ABSTRACT
Tissue precursors and genesis of female reproductive tract carcinoma vis-a-vis its carcinomatous and sarcomatous patterns remain unknown. To determine the clonal origin of 17 female reproductive tract carcinomas, such molecular, genetic and immunohistochemical techniques as PCR-SSCP and/or denaturing gel electrophoresis for K-ras, p53 and PTEN genes; D17S786, CHRNB1, TP53, BAT26 and BAT40 microsatellites and immunostaining for p53 protein were used. Carcinomatous and sarcomatous components were studied separately. Eight tumors were assumed to be monoclonal (combination or conversion tumors), while one--of an obscure origin. Our results suggest that carcinosarcomas were characterized by chromosomal instability. Moreover, it was shown that it is necessary to combine immunohistochemical techniques with a battery of methods including genetic ones to determine clonal origin of immunologically--stained carcinosarcomas.
Subject(s)
Biomarkers, Tumor/analysis , Carcinosarcoma/chemistry , Carcinosarcoma/genetics , Genital Neoplasms, Female/chemistry , Genital Neoplasms, Female/genetics , Adult , Aged , Biomarkers, Tumor/genetics , Carcinoma/chemistry , Carcinoma/genetics , Chromosomal Instability , Electrophoresis, Agar Gel , Female , Genes, p53 , Genes, ras , Genetic Markers , Humans , Immunohistochemistry , Microsatellite Repeats , Middle Aged , PTEN Phosphohydrolase/genetics , Polymerase Chain Reaction , Polymorphism, Single-Stranded Conformational , Receptors, Nicotinic/genetics , Sarcoma/chemistry , Sarcoma/genetics , Tumor Suppressor Protein p53/geneticsABSTRACT
Bacterial RecA protein is the key enzyme in the processes of homologous recombination, post-replication repair and induction of SOS-repair functions. While a significant amount of data on the structure of RecA protein and its functional analogs has been obtained, there is little information about the molecular dynamics of this protein. In this work we present the results of neutron spin-echo measurements of the relaxation kinetics of filaments formed by RecA proteins from E. coli and P. aeruginosa. The results suggest that the protein filaments exhibit both diffusion and internal relaxation modes, which change during the formation of complexes of these proteins with ATP and single-stranded DNA.
Subject(s)
Adenosine Triphosphate/chemistry , DNA, Single-Stranded/chemistry , Escherichia coli Proteins/chemistry , Escherichia coli/enzymology , Neutrons , Pseudomonas aeruginosa/enzymology , Rec A Recombinases/chemistry , Adenosine Triphosphate/metabolism , DNA Repair/physiology , DNA Replication/physiology , DNA, Single-Stranded/metabolism , Escherichia coli Proteins/metabolism , Protein Structure, Quaternary , Rec A Recombinases/metabolism , Recombination, Genetic/physiology , Structure-Activity RelationshipABSTRACT
Bacterial RecA protein is a prototype of ATP-dependent homologous recombinases found ubiquitously from bacteriophages up to human beings. When RecA filament is forming on single-stranded DNA in the presence of ATP, it initiates the strand exchange reaction with homologous double-stranded DNA. Among three phases of the reaction (the search for homology, the three-stranded structure annealing in conjunction with the switch of pairing, and the strand displacement) the first one is the most enigmatic and least studied. As commonly recognized, this phase is directed by a special (stretched) filament structure and does not required any additional consumption of energy in ATP hydrolysis. The novel approaches in the study of strand exchange reaction, using short oligonucleotides as DNA substrates and sensitive methods for a real-time monitoring of the reaction suggest that all three phases of the reaction depend on the ATP hydrolysis.
Subject(s)
DNA/metabolism , Rec A Recombinases/metabolism , Recombination, Genetic , Adenosine Triphosphatases/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , DNA, Single-Stranded/metabolism , Humans , Hydrolysis , Rec A Recombinases/genetics , Transferases/metabolismSubject(s)
Colorectal Neoplasms/diagnosis , Colorectal Neoplasms/genetics , DNA/analysis , Feces/chemistry , Animals , HumansABSTRACT
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.
