucidated, viz., C-T transition in 39 codon (nonsense mutation) and previously unknown G deletion in 82/83 codons.
Asunto(s)
Globinas/genética , Mutación , Talasemia/genética , Azerbaiyán , Codón , Electroforesis en Gel de Poliacrilamida , HumanosAsunto(s)
Mutación , Talasemia/genética , Azerbaiyán , Secuencia de Bases , ADN/sangre , ADN/genética , Sondas de ADN , Genes , Globinas/genética , Humanos , Datos de Secuencia MolecularRESUMEN
A mutation causing beta 0-thalassaemia in Azerbaijanian population is shown, by the polymerase chain reaction followed by Maxam-Gilbert sequencing, to be the deletion of dinucleotide AA from the eight codone of beta-globin gene (the mutation is known to exist also in Turkey and Lebanon). Two other mutations have also been found in beta-globin gene of the same DNA, one of which (transversion C----G at position 16 of intron 2) eliminates the polymorphic AvaII-site and is associated with thalassaemia, and other is transition C----T in the third position of the second beta-globin codon.
Asunto(s)
Deleción Cromosómica , ADN/genética , Amplificación de Genes , Globinas/genética , Talasemia/genética , Análisis Mutacional de ADN , Enzimas de Restricción del ADN , Desoxirribonucleasas de Localización Especificada Tipo II , Electroforesis en Gel de Poliacrilamida , Femenino , Humanos , Masculino , Linaje , Mapeo RestrictivoRESUMEN
The polymerase chain reaction (PCR) method has been used for amplification of two segments of the human beta-globin gene comprising most of pathogenic mutations in the gene.
Asunto(s)
Amplificación de Genes , Globinas/genética , Sondas de Oligonucleótidos , Secuencia de Bases , ADN Polimerasa Dirigida por ADN , HumanosRESUMEN
Cationic proteins--cytochrome c and pancreatic RNAase--possess the apurinic-apyrimidinic DNA-endonuclease activity. The affinity of these proteins for DNA-apurinic sites does not differ from that of specific apurinic DNA-endonucleases described in literature. The main features of the apurinic activity of cationic proteins are as follows: low specific activity, high temperature optimum of the reaction, absence of primer-stimulated activity. The feasibility of participation of cationic proteins and some other nucleophilic compounds in single-stranded breaks production in apurinic DNA is discussed.