Rearrangement of nucleoli in hepatocytes stimulated for proliferation and enhancement of their transcription function

The nucleoli of normally functioning guinea-pig hepatocytes that have a nucleolonemal (strand-like) organization differ from identical nucleoli of other cells. Their nucleolonema consists as a rule of a fibrillar component with 45S RNA and is poor in granulas that contain pre-rNA molecules of an intermediate size and 28S rRNA, a dense fibrillar component with nascent rRNPs in its composition was not revealed. In hepatocytes stimulated by a 2/3 liver resection rearrangements in nucleoli were found. This brought to a conclusion that rRNA metabolism undergoes some changes. In 2.5 and 5 hours after the resection the hepatocytes' nucleoli were characteristic of a greater thickness of strands and a smaller size of vacuoles, appearance of distinct zones of the dense fibrillar component and an increased amount of RNP-granules. All these observations taken together point out at an increased synthesis and processing of rRNA at early stages of the prereplicative period. In 9 hours the character of changes in nucleoli was different: the vacuoles were considerably widened, whereas the thickness of strands that consisted of a well-expressed dense fibrillar, fibrillar and granular components was lesser. Such rearrangement points out at an increased transport of preribosomes from the nucleolus, a high level of synthesis and processing of nascent RNP-product being maintained. The changes of nucleolar RNP-component were followed by appearance of greater blocks of perinucleolar condensed chromatin, which may be connected with "cutting-off" some tissue-specific genes and initiation of functioning of the mitotic operon genes.     

Genetic transformation of mouse cultured cells with the help of high-velocity mechanical DNA injection

NIH 3T3 mouse cells were transfected by the plasmid pSV3neo (G418-resistant) with the help of high-velocity mechanical DNA injection based on the principle of bombarding cells with tungsten particles covered with the DNA. Stable transformants were obtained. Dot-hybridization and Southern analysis revealed the integration into the genome of 5-20 copies per cell of original plasmid DNA. The plasmid DNA was shown to have tandem organization.     

DNA synthesis in heterokaryons obtained by the fusion of polymorphonuclear leukocytes and cultured cells possessing different proliferative potentials

Heterokaryons between terminally differentiated polymorphonuclear leukocytes (PL) and culture cells of different proliferative potentials: mouse and rat embryo fibroblasts (EFM, EFR); immortal cells NIH 3T3 and E2; malignant cells NCC2, L929, He239 and SV 3T3,--were obtained by means of electrofusion. Radioautographic study of 3H-thymidine incorporation in the nuclei of heterokaryons showed that all the cells taken for fusion were able to induce reactivation of DNA synthesis in PL nuclei, however, with different rates: 7-37% for EFM and NIH 3T3 and 20-40% for malignant cells. The presence of oncogenes Elan in E2 cells and ras in NCC2 cells increased the rate of PL reactivation approximately twice as compared with the cells of original lines (EFR and NIH 3T3, correspondingly). In parallel to reactivation of DNA synthesis in PL nuclei inhibition of the synthesis in culture cell nuclei in the same heterokaryons was found. The rate of inhibition was about 70% for non-malignant and 23, 40 and 18% for NCC2, L and SV 3T3 cells, respectively. He239 cells, transformed by a temperature-dependent mutant of virus SV40 showed at permissive temperature the increased capacity of inducing reactivation of PL nuclei, though He239 cells susceptibility to inhibitory action of PL nuclei did not change with temperature. According to the behaviour in heterokaryons PL were found to be similar to chick erythrocytes, but differing from them by a pronounced inhibiting effect upon DNA synthesis in the nuclei of malignant cells.     

Cytogenetic analysis of forms produced by crossing hexaploid triticale with common wheat

Summary Hexaploid triticales were crossed with common wheats, and the resultant froms were selected for either triticale (AD 213/5-80) or common wheat (lines 381/80, 391/80, 393/80). The cytogenetic analysis showed that all forms differ in their chromosome composition. Triticale AD 213/5-80 and wheat line 381/80 were stable forms with 2n = 6x = 42. Lines 391/80 and 393/80 were cytologically unstable. In triticale AD 213/5-80, a 2R (2D) chromosome substitution was found. Each of the three wheat lines had a chromosome formed by the translocation of the short arm of IR into the long arm of the IB chromosome. In line 381/80, this chromosome seems to be inherited from the Kavkaz wheat variety. In lines 391/80 and 393/80, this chromosome apparently formed de novo since the parent forms did not have it. The karyotype of line 381/80 was found to contain rye chromosomes 4R/7R, 5R and 7R/4R. About 15% of the cells in line 391/80 contained an isochromosome for the 5R short arm and also a chromosome which arose from the translocation of the long arms of the 5D and 5R chromosomes. About one-third of the cells in the common wheat line 393/80 contained the 5R chromosome. This chromosome was normal or rearranged. Practical applications of the C-banding technique in the breeding of triticale is discussed.     

