Isolation of a G0-specific ts mutant from a Fischer rat cell line, 3Y1

A ts mutant clone, tsJT60, was isolated from Fisher rat cell line, 3Y1. During the exponential growth at both 34 and 39.5 degrees C, tsJT60 did not appear as ts mutant cells. However, once entered resting state (G0) under serum deprivation at the confluent state, they could re-enter S phase at 34 degrees C but could not at 39.5 degrees C following the stimulation of cells either by the addition of fetal bovine serum or by trypsinization and replating. These and other results suggested that tsJT60 is a G0-specific ts mutant, i.e., the cells have ts defect(s) in the function which is required for the stimulation from the resting state to S phase but not for the progression of the cell cycle in an exponential growth phase.     

A gene function required for cell cycle progression during the G1 portion of the cell cycle and for maintenance of macronuclear DNA synthesis in Paramecium tetraurelia

The ccl mutation in Paramecium tetraurelia reversibly and rapidly blocks cell cycle progression and DNA synthesis at the restrictive temperature. Progression through the cell cycle is blocked during both the G1 and S portions of the cell cycle, while at the restrictive temperature there is neither residual cell cycle progression nor induction of excess delay of subsequent cell cycle events. DNA synthesis activity is reduced to 50% of the normal level in about 5 min and is completely blocked at 30 min after a shift to restrictive temperature. On return to permissive conditions, DNA synthesis is reactivated with similar kinetics.     

Fractionation and characterization of DNA at sites of replication from rat liver nuclei

Regenerating rat liver nuclei when sonicated and centrifuged in a Cs2SO4 gradient were fractionated into three distinct bands. These bands were designated as light band (LB), middle band (MB), and heavy band (HB) according to their density. LB and MB were shown to consist of large granular particles with varying electron densities, but LB also contained remnants of nuclear membrane. When analysed by gel electrophoresis, LB and MB displayed more than 35 bands of proteins. The third fraction, HB, consisted largely of small chromatin fibers and its proteins were predominantly the four histones of the nucleosomal core particle. Following short pulses with [3H]thymidine in vivo, the specific activity of DNA in LB and MB was significantly higher than that of bulk DNA contained in HB. DNA in all three fractions became equally labelled as the duration of the labelling interval increased beyond 30 min. Newly synthesized DNA was characterized by electrophoresis on analytical 1.7% acrylamide -0.5% agarose composite gels. After a 1-min labelling interval in vivo, 17% of the rapidly labelled DNA from LB and MB was stationary at the gel origin like replication forks from E. coli, but only 3% of HB DNA had zero mobility. Electron microscopy confirmed the presence of DNA replication forks in LB, MB, and HB. With increasing time of synthesis the proportion of labelled DNA exhibiting zero mobility decreased in all three fractions. Denaturation of DNA or digestion of single-stranded DNA with S1 nuclease released newly synthesized DNA from the gel origin. Ribonuclease was without effect. DNA recovered from LB and MB also had a higher molecular weight than the HB DNA. Together these results indicate (1) that LB and MB are enriched in newly replicated DNA; (2) that an increased proportion of newly replicated DNA in LB and MB is associated with DNA replication forks; and (3) that the replicating DNA recovered in LB and MB may be associated with other nuclear constituents in situ because this DNA appears to be protected from the more frequent chain breaks introduced into the bulk of chromatin (HB) by sonication.     

Cellular content of ribosomal RNA in relation to the progression and competence signals governing proliferation of 3T3 and SV40-3T3 cells

The method for differential fluorescence staining of cellular RNA and DNA by acridine orange (AO) was optimized for 3T3 and SV40-3T3 cells. Cellular contents of DNA and of ribosomal RNA (rRNA) were determined by dual-channel flow cytometry during cell-density-dependent proliferation and after stimulation of quiescent cells. With increasing density of 3T3 cells, cellular content of rRNA decreases by about 60%, whereas SV40-3T3 cells do not exhibit a significant dependence of rRNA content on cell density. 3T3 cells stimulated early after becoming quiescent resume reaccumulation of rRNA after a delay of only 4 h, whereas cells maintained at quiescence for several days exhibit a delay of about 12 h before a significant rise of rRNA is observed. The extent of rise of cellular rRNA content after different regimens of stimulation of quiescent 3T3 cells does not correlate well with the fraction of cells entering the cell cycle. These and other reported instances of discordance between rRNA content and stimulation into the cell cycle are resolved by showing that of the two signals governing entry into the cell cycle only the progression signal, but not the competence signal is associated with reaccumulation of cellular rRNA. The present results are consistent with the progression function being in essence the achievement of a threshold number of ribosomes per cell, which in conjunction with the competence signal is sufficient for initiation of the cell cycle.     

