ABSTRACT
Our laboratory has focused on defining, localizing, and understanding the mode of action of genes involved in fractionated x-irradiation (FXI) leukemia in susceptible and restraint mouse strains. We have described the genetic and molecular evidence suggesting the existence of multiple independent loci involved in FXI-induced leukemogenesis. These studies indicated that one of these, Ril-1, a locus on the distal portion of chromosome 15, is the major locus influencing susceptibility to the disease. Our data unequivocally place Ril-1 in the gene complex Ly-6--Ril-1--Sis--H-30--Pol-5. Ril-1 appears to be closest to Ly-6 and Sis. We report that in FXI-induced leukemias there are hypomethylation changes in the Ly-6 region as compared to normal thymocytes. In contrast, Sis was found to be hypermethylated and not expressed. In addition, we have noted DNA rearrangements in the Ly-6--Pol-5 region in the majority of tumors examined using the Ly-6 and spleen focus-forming virus (SFFLV) molecular probes. Increased expression of Ly-6 and other surface markers encoded in this region has been noted in FXI-induced thymomas.
Subject(s)
Chromosomes, Human, Pair 15/radiation effects , Genetic Linkage , Leukemia, Radiation-Induced/genetics , Animals , Blotting, Northern , Blotting, Southern , DNA/analysis , Electrophoresis, Gel, Two-Dimensional , Flow Cytometry , Gene Expression/radiation effects , Gene Rearrangement/radiation effects , Humans , Mice , Mice, Inbred Strains , Polymorphism, Restriction Fragment Length , RNA/analysis , Radionuclide Imaging , Thymoma/diagnostic imaging , Thymoma/genetics , Thymus Neoplasms/diagnostic imaging , Thymus Neoplasms/genetics , X-Rays/adverse effectsSubject(s)
Leukemia, Radiation-Induced/genetics , Animals , Chromosome Mapping , Cloning, Molecular , Genetic Linkage , Mice , Mice, Inbred C57BLABSTRACT
We report here that specific T-cell receptor rearrangements were observed in fractionated-X-irradiation-induced murine leukemias. Consistent gamma-chain rearrangements, limited beta-chain rearrangements, and no detectable alpha-chain rearrangements were observed. Gene expression studies revealed that, in comparison with normal thymus tissue, expression of alpha T-cell receptor genes was lower in the thymomas, beta expression was much higher but approximately equal to that of normal thymocytes, and gamma expression was significantly increased. After coupling these data with those from analyses using reagents against other surface markers, such as Lyt-2, L3T4, H-2, IL-2R and MEL-14, we concluded that the target T cells for fractionated-X-irradiation-induced transformation resemble fetal thymocytes from days 15 and 16 of gestation.
Subject(s)
Neoplasms, Radiation-Induced , Receptors, Antigen, T-Cell/genetics , Thymoma/genetics , Thymus Gland/embryology , Thymus Neoplasms/genetics , Animals , Antigens, Surface/analysis , DNA Restriction Enzymes , DNA, Neoplasm/genetics , Gene Expression Regulation , Gestational Age , Mice , Mice, Inbred C57BL , Nucleic Acid Hybridization , RNA, Neoplasm/genetics , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Thymoma/etiology , Thymus Gland/metabolism , Thymus Neoplasms/etiology , Time FactorsABSTRACT
Positioning of nucleosomes was examined in a reconstituted system using a plasmid DNA and histones from normal human and xeroderma pigmentosum, complementation group A (XPA), lymphoblastoid cells. The present studies indicate that the arrangement of nucleosomes, composed of normal human histones, in a region near the SV40 origin of replication on the plasmid DNA, is nonrandom. The alignment of nucleosomes in this region was not affected by the presence of histone H1. No difference in nucleosome positioning was observed when the nucleosomes were composed of histones from XPA cells.
Subject(s)
Histones/analysis , Nucleosomes/analysis , Xeroderma Pigmentosum/genetics , DNA/analysis , DNA Repair , DNA Replication , Humans , Plasmids , Simian virus 40/geneticsABSTRACT
Histones from normal human and xeroderma pigmentosum, complementation group A (XPA), lymphoblastoid cells were compared both quantitatively, qualitatively and for binding affinity for DNA. Electrophoretic examination of the histones showed that all five major histone species were present in both cell groups and that there were no quantitative differences between normal and XPA histones. Binding affinity to [3H] mammalian DNA of the histones was determined. No significant differences were observed in binding of either normal or XPA histones to DNA.
Subject(s)
Histones/isolation & purification , Xeroderma Pigmentosum/metabolism , Animals , Cell Line , Chromatography, Ion Exchange , DNA, Neoplasm/metabolism , Electrophoresis, Polyacrylamide Gel , Histones/metabolism , Humans , Melanoma, Experimental/metabolism , Mice , Protein Binding , Reference ValuesABSTRACT
The influence of nucleosomes on the activity of two chromatin-associated apurinic/apyrimidinic (AP) DNA endonuclease activities, pIs 9.2 and 9.8, from normal and xeroderma pigmentosum, complementation group A (XPA), lymphoblastoid cells was examined. These AP endonuclease activities were studied on non-nucleosomal and nucleosomal plasmid pWT830/pBR322 DNA which had been reconstituted with core (H2A, H2B, H3, H4) or total (core plus H1) histones from normal or XPA cells. Both nucleosomal and non-nucleosomal DNA was rendered partially AP by alkylation with 12.5 mM methyl methanesulfonate, followed by heating it at 70 degrees C, to produce approximately three AP sites per DNA molecule. The activities of both normal lymphoblastoid AP endonuclease activities on nucleosomal AP DNA, reconstituted with core histones, was approximately 2.5 times greater than that on non-nucleosomal AP DNA. When histone H1 was added to the system, this increase was reduced. XPA AP endonuclease activities, on the other hand, did not show any increase in activity on nucleosomal AP DNA reconstituted with core histones. These differences between normal and XPA endonuclease activities on AP nucleosomal DNA were the same regardless of whether histones from normal or XPA cells were used in the reconstituted system.
