Xenopus Erythrocytes Research Articles

Sub-nuclear fractionation: I. Procedure and characterization of fractions

A procedure for fractionation of nuclei from rat liver, Xenopus liver and Xenopus erythrocytes is described. It is based on mild sonication of isolated nuclei for 7–12 sec in a nearly isotonic medium, separation of nuclear sap and centrifugation on a discontinuous sucrose density gradient containing Na and K citrate. Nuclei are thus separated in a single operation into 8 fractions representing nucleoplasm, euchromatin, nucleoli, heterochromatin and nuclear membranes. The sub-nuclear fractions were characterized by chemical composition (DNA, protein, RNA and phospholipid), electron microscopy, thermal denaturation properties of chromatin, relative binding of 3 H-actinomycin D , polyacrylamide gel electrophoresis of nuclear proteins and titration of membranes against Triton X-100. Approx. 10% of total DNA was recovered as heterochromatin associated with membranes but the bulk of nuclear membranes co-sedimented with the major euchromatin zones. Subnuclear fractions prepared in this way retain virtually all the RNA polymerase activity bound to chromatin [41].

RNA synthesis in Xenopus erythrocytes

Synthesis of RNA has been studied in erythrocytes from Xenopus laevis, both in cultures of washed cells and in heparinised whole blood. RNA synthesis has been assessed by incorporation of tritiated uridine and detected both by autoradiography of the cells and scintillation counting of cell extracts. On average, 12% of the erythrocytes from a normal, adult Xenopus retain some RNA synthetic activity. Extraction of RNA from the crythrocytes followed by its characterisation by sucrose gradient centrifugation and polyacrylamide electrophoresis, reveals that most of the RNA is ribosomal (18 and 28S) and transfer (4S) and that the latter peak has the highest specific activity. The nucleus of the Xenopus erythrocyte probably makes the nearest approach to being metabolically inert of any known nucleus.

Observations on aspects of nutrition in Xenopacarus africanus (Ereynetidae: Trombidiformes).

Xenopacarus africanus is found in the nasal passages of the African clawed toad, Xenopus laevis. The alimentary canal of the mite includes a powerful, muscular, sucking pharynx, a long narrow oesophagus and a ventriculus from which arise a number of paired lateral caeca. The food of the mite consists of blood obtained from vessels in the head of the toad. Since no intact Xenopus erythrocytes were found in the gut of the mite, their breakdown must either be rapid or take place outside the body as a result of the salivary activities of the mite. Although some digestion may occur within the lumen of the caeca it is thought that the haemoglobin or a breakdown product of this is absorbed by the cells lining the gut caeca and that within these cells, or parts of them which become detached and pass into the lumen, the main stages of digestion occur. The degradation of haemoglobin results in the production of large amounts of haematin.

Histone differentiation and nuclear activity.

When fast green and eosin are used in combination to stain histones, nuclei display different affinities toward the dyes, some binding fast green exclusively, others binding eosin exclusively, and still others, both stains. In a given tissue, the frequencies of nuclei exhibiting the different colors remain fairly constant over a wide range of staining conditions. Nuclei of cells of the same type may stain differently, but when they are in the same stage of development or state of activity they tend to stain alike. Xenopus erythrocyte nuclei stain bright pink. Condensed mitotic and meiotic chromosomes stain purple. In the grasshopper spermatocyte, the main body of the interphase nucleus stains bright green, but the condensed chromosome stains purple. The mole crab sperm contains several distinct histone-like proteins, that differ in their amino acid compositions, within separate areas of the cell. In these sperms, the lysine-rich histones bind eosin, while the protamine-like protein and arginine-rich histone bind fast green. In general, the eosin and fast green bind preferentially to the lysine and arginine rich histones respectively, when the dyes are permitted to compete with one another. In several systems, including spermiogenesis and erythropoiesis, the aquisition of an eosinophilic component by the nuclei accompanies the slowing of RNA synthesis, and it is suggested that there may be a causal relationship between the two events, the eosinophilic histone effecting RNA synthesis within the nucleus as a whole.