Synthesis and degradation of H1 histone subtypes in mouse lymphoma L5178Y cells

H1 histone of mouse lymphoma L5178Y was fractionated into five subtypes, I-V, by Bio-Rex 70 column chromatography. The rates of synthesis of subtypes III and V were higher than those of I, II, and IV, as determined by the measurement of [3H]lysine incorporation. The degradation of the subtype was estimated assuming first order kinetics; subtypes III and V had half-lives of 18 h and 25 h, respectively, and the three other subtypes all had half-lives of 63 h. The syntheses of these subtypes during the cell cycle were examined using synchronized cultures. The syntheses of subtypes I, II, and IV started at the beginning of S phase, whereas those of III and V started in mid-S phase. The syntheses of III and V were at least 1.5-2 times more rapid than those of I, II, and IV, and their active synthesis was accompanied by their rapid degradation. The five subtypes of H1 were further characterized in relation to phosphorylation. Each showed characteristic differences in its synthetic pattern or phosphorylation, and we concluded that each H1 subtype has its own specific function at least in the process of replication of chromatin.     

Behavior in two-phase aqueous polymer systems of L5178Y mouse leukemic cells : II. The lag and exponential phases of growth

The behavior of lag and exponential growth phase L5178Y mouse leukemic cells under normal and prolonged lag phase conditions with respect to partition in aqueous dextran — polyethylene glycol polymer systems has been studied. ‘Backculture’ of early stationary cells into fresh growth medium is accompanied by a decrease in partition ratio from 0.52 to 0.11. The partition ratio remains depressed for a time considerably longer than the duration of lag phase but rises rapidly and returns to its former value as the cells reach late exponential/early stationary phase. If lag phase is prolonged, the time for which the partition ratio remains depressed is also prolonged. In the exponential phase following a prolonged lag phase, the partition ratio rises at a rate slower than during a normal exponential phase and does not reach the same magnitude for the same position in the cycle. Net negative surface charge as measured by particle microelectrophoresis does not change appreciably throughout the growth cycle. The results suggest that the sequence of events at the cell surface on a populational basis which contribute to the partitioning behavior is possibly predetermined or programmed at the time of transfer into fresh medium. The results further substantiate the technique of aqueous polymer partitioning as being the most sensitive method available for monitoring subtle changes in plasma membrane properties during the cell growth cycle.     

Amino acid content of L5178Y and L5178Y/L-ASE cells after L-asparaginase treatment

The amino acid contents of tumor cells that are either sensitive or resistant to treatment with L-asparaginase were measured. These amino acid concentrations were measured as a function of incubation time with L-asparaginase or as a function of the L-asparaginase dose. The cell types compared were the mouse leukemia lines L5178Y (sensitive to L-asparaginase treatment) and L5178Y/L-ASE (resistant to L-asparaginase treatment). Upon L-asparaginase treatment both cell lines lost most of their cellular asparagine but, whereas the resistant cells exhibited the ability to rebound to about 50% of initial values, the sensitive cells did not. While previous work had suggested that asparagine-dependent glycine synthesis was essential for sensitive cells (but not in resistant cells), we found no difference in the glycine content of either of the two cell lines as a function of either time or dose that would support this hypothesis. Major differences between the two cell lines were seen in the content of the essential amino acids before treatment with L-asparaginase. After incubation without L-asparaginase the contents of the two cell lines became similar. These results are discussed in terms of possible mechanisms of L-asparaginase sensitivity and resistance.     

Isolation and characterisation of five histone H1 subtypes from adult rat liver

A procedure is described for quantitative purification of H10 and five H1-1 subtypes--named H1-1a to e--from adult rat liver by reverse-phase high-pressure liquid chromatography. Milligram amounts of each fraction have been obtained. The H1-1a subtype shows a very high lysine content (34%) and H1-1d subtype has an amino-acid composition close to that of H10, but its electrophoretic mobility is different. Salt dependent folding of these subtypes has been studied by circular dichroism. In the presence of 2 or 10 mM sodium phosphate buffers at pH 7.5, H1-1a shows the lowest alpha-helix content. In phosphate-buffer containing 1 M NaCl the number of residues in alpha-helix for all the subtypes rises to 9-10%. Partial cleavage of these subtypes by endoproteinase Glu-C produce three main peptides arising from C-terminal domains. The interaction of the H1-1 subtypes with 196 basepairs linear DNA, purified from rat liver chromatin by high-pressure ion-exchange liquid chromatography, has for consequences a modification of the patterns of digestion: partial proteolysis of the H1-1a and H1-1b subtypes shows differences in the presence or in absence of DNA; on the contrary, H1-1c and H1-1d seem to have the same organization. So these subtypes may play a role in the differential packing of specific region of chromatin.     

Sequence complexity of histone H1 subtypes

H1 subtypes are involved in chromatin higher-order structure and gene regulation. H1 has a characteristic three-domain structure. We studied the length variation of the available H1 subtypes and showed that the length of the N-terminal and C-terminal domains was more variable than that of the central domain. The N-terminal and C-terminal domains were of low sequence complexity both at the nucleotide and at the amino acid level, whereas the globular domain was of high complexity. In most subtypes, low complexity was due only to cryptic simplicity, which reflects the clustering of a number of short and often imperfect sequence motifs. However, a subset of subtypes from eubacteria, plants, and invertebrates contained tandem repeats of short amino acid motifs (four to 12 residues), which could amount to a large proportion of the terminal domains. In addition, some other subtypes, such as those of Drosophila and mammalian H1t, were only marginally simple. The coexistence of these three kinds of subtypes suggests that the terminal domains could have originated in the amplification of short sequence motifs, which would then have evolved by point mutation and further slippage.     