Subject(s)
Chlamydomonas reinhardtii/metabolism , DNA-Binding Proteins/metabolism , Recombination, Genetic , Adenosine Triphosphate/chemistry , Animals , Chlamydomonas reinhardtii/genetics , Cloning, Molecular , DNA Repair/genetics , DNA, Algal/chemistry , DNA, Algal/genetics , DNA, Complementary/genetics , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , Expressed Sequence Tags , Hydrogen-Ion Concentration , Hydrolysis , Rad51 Recombinase , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolismSubject(s)
Colorectal Neoplasms, Hereditary Nonpolyposis/genetics , Mutation , Adaptor Proteins, Signal Transducing , Carrier Proteins , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/metabolism , Colorectal Neoplasms, Hereditary Nonpolyposis/diagnosis , Colorectal Neoplasms, Hereditary Nonpolyposis/metabolism , DNA Repair/genetics , Disease Progression , Genomic Instability/genetics , Humans , Microsatellite Repeats/genetics , MutL Protein Homolog 1 , Neoplasm Proteins/genetics , Nuclear ProteinsSubject(s)
DNA-Binding Proteins/chemistry , Fungal Proteins/chemistry , Pichia/chemistry , Adenosine Triphosphate/metabolism , Bacteriophages/genetics , DNA, Single-Stranded/chemistry , DNA, Single-Stranded/metabolism , DNA, Viral/chemistry , DNA, Viral/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Fungal Proteins/genetics , Fungal Proteins/metabolism , Hydrolysis , Pichia/genetics , Pichia/metabolism , Polymers/chemistry , Rad51 Recombinase , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Temperature , ThermodynamicsSubject(s)
Escherichia coli/genetics , Genes, Bacterial , Pseudomonas aeruginosa/genetics , Rec A Recombinases/genetics , Amino Acid Substitution , Rec A Recombinases/chemistry , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombination, Genetic , SOS Response, GeneticsSubject(s)
Rec A Recombinases/chemistry , Tryptophan/chemistry , Tyrosine/chemistry , Adenosine Diphosphate/metabolism , Adenosine Triphosphate/analogs & derivatives , Adenosine Triphosphate/metabolism , DNA, Single-Stranded/metabolism , Escherichia coli/chemistry , Ligands , Protein Binding , Protein Conformation , Rec A Recombinases/metabolism , Spectrometry, FluorescenceABSTRACT
The previously offered procedure for detecting. Mycobacterium tuberculosis, which is based on the polymerase chain reaction (PCR), has been tested for diagnosing genital tuberculosis in females. PCR was used to examine endometrial curettage specimens in 44 patients with different nosological entities, which showed a high sensitivity (80%) and a high specificity in the diagnosis of genital tuberculosis.
Subject(s)
DNA, Bacterial/analysis , Mycobacterium tuberculosis/genetics , Polymerase Chain Reaction , Tuberculosis, Female Genital/diagnosis , Diagnosis, Differential , Female , Humans , Mycobacterium tuberculosis/isolation & purification , Pregnancy , Pregnancy Complications, Infectious/diagnosis , Pregnancy Complications, Infectious/microbiology , Sensitivity and Specificity , Tuberculosis, Female Genital/microbiologyABSTRACT
In order to compare the frequency of damage to the transforming growth factor TGF-beta receptor type II gene (RII gene) and microsatellite instability (MIN) in oncogenesis of sporadic and hereditary cancer of gastrointestinal tract (GIT), 4 groups of carcinomas were analyzed. They included sporadic gastric (GC), family gastric (FGC), sporadic colorectal (CC) and hereditary nonpolyposis colorectal (HNPCC) carcinomas having appropriate clinical and pathological characteristics. Each group consisted of two types of carcinomas, one of them showing MIN. The RII gene damage occurred in 89% of GC (8 cases out of 9), 86% of CC (6 out of 7), 71% of FGC (5 out of 7), 50% of HNPCC (3 out of 6) for carcinomas coupled with MIN, whereas only in 6% (1 out of 18) of GC and 5% (1 out of 22) of CC for carcinomas without MIN. No damage to RII gene was found in the cases of hereditary carcinomas which did not show MIN though the number of cases analyzed was not sufficient for final conclusions (3 cases of FGC and 3 HNPCC). The data revealed a correlation between the MIN phenotype and mutations in RII gene both for sporadic (p < 0.001) and for hereditary (p < 0.02) cases. For all 4 groups the frequency of RII gene damage was found for early and advanced carcinomas. This suggests that the deficiency of TGF-beta receptor complex in both sporadic and hereditary carcinomas of GIT is revealed at early stages of tumor development and consequently may be responsible for tumor progression. The correlation between RII gene damages and MIN in GIT carcinoma cells suggests that genetic change predetermined the neoplasia of colorectal and gastric epithelium and partially overlapped for both sporadic and hereditary cases.
Subject(s)
DNA Damage/genetics , DNA, Neoplasm/genetics , Gastrointestinal Neoplasms/genetics , Microsatellite Repeats/genetics , Receptors, Transforming Growth Factor beta/genetics , Colorectal Neoplasms/genetics , Colorectal Neoplasms, Hereditary Nonpolyposis/genetics , Disease Progression , Gastrointestinal Neoplasms/metabolism , Humans , Mutation , Phenotype , Stomach Neoplasms/geneticsABSTRACT
Gene recA from Pseudomonas aeruginosa was cloned into pUC19 vector under lacZ promoter. The expressed protein appeared to be modified, the aminoterminal part of deduced amino acid sequence of the RecAPa protein was found elongated by a polypeptide of 10 amino acids. The modified protein named RecA*Pa completely replaces RecAEc from E. coli in vivo recombination. In vitro RecA*Pa promotes the homologous strand transfer from a short linear duplex DNA fragment (346 bp) into circular single-stranded DNA (8196 n) being 5 times more active than RecAEc. However, when the length of dsDNA increased the difference between two proteins becomes negligible. To understand the reasons, some properties of RecA*Pa and RecAEc were compared. The former was shown to be more active both in binding to ssDNA in ssDNA-dependent ATP hydrolysis. The Rec*Pa protein showed also a high affinity to dsDNA, even at a physiological pH which is known to be unfavorable for RecAEc/dsDNA binding. However, both proteins equally catalyzed the dsDNA-dependent ATP hydrolysis; we suggest that this is crucial for a full-length DNA strand transfer recombination reaction.
