A Partial Sequence of Nuclear Events in Regenerating Rat Liver

Abstract

When two lobes of the liver of the adult rat are removed, the cells of the remaining lobe are aroused to renewed cell division. We have studied the early events in such regeneration. The first observable response to such partial hepatectomy is the production in the liver nuclei of rapidly-labeled high-molecular-weight RNA of sequences not produced by normal liver. This is followed, with a lag of about 1 hr, by the appearance of increased (above normal) amounts of chromosomal RNA, again of sequences not produced by normal liver. With a lag of another hour, the template activity in support of RNA synthesis of the liver chromatin increases substantially. These events occur before initiation of DNA synthesis in the cells of thAe regenerating liver.Chromosomal RNA (eRNA) is a small nuclear RNA that comprises a significant fraction of the total nuclear RNA in all higher eukaryotes investigated (1-3). It hybridizes to the repetitive DNA sequences (4, 5) and is distinguished by a high content of dihydropyrimidine (6). Several experiments have suggested that cRNA is involved in the processes of gene activation (7,3). In addition, it has recently been shown that cRNA and high-molecular-weight rapidly-labeled nuclear RNA (Hn RNA) share a large proportion of their nucleotide sequences, a result that strongly suggests a precursor-product relationship between them (5). To shed further light on these matters, we have investigated the temporal relationships between Hn RNA, cRNA, and template activity in regenerating rat liver. MATERIALS AND METHODSRegenerating Livers. The livers of 200-400 g male, albino Sprague-Dawley rats were caused to regenerate by surgically removing their two largest lobes (9). All operations were performed on rats anesthetized with ether, and timed so that the regenerating livers were harvested between 6 p.m. and 10 p.m. to minimize circadian effects. The rats were decapitated and their livers were quickly frozen in liquid nitrogen.Chromatin. 20-40 g of frozen livers in saline-EDTA buffer (75 mM NaCl-24 mM EDTA, pH 8) were homogenized in a Waring blendor (85 V for 10 sec, 30 V for 3 min). This solution was then filtered through Miracloth (Chicopee Manufacturing Co., Milltown, N.J.) and centrifuged at 3500 rpm in the Sorvall centrifuge (SS-34 rotor) for 10 min. Crude chromatin was made from the pellet by three or four cycles of glass-teflon homogenization and centrifugation at 10,000 rpm (SS-34 rotor) for 10 min in 0.01 M Tris buffer (pH 8) (8). Chromatin for the template assays was purified by centrifugation through 1.7 M sucrose. The pellet was washed once by resuspension in 0.01 M Tris buffer (pH 8) and centrifugation at 10,000 rpm for 10 min. It was then sheared in a Virtis homogenizer at 45 V for 90 sec at a concentration of 10 A260/ ml. This solution was centrifuged for 30 min at 10,000 rpm in the Sorvall centrifuge and the supernatant was carefully removed and frozen in liquid nitrogen (8).Preparation of Chromosomal RNA. Chromosomal RNA was prepared by one of two previously described method...

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Analysis of Cell Cycle Compartments of Hepatocytes After Partial Hepatectomy

“…Specific biochemical differences exist between the prereplicative GI which is characterized by profound changes of the transcriptional pattern of chromatin (Church & McCarthy, 1967;Mayfield & Bonner, 1972) and RNA synthesis (Fujioka, Koga & Lieberman, 1963;Rabes & Brandle, 1969), and postmitotic GI of the second postoperative cycle which shows a continued elevation of replicative enzymes after cessation of the first wave of DNA synthesis (Bollum & Potter, 1959). Despite of these differences, tcl between two replicative cycles is similar to the duration of the prereplicative period between partial resection of the liver and start of DNA synthesis.…”

The problem of automation in animal development: confrontation of the concept of cell sociology with biochemical data

“…Giant HnRNA has repetitive sequences in common with cRNA (Holmes and Bonner, 1973). During regeneration of the rat liver increased amounts of cRNA appear after the production of rapidly labelled heavy molecules (both classes of molecule are undetectable in the normal liver) but before new template activity of RNA starts (Mayfield and Bonner, 1972). The authors emphasize that there is a 'precursor-product relationship' between HnRNA and cRNA and that the cRNA triggers gene activity.…”