Protein synthesis during the cell cycle of L5178Y cells

There are two peaks of ³H-leucine incorporation in the cell cycle of L5178Y cells. The first, during S stage, corresponds to a peak of ³H-leucine incorporation into the nuclear fraction. The second, during S or early G2, corresponds to a peak of ³H-leucine incorporation into the mitochondrial fraction. The rate of protein synthesis is unique for the proteins from each of the four fractions, nuclear, mitochondrial, microsomal, and soluble.The SDS polyacrylamide-gel electrophoretic patterns of ³H-leucine incorporation were different among three subcellular fractions: nuclear, mitochondrial, and microsomal + soluble. However, the incorporation pattern for each fraction remains qualitatively the same throughout the cell cycle.     

Cellular growth control: properties of a unique adhering derivative of L5178Y cells

A new cell line derived from L5178Y murine lymphoma cells with unique properties is described. The cells, L5178Y Adh, grow simultaneously in suspension culture and attached to a substratum but not in contact with each other. Limitation of growth of the cells in either the suspended state or attached to substratum is not linked to a limiting surface or cell density in a manner analogous to “contact inhibition.” The adhering and non-adhering cells grow with different doubling times. Cell size analysis indicates that initial gravity settling of the inoculum determines which cells attach. Contact of these cells' external surface with substratum may then initiate events which ensure attachment of adhering populations.     

Effects of temperature on growth rate of cultured mammalian cells (L5178Y)

L5178Y cells were cultured in vitro at various temperatures. When the cells were in the exponential growth phase, the cells were in the "steady state of growth," i.e., the fraction of cells in the G₁, S, G₂, and M stages and the durations of each stage were constant. The life cycle analysis of the cells in the steady state of growth demonstrated that the G₁ stage and the S stage were affected the most by variation of temperature, and suggested that these two stages have considerable influence on the growth rate of the L5178Y cells. The calculated activation energies were positive in each stage of the life cycle, whereas the entropies of activation were negative throughout. The possible significance of these findings in our search for the regulatory mechanisms of cell growth is discussed.     

Genotoxicity of gamma-irradiation in L5178Y mouse lymphoma cells

The ability of gamma-irradiation to induce gene mutation at the thymidine kinase locus and gross chromosome aberrations in L5178Y TK+/- 3.7.2C mouse lymphoma cells was evaluated. Positive results were obtained for both end-points. The majority of mutants were found to be small-colony mutants which correlated with the induction of gross chromosome aberrations.     

Studies on three mutagen-sensitive mutants of mouse L5178Y cells. I. Characterization of the hybrids between L5178Y and mutagen-sensitive mutants

Three mutagen-sensitive mutants, MS-1, M10 and Q31, have been isolated from mouse L5178Y cells. MS-1 cells are sensitive to methyl methanesulfonate (MMS), M10 cells are cross-sensitive to X-rays, MMS and 4-nitroquinoline 1-oxide (4NQO), and Q31 cells are cross-sensitive to UV and 4NQO. Lines resistant to 6-thioguanine (TGr) and 5-bromo-2'-deoxyuridine (BUr) were isolated from L5178Y and these three mutagen -sensitive mutants. All the TGr lines were sensitive to 5-bromo-2'-deoxyuridine and HAT medium and all the BUr lines were sensitive to 6-thioguanine and HAT medium. The hybrids homozygous for the mutagen-sensitive markers showed nearly the same sensitivity to UV, 4NQO, X-rays and MMS as their parental TGr and BUr lines. The hybrids constructed by fusing L5178Y BUr and TGr lines from each of MS-1, M10 and Q31 displayed the normal UV, X-ray and MMS resistancy of L5178Y cells. Thus the UV-, X-ray- and MMS-sensitive markers in MS-1, M10 and Q31 were recessive in somatic cell hybrids. The 4NQO-sensitive phenotype, however, behaved codominantly in somatic cell hybrids.     

Specificity of transport of bleomycin and cobalt-bleomycin in L5178Y cells

The mechanism of transport of [³H]peplomycin (PEP), a new member of bleomycin group antibiotics, was studied in cultured L5178Y mouse leukemic cells. Cobalt ions enhanced the uptake of PEP, but Cu, Zn, Fe(II) and Fe(III) had no effect. The initial rate of uptake of cobalt chelated PEP [PEP(Co)] was several times higher than that of free or Cu-chelated PEP and was temperature independent. A double reciprocal plot of the data demonstrated both saturable (K_m = 4.5 μM, V_max = 1.3 × 10^?18 mole/min/cell) and non-saturable components of the uptake of PEP(Co). The saturable component was inhibited specifically by cobalt chelated bleomycin analogs. PEP-chelates with metals other than cobalt, such as PEP(Cu) were metabolically unstable. These results suggest that bleomycin enters into cells as a metal chelate through a specific transport site.