Avian Erythroid Cells Research Articles

Nucleotide polymerases in the developing avian erythrocyte.

Avian erythroid cells were separated into five developmental stages by sedimentation on discontinuous isotonic albumin gradients. Solubilized enzyme activities from whole cells were partially purified and characterized by ion exchange and ion filtration chromatography and velocity sedimenttation analysis. Three nucleotide polymerase types were investigated: (a) DNA-dependent RNA polymerases; (b) RNA-dependent terminal ribonucleotidyltransferases, and (c) DNA-dependent DNA polymerases. The two characteristic forms of eucaryotic DNA-dependent RNA polymerases, polymerase I (nucleolar) and polymerase II (nucleoplasmic), were identified. Polymerase III was only marginally detectable even in the earliest developmental populations. At least two species of RNA-dependent terminal ribosyltransferases were present. One apparently was the poly(A) polymerase observed in other systems. The other terminal transferase was present in two chromatographic forms, required an RNA primer, and used UTP and/or CTP as particularly efficient substrates. Three DNA polymerase activities were resolved, two of which were characteristic of the alpha and beta DNA polymerases described in other eucaryotic systems. The third polymerase was not the gamma polymerase but a separate entity. Poly(dC)-dependent RNA polymerase activity, associated with the alpha polymerase, was relatively enriched in the third DNA polymerase species. The activity levels of the nucleotide polymerases were monitored as a function of red cell maturation. Characteristic declining patterns of activity were obtained for each enzyme which correlate well with the synthetic rates of their in vivo products where these are known. These results correlate well with the synthetic rates of their in vivo products where these are known. These results are consistent with the postulate that the general transcriptive and replicative control processes operating during development may involve changes in the level of the requisite polymerases.

Selective synthesis and modification of nuclear proteins during maturation of avian erythroid cells

The synthesis of the nuclear proteins of duck erythroid cells at different stages of maturation has been investigated. Synthesis of histone fractions H1, H2a, H2b, H3, and H4 is restricted to the erythroblasts, while synthesis of H5 can be detected even at later stages of maturation after DNA synthesis has ceased. The synthesis of nonhistone nuclear proteins (NHNP), on the other hand, occurs in cells at all stages of maturation although their rates of synthesis decline as the cells mature. The same size classes of NHNP appear to be synthesized in erythroblasts and in early- and midpolychromatic erythrocytes. In late polychromatic erythrocytes the synthesis of a new group of NHNP of molecular weights ranging from 54,000 to 130,000 was observed. This group of proteins does not accumulate in the mature erythrocyte, indicating that their relative proportions are very small. Turnover of histone-bound phosphate was found to occur mainly at the erythroblast stage, except for histone H2a which was actively phosphorylated even at more advanced stages of maturation. Phosphorylation of most of the histones appears to be coupled to histone (and coordinate DNA) synthesis. Incorporation of radioactive acetate into histones occurs at all stages, but the rate of acetylation decreases four- to fivefold with maturation. Although the RNA synthetic activity of erythroid cells also decreases with age, experiments involving the use of RNA polymerase inhibitors suggest that the mechanisms that control RNA synthesis and histone acetylation are not tightly coupled.

Histone proteases of avian erythroid cells

Protease activity associated with avian erythroid chromatin has been studied by gel electrophoresis of histones. Histone degradation is minimal at neutral pH, but is readily detected when chromatin is incubated at pH 3, and is evident to a lesser extent at pH 9. As a result of the pH 3 activity, the f1 and f2 chistones are preferentially degraded when the histone complement is DNA-bound, but these histones are relatively resistant to attack when present as free histone. The pH 3 activity reported here has properties similar to those of neutral histone proteases from other tissues, except that it is not inhibited by bisulphite. Added exogenous proteins are not degraded. The activity of avian erythroid histone protease decreases as maturation of the cells proceeds. Since we have previously shown that turnover of DNA-bound f2c histone occurs in reticulocytes and histone synthesis is absent in erythrocytes, it is possible that the histone protease described here may be involved in f2c histone turnover.

Preparation of membrane-free chromatin bodies from avian erythroid cells and analysis of chromatin acidic proteins.

A primary objective, realized in this study, was the preparation from avian erythroid cells of chromatin free of contaminating membrane, as a prerequisiste to the study of chromatin acidic proteins from cells throughout the maturation pathway. Cells are lysed in saponin (S), washed in Nonidet-P40 (N), and plasma membrane removed by high-speed rotating knives (K). Purified SNK nuclear bodies are recovered free of membrane after centrifugation through 2.35 M sucrose. The chromatin acidic proteins from such preparations of the three major avian erythroid cell types were studied. Reticulocyte SNK chromatin was compared with reticulocyte chromatin derived from saponin lysis of cells and subsequent dispersion in EDTA solutions (Harlow et al. (1972), Cell Differ. 2, 341). The dispersed preparation has a lower acidic protein/DNA ratio, but the pattern of these proteins is more complex, presumably due to the contaminating membrane. In examining SNK acidic proteins throughout the maturation pathway it is clear that there are quantitative and qualitative differences. In the dividing erythroblast, the pattern of proteins is complex and the amount relative to DNA is 1.25:1.0. For nondividing, but transcriptionally active reticulocytes and also for transcriptionally inactive erythrocytes, the pattern is very much simpler, being dominated by three bands visible on sodium dodecyl sulfate polyacrylamide gels. The ratios of chromatin acidic proteins in these preparations relative to DNA are 0.69:1.0 and 0.36:1.0, respectively. These results, obtained with purified populations of cells from a single cell line, indicate a strong positive correlation between the transcriptional activity of the cell and both the amount and complexity of non-histone proteins associated with chromatin. The correlation does not indicate whether the proteins are the cause or result of increased transcription.

