To establish the conditions for the efficient transfer of foreign genes to baby hamster kidney (BHK) cell lines, we investigated the factors that affect the efficiency of transformation of thymidine kinase deficient (tk^-) BHK cells to the tk⁺ phenotype. Plasmid pAGO, which carries the herpes simplex virus (HSV) tk gene, was used as the donor DNA, and the frequencies of transformation of BHK and mouse L cells under various conditions were compared.
Although the optimal times of exposure to DNA were almost the same, the two kinds of cells had different cell density dependencies; transformation of BHK cells occurred only when a limited number of cells were seeded prior to DNA transfer, whereas there was a high frequency of transformation with mouse L cells over a broader range of cell density. The frequency of transformation increased as the amounts of pAGO DNA increased, and a clear dose dependence was found. The efficiency of the transformation for BHK cells was about 80 tk⁺ transformants per 4 × 10⁵ cells per 400 ng of pAGO DNA; about 50 times lower than the efficiency of transformation for mouse Ltk^-cells.

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Mouse myeloid leukemia (Ml) cells that were induced to differentiate with dexamethasone showed at least a 100-fold increase in lysozyme activity. This induced activity was distributed in various subcellular fractions, the molecular weights of which were nearly identical. The activity detected in the nuclear fraction (nearly one-third that of the total number of cells) could be extracted completely with 0.3 M NaCl or 0.4 N H₂SO₄, but not with 0.74 M perchloric acid. When nuclei were incubated with DNase I or micrococcal nuclease, the release of lysozyme showed a positive relation to increased digestion of DNA. The lysozyme, released by treatment with micrococcal nuclease, was distributed throughout the region of the multimers of nucleosomes after centrifugation on a 5-20 % linear sucrose gradient. Our results suggest that a part of the lysozyme activity induced during M1 cell differentiation may be associated with chromatin DNA.

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Cultures of a cloned epithelial cell lines derived from rat liver that synthesizes α-fetoprotein were used. Time courses of the appearance and distribution of types I and IV collagen, laminin and fibronectin in these cultures were assayed by the unlabeled peroxidase anti-peroxidase technique. Fibrillogenesis was monitored by the conventional phosphotungstic acid aniline-blue staining method for collagen fibrils.
In the initial stage of cell proliferation, the antigenecities of these four matrix proteins were found only in the interior of most cells. Later, laminin and fibronectin began to appear in the intercellular spaces, although types I and IV collagens still were confined within the cells. With the onset of fibrillo-genesis, extracellular deposition of types I and IV collagen began together with a relative decrease in the intensity of cellular staining. As fibrillogenesis progressed, these four matrix proteins gave more and more intense immuno-cytochemical reactions in the extracellular spaces, developing into the charac-teristic circular pattern of distribution. This process usually stopped when fibrillogenesis reached what appeared to be a static state. Immunocytochemical staining for the type I collagen of the preparations, then phosphotungstic acid aniline blue staining showed that two types of staining coincide. In contrast, none of three other matrix proteins showed complete codistribution with aniline blue positive fibrils. They were, however, located close to each other.
Our results show that cultured epithelial liver cells produce these matrix proteins from a time early in culture, and that the extracellular depositions of these proteins take place successively with some interrelation.

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Histone fractions prepared from various sources stimulated DNA synthesis in quiescent BALB/ 3T3 cells in sparse culture. Of the various sources investigated, the stimulatory activity was highest in the histone fraction prepared from rat Rhodamine fibrosarcoma. The maximal activity at optimal concentration of the histones from the tumor was about half that in the presence of an excess concentration of calf serum. The histone fraction was fractionated by column chromatography on Bio-Gel P-60. All histone species thus obtained had stimulatory activity for DNA synthesis.
Although protamine, poly-L-lysine and poly-L-arginine also stimulated DNA synthesis in BALB/ 3T3 cells, the former two polypeptides were less effective than any of the histones.
In addition, we found that the nonhistone protein fraction from tumor chromatin stimulated DNA synthesis in BALB/ 3T3 cells. The maximal activity at optimal concentration of nonhistone protein corresponded to that achieved by excess calf serum. From these findings, we postulate the role of a growth factor located in cell nuclei in controlling cell proliferation.

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Macromolecules which eliminate the effects of bovine serum albumin (BSA) to inhibit cell adhesion, were investigated using serum-free medium supplemented with 5 g/1 of BSA. Fibronectin (FN) produces adhesion in various types of cells-BHK-21, TE-1, FL, HeLa-S₃ and human diploid fibroblasts, while asdoes gelatin in fibroblasts and TE-1 cells. These two types which adhered to gelatin-coated dishes secreted sufficient amounts of cellular FN into the cultured medium to allow such adhesion. The dual effects of both FN and gelatin were exhibited only when added prior to BSA. This indi-cates that further adsorption of either FN or gelatin is inhibited when BSA has already been adsorbed on the substratum. FN may also act as an adhesive bridge between cells and gelatin-coated ornon-treated dishes.

