Nucleoproteins can be extracted and isolated from the nuclei of animal and plant tissues by the same procedure, somewhat modified, that is effective in preparing the nucleoprotamines of sperm (1, 2). In this paper the preparation of nuclear nucleoproteins from animal and plant tissues and from bacterial cells is described and some results of an investigation of these substances are given. Nucleoproteins are also present in the cytoplasm of animal and plant tissues. It is an important step in the isolation of nucleoproteins to separate those occurring in the nucleus from those of the cytoplasm. This can be accomplished in two ways: by chemical procedures of fractionation and by methods in which well formed nuclei or chromosomes are isolated from the cytoplasm before extraction is begun. These two methods of preparation have in practice much in common. Although many bacteria may not have distinctly formed nuclei, they do have both the nucleoproteins found in the nuclei and those found in the cytoplasm of cells in which the distinction between nucleus and cytoplasm can be made morphologically. In this paper the preparation of what might be called the nuclear nucleoproteins of bacteria is described and it will be seen that this preparation was made possible by the experience gained by working with animal and plant ceils. Although this paper is concerned primarily with nuclear nucleoproteins, the first steps in the isolation of cytoplasmic nucleoproteins are outlined.Animal and plant tissues in general have not appeared to be favorable material for investigation of nuclear constituents because in so many tissues nuclei are embedded in relatively large masses of cytoplasm. For this reason Miescher (8), Kossel (3), and other leading investigators preferred to work on sperm, cells in which there is little cytoplasm. The thymus gland is an animal tissue in which nuclei form an unusually large fraction of the total volume, and on the nuclear constituents of the thymus there are the important investigations of Lilienfeld (27), Huiskamp (7), Bang (7a), Hammarsten (22), and others. These investigators did not use strong acid and alkali, the reagents by means of which the somewhat disintegrated nuclear constituents of sperm had been isolated; instead, in extractions from the thymus nothing more drastic than distilled water was used. The consequences of this mild treatment were that nucleic
The history of the cytoplasmic components in the spermatogenesis of Gerris is, in general, like that which has been described in the Pentatomidae. The observations of fixed material have been checked by extensive studies of freshly teased preparations. During the spermatocyte growth period the chondriosomes undergo considerable increase in mass. During the maturation divisions the chondriosomes are remarkably constant in orientation with respect to the centrioles. The nebenkern arises by fusion of chondriosomes differentiated into chromophilic and chromophobic portions. The Golgi bodies of the earlier spermatocytes are vesicular bodies, the peripheries of which are osmiophilic. These are not visible in fresh preparations, but the masses resulting from their fusion in the late prophase of the first division are visible in the unfixed cells. The non‐osmiophilic material inside these masses stains with neutral red in fresh preparations. Only the osmiophilic part of the Golgi masses is involved in the fragmentation to form dictyosomes. There is very suggestive evidence that the process of acrosome synthesis largely takes place inside the sac‐like acroblast (Golgi apparatus). In the spermatid, material which stains, in fresh preparations, like the acroblast is never seen, except inside or attached to the acroblast, where it appears in the form of small spheres of ‘pro‐acrosomic’ material, which fuse to form the acrosome.
We have prepared nucleoproteins from a wide variety of animal cellsfrom mammalian liver, kidney, pancreas, spleen, thymus, brain, from the liver, spleen and blood cells of the dogfish, and from the sperm of the trout, shad, frog and sea urchin. These nucleoproteins are located in the nuclei of the cells from which they are derived. In this paper we shall briefly describe the method of preparation, some properties of the nucleoproteins and the evidence that they are in fact derived from cell nuclei.I. Preparation.-To extract these nucleoproteins from the cell and to separate them from other cellular constituents nothing more drastic is used than neutral sodium chloride solutions of varying concentrations. Before extraction, much cytoplasmic material is removed by thoroughly washing the minced tissue with physiological saline. From liver more than 60 per cent of all the protein present can be removed in this manner without destroying the main outlines of cell structure. The washed tissue is then extracted with 1 M NaCl (2 M NaCl is needed for extraction of seaurchin sperm). As soon as the more concentrated salt solution is added the mixture becomes exceedingly viscous. By centrifugation at high speed (10,000 to 12,000 r. p. m.) a viscous, slightly opalescent supernatant fluid is obtained. The supernatant fluid is viscous because of the nucleoprotein dissolved in it. When this solution is added to six volumes of water the nucleoprotein precipitates in a fibrous mass, settling rapidly so that the supernatant fluid can be syphoned off. The precipitate is washed with 0.14 M NaCl and then redissolved in 1 M NaCl. The solution is centrifuged at high speed to remove any suspended material. The nucleoprotein is reprecipitated by pouring into six volumes of water. If the mixture is stirred with a rod having a crook at its end, the fibrous material generally winds around the rod and adheres to it when the rod is transferred to another vessel (Plate I). The nucleoprotein is again dissolved, centrifuged and precipitated. At this point the preparation is frequently considered to be finished, although purification can be carried further.For further purification advantage is taken of the unusual solubility of the nucleoprotein. It is soluble in 1 M NaCl, insoluble in 0.14 M NaCl and soluble again when the salt concentration is reduced to approximately 0.02 M. When a solution of nucleoprotein in 1 M NaCl is placed in a cellophane tube and dialyzed against water the nucleoprotein first precipitates and then tends to redissolve as the salt concentration within the 344 PROC, N. A. S.
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