Characterization of chromatin-bound erythrocyte histone V (f2c). Synthesis, acetylation, and phosphorylation

Synthesis and enzymatic modification of histone V was 1 order of magnitude lower in mature gander erythrocytes as compared with immature enriched cells hwich were capable of DNA synthesis. Application of shallow, linear gradient chromatography was used to demonstrate qualitative changes as well. This technique permitted the separation of newly synthesized and phosphorylated histone V from older, less phosphorylated molecules but did not discriminate between acetylated species. The most easily eluted fractions were those most recently synthesized, acetylated, and phosphorylated. While lysine chased into the other subfractions of histone V, phosphate did not, indicating a dephosphorylation step in the immature cells. Acetylation of histone V which occurs at a very low level was closely related to its synthesis. No differences in molecular weights or amino acid compositions were apparent, and behavior on polyacrylamide gels was similar to whole histone V. It is proposed that phosphorylation of histone V may play an important role in the modulation of the effect of histone V in immature cells on condensation and template restriction of chromatin which occurs in the terminal stages of differentiation of the avian erythroid cells.

Changes in Histone Acetyl Content and in Nuclear Non-Histone Protein Composition of Avian Erythroid Cells at Different Stages of Maturation

Abstract Duck erythroid cells were fractionated by centrifugation in bovine serum albumin density gradients. Differences in cell density were correlated with the stage of maturation as determined by biochemical and morphological parameters. Avian erythroid cells increase in density as they mature. Cell populations corresponding to known stages in differentiation were compared with regard to histone composition and degree of acetylation, nature and amount of the non-histone nuclear proteins, and DNA and RNA synthetic capacities. Although the total histone to DNA ratio of the chromatin does not vary appreciably during maturation, the relative proportion of the erythroid cell-specific histone F2c increases at least 2-fold with the increasing age of the cell, whereas the other histones, except F2a2, decrease. The degree of acetylation of histones F2a1 and F3 decreases with the increasing age of the cell. The non-histone protein complement of the erythroid cell nucleus decreases 6-fold during maturation. Analysis of the non-histone proteins in polyacrylamide gels containing sodium dodecyl sulfate shows a limited heterogeneity and a remarkable similarity in their electrophoretic patterns at different stages in maturation. Quantitative rather than qualitative changes predominate as maturation proceeds. The decline in non-histone protein content with age is striking particularly in the lower molecular weight components. However, a protein band of molecular weight 55,000 is conserved selectively during maturation and it represents the predominant component of the non-histone nuclear proteins of the mature erythrocyte. The nucleoprotein changes occurring during erythrocyte maturation parallel a decrease in the amount of chromatin-associated RNA and the rate of RNA synthesis. The average RNA synthetic capacity of the isolated erythroblasts and early polychromatic erythrocytes is about 8 times higher than that of the mature erythrocyte fraction.

Membrane — A major source of chromatin-associated RNA and non-histone proteins in avian erythroid cells

Crude chromatin (CC) from avian reticulocytes and erythrocytes has been fractionated on 1.7 M sucrose into a membrane component (M) and purified chromatin (X) by standard procedures. As expected, histone behaves as a DNA-associated entity, being found predominantly in purified chromatin. In contrast, the non-histone components, both RNA and protein, fractionate as membrane-associated moieties in vitro. For reticulocyte chromatin 70–80% of membrane (cholesterol and phospholipid estimations), of non-histone protein and chromatin-associated RNA are found in fraction M and the remainder in X. For erythrocyte chromatin about 50% of membrane and non-histone protein is in each of the fractions M and X. Most, if not all of the non-histone components of purified avian erythroid chromatin can be accounted for as membrane-associated components.

DNA polymerase in avian erythroid cells.

ABSTRACT The level and properties of DNA polymerase activity assayable in extracts of avian erythroid cells was studied. The enzyme was detectable in the dividing cells (erythroblasts) of the erythropoietic series and also the immature non-dividing erythrocytes. It could not be assayed in mature erythrocytes. Investigations showed that activity began to decline at the time of the last cell division of the erythroid series. Properties of the enzyme did change in different cell types; however, the changes did not correlate with cessation of DNA synthesis. Some preliminary results on DNA synthesis by isolated nuclei are also reported and these showed that only nuclei from erythroblasts could synthesize DNA in vitro in the absence of primer.