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A nuclear matrix obtained from bovine lymphocytes contained a large number of small nuclear RNAs (snRNAs) : 30 % Ul +5.8SRNA, 65 U2RNA, 82 % U3RNA, 44 % U4RNA, 45 % U5RNA and 55 % U6RNA from isolated nuclei. These RNAs were tightly associated as a small nuclear RNA-protein complex (snRNPs) on the nuclear matrix, but they could be dissociated by treatment with high salt buffer or by sonication. When dissociated by this treatments, subsets of RNPs containing differentsnRNAs were found in the same fractions after centrifugation of a sucrose density gradient.
In contrast, snRNPs could be dissociated effectively by treatment with a low salt buffer containing 1 mM ATP, 1 mM DTT, 0.4 mM CaC1₂, 0.2 mM EDTA and 10 % (v/v) formamide. A large portion of the stractural proteins of this nuclear matrix were depolymerized and released from the matrix by this treatment. The dissociated snRNPs had different sedimentation velocities in the sucrose density gradient among snRNPs. When, instead of CaC1₂ the same concentration of MgCl₂ was added, the amount of released snRNPs decreased greatly. These results suggest that snRNPs bind to proteinaceous filaments of the nuclear matrix in diverse ways in spite of the similarities of the snRNP "core particles" themselves.

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Clean and large dividing cells of cultured lung epithelium of the Japanese newt were used to study physiological and morphological differences that exist between the metaphase and anaphase of mitosis. The response of the mitotic apparatus to pulse treatment with mitotic poisons (10μM of Colcemid or vinblastine) at room temperature was followed with polarization and phase-contrast microscopy.
In general, the length and birefringence of the spindles in both metaphase and anaphase gradually decreased on the pulse application of these drugs and finally disappeared. Chromosomes in metaphase in treated cells always maintained their shape and structure for a longer time than did those of the control. Chromosomes in anaphase in treated cells tended to change their structure and formresting nuclei or karyomeres.
These results support the belief that the physical properties of chromosomes are altered during the transition from metaphase to anaphase.

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Various inhibitors were microinjected into the cytoplasm of the plasmodium of Physarum polycephalum, and their effects were monitored by measurements of contractile activity, and the intracellular ATP and cytoplasm-ic Ca²⁺ concentrations. Fluoride and monoiodoacetate (glycolytic inhibitors) reduced the ATP by 50 %, but the contractional rhythm was unaffected. In contrast, azide and arsenate (respiratory inhibitors) and 2, 4-dinitrophenol (uncoupler) induced a transient decrease of 50 % in the ATP value and a 2-3-fold transient prolongation of the rhythm. A combined injection of glycolytic and respiratory inhibitors reduced the ATP to 10 % and led to complete ces-sation of the rhythm. P-chloromercuribenzoate (SH-blocking reagent) had similar effects as those of a respiratory inhibitor, but N-ethylmaleimide had no affect. Ruthenium red (inhibitor of mitochondrial Ca²⁺-uptake) did not affect the ATP value, but did bring about gradual prolongation of the rhythm accompanying an increase in the Ca²⁺ efflux into the cytoplasm.
These results are evidence that the time-keeping of contractional rhythm inPhysarum plasmodium is related primarily to mitochondrial activity.

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Characteristics of the (H⁺+K⁺)-ATPase from hog gastric mucosa were examined in relation to ATPase activity and proton uptake as functions of the osmotic states of the gastric vesicles. When osmotically expanded by the presence ofan internal, hyper concentration of NaCl, choline Cl, or sucrose, ATPase activity increased as the internal osmolality was increased with 15 mM of external KCl;but, no proton uptake was found. When the gastric vesicles had expanded because of the internal KCl, ATPase activity increased and was accompanied by proton uptake. This indicates that an idle, uncoupling mechanism of the ATPase worked in the absence of internal, transportable K⁺. Shrinkage of vesicles, caused by external sucrose, induced a decrease in ATPase activity in the presence of 15 mM of external KCl (the internal medium was sucrose only) that was not accompanied by proton uptake. Shrinkage due to NaCl induced greater inhibition than did sucrose, evidences that competitive binding of Na⁺ to the ATP site as well as through itsshrinking effect. The results of this study are important because the osmotic expansion of vesicles is thought to produce morphological changes in the apical membrane of parietal cells and the onset of acid secretion.

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At a nonpermissive temperature, premature chromosome con-densation is induced in the temperature-sensitive mutant from BHK21, tsBN2 (4). We fused tsBN2 cells that showed premature chromosome condensation with interphase tsBN2 cells and found that premature chromosome condensa-tion was induced in the interphase nuclei. This suggests that a "chromosome condensing factor(s)" which has been known as a mitotic factor(s) (1, 2) is produced in tsBN2 cells that have premature chromosome condensation.

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