DNA Synthesis in Circulating Erythroblasts of Anemic Duck

Abstract

In the circulating blood of anemic ducks, 5 % of all erythroid cells synthesize DNA. Immature erythroblasts, at all stages of differentiation, synthesize DNA although to a varying degree, while reticulocytes and erythrocytes do not. In the erythroid cell population labeled in vitro 2 h with 32Pi, half of the labeled DNA sediments as small-molecular-weight molecules, suggesting that these molecules fail to integrate into the high-molecular-weight components.Labeled DNA is found in the cytoplasmic postmitochondrial fractions and it is in a form of deoxyribonucleoproteins which cosediment with ribosomes as well as subribosomal particles in sucrose gradients. However, fixation with HCHO and centrifugation to equilibrium in CsCl gradient of these particles shows that the deoxyribonucleoprotein bands at the density different than the ribosomes and, thus, not physically linked to them. In EDTA-dissociated ribosomes, the deoxyribonucleoprotein particles cosediment with ribosomal subunits in such a way that the larger the particle, the larger the molecular weight of the DNA cosedimenting with it.The specific radioactivity of the cytoplasmic ribosome-derived and postribosomal-particlederived DNAs and the small molecular-weight nuclear DNA is similar and 10-20-fold higher than that of the bulk nuclear DNA. The former three DNA species sediment between 4-14 S. It is concluded that the cytoplasmic nonmitochondrial DNA species are of the nuclear origin. Less than 0.5 % of the total cellular nonmitochondrial DNA can be purified from the nucleus and the cytoplasm as fast-labeled small-molecular-weight components. All of the cellular nonmitochondrial DNA species band at the same mean buoyant density in Cs2S0,/urea gradients. All behave as native structures in hydroxyapatite and contain less than 5 "/, of their length as single-stranded regions. down product of the nuclear DNA. Furthermore, Fromson and Nemer [7] have attributed the presence of DNA in cytoplasmic nonmitochondrial fractions to contamination by a nuclear leakage. The problem, however, remains unsolved due to recent findings by Koch [8,9] that when compared to the nuclear DNA, the cytoplasmic DNA seems to be relatively enriched in unique sequences.The transfer of the products of transcription from the nucleus to the cytoplasm in the form of mRNA and its translation on ribosomes has been extensively investigated, for instance in avian erythroid cells [lo, 111. The primary transcription product of the globin genes is of the size 5 x lo6 daltons [12,13] and is suggested to be the precursor of the cytoplasmic mRNA (pre-mRNA). Although there is little doubt about the role of this pre-mRNA as intermediary in cellular information transfer, a straightforward scheme of direct

Ultraviolet light-induced crosslinking of mRNA to proteins

“…In order to demonstrate crosslinking we isolated polyribosomes from cells which had been labeled with 3H-uridine in the presence of 0.08 ig/ml of actinomycin D. These labeling conditions were chosen so that mRNA would be labeled, but labeling of rRNA would be inhibited (11). The polyribosomes were irradiated at 254 nm and centrifuged in Cs2SO4 density gradients containing 5 M urea (12) in order to resolve crosslinked RNA-protein complexes from RNA and protein. Urea was included to prevent precipitation of RNA and protein, and also, to enhance the dissociation of noncovalent complexes.…”

Direct evidence that ribosome bound RNA-dependent RNA polymerase does not play a role in globin messenger RNA replication

“…4) which significantly (65% nuclease resistance in RNA excess) and consistently hybridized to the labelled cDNA.28S rRNA (as shown in Fig. 4) Modak et al (10) have speculated that the double stranded low molecular weight DNA, shown by the same authors to be present in the ribosome fraction of immature duck erythrocytes, might act as a template for the synthesis of specific mRNAs. The lack of any detectable, radioactively labelled globin mRNA in [3H]-pRNA (Table 1) argues against the synthesis of globin mRNA from such a cytoplasmic DNA template.…”

Analysis of DNA double- and single-strand breaks by two dimensional electrophorresis: action of micrococcal nuclease on chromatin and DNA, and degradation in vivo of lens fiber chromatin

“…DNA or chromatin samples were suspended in buffer containing 60 mM KCl, 3 mMl NaCl, 15 mM Tris-HCl (pH 7.4), 0.34 M sucrose, 0.1 mM EDTA (17) which was supplemented with 0.1 mM EGTA, 5 mM 2-mercaptoethanol and 0.2 mM phenylmethylsulfonylfluoride (Sigma). Nuclei from 6-day old duck embryos and IF.-day old chick embryo liver were isolated and purified as described before (18) and suspended in the buffer described above. Samples were treated with micrococcal nuclease (Worthington grade NFCP, electrophoretically pure) at 370 C (duck and chick nuclei), or at 00 C (SV40 chromosomes and DNA) in the presence of 2 mM CaCl2.…”