Phosphate incorporation into “nuclear” residual proteins of goose erythrocytes

Minor fractions of “residual proteins” from erythroblast-enriched avian erythroid cells were found to incorporate significantly larger amounts of radiophosphate than similar fractions from mature erythrocytes. This higher level of incorporation could not be detected in the bulk, unfractionated residual proteins from cell populations, respectively, enriched in erythroblasts, reticulocytes, or mature erythrocytes, probably because of variable amounts of phosphate-free protein. It was confirmed that when avian erythroid cells incorporated radiophosphate, specific activities of chromosomal “residual proteins” were orders of magnitude greater than those of histones, although levels of alkali-labile phosphate were only a fewfold higher. The phosphate incorporated into the residual proteins was not in the form of free nucleotide, inorganic phosphate, phospholipid or nucleic acid. The prospect of contamination by unextracted histones was reduced by washing the nuclear preparations with acidified organic solvents, in which some histones are soluble. Furthermore, histones and hemoglobin can be distinguished from residual proteins by characteristic mobilities in polyacrylamide gel electrophoresis with sodium dodecylsulfate. The radiophosphate bound by residual proteins was distributed among virtually all of the components resolved by zonal electrophoresis, including bands characteristic of erythrocytes. At least one major radioactive peak, extracted in phenol at pH 9.4, did not migrate with a pronounced band of protein and other components of high specific activities may be resolved from concomitant proteins on sequential electrophoresis in different systems. The role of increased protein phosphorylation in immature erythroid cells is unknown.

Deoxythymidine metabolism in avian erythroid cells.

ABSTRACT The ability of dividing and non-dividing cells of the avian erythropoietic series to phosphorylate [3H]deoxythymidine and [3H]uridine has been studied. Enzyme levels of deoxythymidine kinase were also assayed. Both dividing and immature non-dividing erythroid cells phosphorylated uridine to uridine triphosphate (UTP). However, only cells able to synthesize DNA could phosphorylate deoxythymidine to the triphosphate; also these cells were the only ones to show detectable deoxythymidine kinase activities.

Synthesis and turnover of DNA-bound histone during maturation of avian red blood cells

We have investigated the possible relation between histone metabolism and the gradual decline in capacity for RNA and protein synthesis during the maturation of non-dividing avian erythroid cells. The central point examined was whether net addition of histone to DNA occurred during this process. If histone did accumulate on DNA during erythroid cell maturation, we predicted that radioactivity (from 14C-labelled amino acids) incorporated into histone by polychromatic erythrocytes (non-dividing cells still active in RNA and protein synthesis and destined to mature to inactive erythrocytes) should be metabolically stable. Pulse-chase experiments with cells cultured in vitro were carried out to test this prediction. In dividing avian erythroid cells (erythroblasts), 14C-labelled amino acids were incorporated into all histone species. However, in non-dividing polychromatic erythrocytes the only histone labelled was the f2c histone which is tissue-specific to avian erythroid cells. In pulse-chase experiments the radioactivity in DNA-bound f2c histone was found to decay (in the chase period) at a rate which approximated the rate at which it was initially incorporated. Radioactivity in cytoplasmic proteins (mainly haemoglobin) was completely stable during the entire chase period. We suggest that the metabolism of f2c histone in non-dividing avian red blood cells may be an example of a more general phenomenon for other histones (particularly f 1 histones) in other tissues, and that the important feature which relates histones to the over-all potential of chromatin for transcription is not the static level of histone but rather the dynamic state of its synthesis and its release from DNA.

DNA synthesis in purified populations of avian erythroid cells.

ABSTRACT By the use of equilibrium density-gradient centrifugation erythroblasts and early polychromatic erythrocytes have been isolated from avian anaemic bone marrow. Cells from both the unfractionated and purified preparations have been characterized in terms of their histological type, size, haemoglobin content and ability to synthesize DNA. Erythroblasts were the only cells to synthesize DNA and it appeared that their progeny, the polychromatic erythrocyte, failed to enter a new S’ phase. The experimental system described allows biochemical characterization of earlier stages of avian erythropoiesis than has previously been possible.

Characterization of DNA-bound histone in the cells of the avian erythropoietic series.

ABSTRACT The distribution of DNA-bound histone from purified cell populations of the avian erythroid cell series was studied to examine the possible relationship between these molecules and the in vivo activity of cells. High-resolution polyacrylamide gel electrophoresis of histones indicated that the 3 main cell types, namely, erythrocytes (inactive in macromolecular synthesis), polychromatic erythrocytes (active in RNA and protein synthesis) and erythroblasts (dividing cells) all contained the same histone components. This result is contrary to previous reports that the f2c histone (characteristic of avian erythroid cells) was absent from erythroblasts; in addition it does not support the proposition that dividing cells contain a uniqueμ histone component. Quantitation of histone analyses showed that erythroblasts contain relatively less f2c histone than the non-dividing cells of the series and that there was a slight redistribution ofμ histone components between polychromatic and mature erythrocytes.