Alicia De María1, Cristina Arruti. 1. Laboratorio de Cultivo de Tejidos, Sección Biología Celular, Departamento de Biología Celular y Molecular, Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay.
Abstract
PURPOSE: Nuclear loss is a most remarkable organelle disappearance during terminal differentiation of lens fiber cells given that it implicates the full degradation of a major molecular component, DNA. Consequently, to gain insight into the progression of DNA cleavage we analyzed the appearance of single strand breaks in relationship with chromatin condensation. To assess a possible involvement of DNase I in DNA fragmentation we explored its localization in lens fibers having different degrees of nuclear breakdown, evaluated by the state of chromatin, nuclear envelope, and DNA. METHODS: Whole mounts of adult bovine lens epithelium as well as lens cryosections were utilized to examine, using antibodies or specific molecular probes, the localization of DNase I, nuclear membrane, lamins, and DNA 3'-OH-free termini. Nuclease activity gel and western blot assays were used to characterize DNase I in different lens fiber extracts. RESULTS: Nuclear morphology was found to undergo significant changes from the onset of fiber differentiation. Initial spherical nuclei present at early fibergenesis stages evolve to elongated ones in mature fibers. Chromatin did not present signs of condensation in these nuclei. However, nuclei from fibers located deeper in lens volume exhibited some chromatin condensation and fragmentation while the nuclear lamina appeared undamaged. At more advanced stages, different patterns of nuclear envelope integrity and chromatin condensation and cleavage were observed. DNase I was found in the cytoplasm in the very initial fibers and then in the nuclear territory. DNase I appeared closely associated with fully condensed and fragmented chromatin at the final phases of nuclear breakdown. CONCLUSIONS: DNase I is a nuclease present in bovine lens fibers and can be considered as an enzyme producing final DNA cleavage since it is closely associated with highly fragmented DNA in disintegrating nuclei.
PURPOSE: Nuclear loss is a most remarkable organelle disappearance during terminal differentiation of lens fiber cells given that it implicates the full degradation of a major molecular component, DNA. Consequently, to gain insight into the progression of DNA cleavage we analyzed the appearance of single strand breaks in relationship with chromatin condensation. To assess a possible involvement of DNase I in DNA fragmentation we explored its localization in lens fibers having different degrees of nuclear breakdown, evaluated by the state of chromatin, nuclear envelope, and DNA. METHODS: Whole mounts of adult bovine lens epithelium as well as lens cryosections were utilized to examine, using antibodies or specific molecular probes, the localization of DNase I, nuclear membrane, lamins, and DNA 3'-OH-free termini. Nuclease activity gel and western blot assays were used to characterize DNase I in different lens fiber extracts. RESULTS: Nuclear morphology was found to undergo significant changes from the onset of fiber differentiation. Initial spherical nuclei present at early fibergenesis stages evolve to elongated ones in mature fibers. Chromatin did not present signs of condensation in these nuclei. However, nuclei from fibers located deeper in lens volume exhibited some chromatin condensation and fragmentation while the nuclear lamina appeared undamaged. At more advanced stages, different patterns of nuclear envelope integrity and chromatin condensation and cleavage were observed. DNase I was found in the cytoplasm in the very initial fibers and then in the nuclear territory. DNase I appeared closely associated with fully condensed and fragmented chromatin at the final phases of nuclear breakdown. CONCLUSIONS:DNase I is a nuclease present in bovine lens fibers and can be considered as an enzyme producing final DNA cleavage since it is closely associated with highly fragmented DNA in disintegrating nuclei.