Changes in redox potentials during transitional processes in pure and mixed cultures of Escherichia coli and Serratia marcescens

There were studied transitional processes accompanying the beginning of growth under glucose addition and stopping of growth under glucose exhaustion in pure and mixed aerobic cultures of Escherichia coli and Serratia marcescens. Continued record of Eh, pH, and CO2 showed that these processes sharply differ from each other in their character in pure and mixed cultures, it is particularly related to the changes of the redox potential. There is no characteristic change in the redox potential in pure culture of E. coli at growth termination in the case when S. marcescens cells are present in the culture.     

A simple method of determination of antigenic determinants in proteins with known primary structure

A simple and rapid method is proposed for the localization of antigenic determinants in proteins of known primary structure exemplified by human myoglobin. The polypeptide chain of myoglobin was cleaved with BrCN (at Met residues) or with bromosuccinimide (at Trp and Tyr residues) under conditions which on average gave less than one scission per myoglobin molecule. The "single-hit" cleavage products were separated by gel electrophoresis and transferred to nitrocellulose by electroblotting. The peptides containing intact antigenic determinants were vizualized by immuno-peroxidase staining with four monoclonal anti-myoglobin antibodies. Comparison of the lengths of the immuno-reactive peptides with the known positions of methionine, tryptophan and tyrosine residues suggested that the four monoclonal antibodies were bound by myoglobin over the region Trp-14 to Met-55. As compared with other methods of localization, the method proposed is much faster and takes much lesser amount of protein.     

The short-term increase in extracellular potassium concentration causes DNA replication onset in density-inhibited cells.

The high potassium concentration effect on the human diploid fibroblasts (HDF) and 3T3 cells was investigated. The incubation of confluent cultures of HDF or 3T3 Swiss cells in the medium with 50 mM K+ for 35 min induced, 12 h later, the onset of DNA replication in a significant proportion of culture cell population. The same treatment had no effect upon the sparce cell cultures. No stimulation of DNA replication was observed in the absence of serum in culture medium.     

Increased susceptibility of activated rat liver chromatin to DNAse I

Rat liver chromatin activated by partial hepatectomy is more susceptible to the action of DNAse I than control chromatin isolated from intact liver. The study on the transfer of chromatin material to the acid-soluble fraction reveals a higher rate of activated chromatin degradation. Activated chromatin shows also an increased capacity for ethidium bromide (EB) binding as estimated from the isotherms of adsorption. The difference in EB binding between activated and control chromatin is abolished after DNAse I treatment. Conditions of mild digestion with DNAse I have been found under which the number of binding sites for EB per nucleotide decreases to almost the same level in activated and non-activated chromatin. The results suggest a preferential degradation of those DNA sequences in activated chromatin that are responsible for the increase in the ligand binding.     

Early changes in liver chromatin in response to partial hepatectomy]

Guinea-pig and mouse liver chromatin responds to the partial hepatectomy by an increase in binding of a basic dye acridine orange (AO) and by a decrease of its stability to heat in thermal denaturation test in situ. Degree of the changes in AO chromatin binding is identical in the cells of different ploidy and proportional to their DNA content. Treatment of the preparations by 0.6 M NaCl solutions under conditions bringing about the selective removal of histone H1 from the cells produces in vitro changes in DNA properties taking place in cells in vivo in the course of their activation. The treatment of cells with 0.35 M NaCl solution results in the disappearance of changes occurring in the chromatin of activated cells whereas the properties of control cells remain unchanged. The data obtained are interpreted as a result of the removal of some non-histone regulatory proteins from the chromatin of activated cells that is accompanied by changes in the character of DNA-histone interaction. At the time of maximum increase of AO binding a significant intensification of endogenous RNA polymerase activity was found, the incorporation of [3H] UTP in the nucleolus being higher than that in the extranucleolar part of the nucleus. High ionic strength in the incubation medium (0.4 M (NH4)2SO4) results in drastic increase of radioactive label in the nucleus and in the disappearance of differences between activated and non-activated chromatin. It is concluded that the intensification of RNA synthesis under the influence of proliferative stimulus is more likely dependent on the additional opening of DNA-matrix than on the direct activation of the enzyme.