In mouse cells (line P815), newly synthesized DNA labeled for [20][21][22][23][24][25][26][27][28][29][30] sec during exponential growth is found by electron microscope autoradiography at sites throughout the cell nucleus. These sites are relatively more concentrated in the peripheral region of the nucleus (averaged over a random population of S-phase cells), probably reflecting a higher local concentration of DNA in this region. Newly synthesized DNA is not preferentially associated with purified nuclear envelopes, but is found in a fraction of the chromosomal deoxynucleoprotein whose buoyant density in CsCl after formaldehyde treatment is about 1% lower than that of the deoxynucleoprotein peak.Kinetics experiments suggest that this material is a precursor of mature deoxynucleoprotein; it may represent regions of deoxynucleoprotein containing replicating DNA and the additional proteins involved in DNA replication. Other complexes of newly replicated DNA that are found in the interphase after phenol extraction of nuclei are formed during the extraction procedure, probably due to the partially single-stranded nature of replicating DNA, and do not appear to exist in vivo.The many studies on the location of replication sites of chromosomal DNA in the eukaryotic nucleus have led to divergent conclusions. Electron microscope autoradiography (1-3) shows that replication sites of chromosomal DNA are distributed throughout eukaryotic nuclei, and do not appear to be associated with any specific morphological structure. The peripheral replication sites in cells released from synchronizing inhibitors (4) may not represent normal S-phase initiation (5).In contrast, most (6,7,8) [but not all (9)] biochemical studies show that newly replicated DNA is preferentially associated with the nuclear envelope. Newly replicated DNA is often (6, 10-12), but not always (13,14), localized in the interphase after phenol or chloroform-isoamyl alcohol extraction. It sometimes associates preferentially with crystals of detergent in sucrose gradients (7,30). The interpretation of these observations has been influenced by models of membrane-associated replication of bacterial (15) and phage (16) DNA; the phage DNA-membrane association may, however, be related to transcription rather than replication (17).We present here studies designed to resolve these discrepancies and to identify by two independent methods, electron microscope autoradiography and cell fractionation, the sites of chromosomal DNA synthesis in exponentially growing mouse cells. Eukaryotic chromosomal DNA replicates at 0.5 (18) to 1-2 (19) Mum/min; within the nucleus, therefore, a labeled precursor newly incorporated into DNA could be displaced as much as 1 Mm away from its site of incorporation after 1 min. We have used labeling periods of 20-30 sec, which are about the lower practical limit for present techniques of electron microscope autoradiography; labeled molecules incorporated at the beginning of this period are unlikely to be displaced more than 0.5 ,m from the...
Electrophysiological investigations were performed in patients with inflammatory eye disease characterized by the presence of vitreous cells. The eyes were classified into four categories on the basis of fluorescein angiography: 1) no fluorescein leakage from retinal vessels, 2) fluorescein leakage from peripheral retinal vessels, 3) fluorescein leakage from the disc or macular vessels, and 4) fluorescein leakage from retinal vessels associated with pigment epithelial and choroidal changes. The electro-oculogram light rise was abnormally increased in the eyes in category 1, but it progressively declined for those in the other categories. The ratio of the b-wave (postreceptoral component) and a-wave (receptoral component) of the flash electroretinogram was unchanged in all categories, but the electroretinographic amplitudes progressively declined from a somewhat supernormal level in category 1 to subnormal in the other categories. Thus, in inflammatory eye disease, changes in the electrical potentials arising in the pigment epithelium and photoreceptors are the earliest detectable signs. Some biochemical changes in the choroid, pigment epithelium, and the photoreceptors appear to take place before any pathological changes in these structures or in the retinal vessels are detectable by ophthalmoscopy or fluorescein angiography.
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