Regulation of low-density lipoprotein receptors in the human hepatoma cell line Hep G2

The human hepatoma cell line Hep G2 can be maintained in continuous culture and secretes numerous plasma proteins and lipoproteins into the medium. To better characterize cholesterol homeostasis in these cells we have examined the binding, internalization and degradation of [125I]LDL by cultured Hep G2 cells. Hep G2 cells express high-affinity low-density lipoprotein (LDL) receptors which facilitate the binding, internalization and degradation of [125I]LDL; these receptors can be induced by growth in LDL-depleted medium and repressed by further incubation in medium supplemented with LDL. The degradation of [125I]LDL by derepressed Hep G2 cells was inhibited by greater than 90% by monensin. Incubation of Hep G2 cells in the presence of increasing concentrations of LDL also inhibited cholesterol biosynthesis. Our results indicate that Hep G2 cells possess high affinity LDL receptors which are subject to metabolic regulation and suggest that this cell line affords a valuable model to further examine cholesterol and lipoprotein metabolism in human liver cells.     

Plasmid replication in DNA Ts mutants of Bacillus subtilis.

In an attempt to increase our understanding of plasmid replication in Bacillus subtilis we determined the effect of various dna Ts mutations [Gass, K. B., and Cozzarelli, N. R. (1973). J. Biol. Chem. 248, 7688–7700; Gross, J. D., Karamata, D., and Hempstead, P. G. (1968). Cold Spring Harbor Symp. Quant. Biol.33, 307–312; Karamata, D., and Gross, J. D. (1970). Mol. Gen. Genet.108, 277–287] on pUB110 replication. pUB110 is a kanamycin resistance plasmid originally isolated in Staphylococcus aureus and introduced into B. subtilis by transformation. At temperatures nonpermissive for chromosomal DNA synthesis dnaA13, dnaB19, dnaC6, dnaC30, dnaD23, dnaE20, and dnaI102 permit replication of the plasmid. In several cases this “amplification” continues until approximately equal amounts of plasmid and chromosomal DNA are present. dnaG34, dnaH151, dnaF133, mut-1, and polC26 affect both pUB110 and host DNA synthesis at nonpermissive temperatures. The last three mutations are known to affect the activity of DNA polymerase III (PolIII). When polC26 is incubated at a nonpermissive temperature, there is an accumulation of plasmid DNA with a density on EtBr-CsCl gradients intermediate between that of covalently closed circular (CCC) and open circular DNA. pUB110 can replicate in a strain which is deficient in DNA polymerase I (PolI). Finally, chloramphenicol (Cm) inhibits the replication of pUB110 as well as of chromosomal DNA.     

Cell division cycle in mammalian cells : VIII. Mapping of G1 into six segments using temperature-sensitive cell cycle mutants

The G1 blocks in three temperature-sensitive (ts) Syrian hamster cell-cycle mutants have been mapped in relation to other G1 landmarks. Two mutants reported here, ts-559 and ts-694, show defective progression only in G1. When shifted from the permissive temperature of 33 degrees C to the non-permissive temperature of 39 degrees C, G1 cells of these two mutants show no further cell cycle progression, while cells in S, G2 and mitosis progress through the cell cycle but become blocked after entering G1. The two mutants complement each other, and also complement the previously reported mutant ts-550C with blocks in both G1 and G2 of the cell cycle. The locations of the G1 blocks in both ts-559 and ts-694 are before the hydroxyurea arrest point. The G1 ts point in ts-694 is prior to the isoleucine deprivation and serum starvation points, while the G1 block in ts-559 is after the serum starvation point but before the isoleucine block. Other G1 block points which have been reported are in mutants of different species and isolated in different laboratories, causing difficulties for relative positioning of the blocks in G1. The mutants for mapping in this study have been isolated from the same cell line. The G1 ts arrest points of ts-559 and ts-694, and that found in ts-550C, together with nutritional deprivations and metabolic inhibitors, provide seven reference points which divide G1 into six segments, each of which is bracketed by two adjacent points: mitosis, ts-694 block, serum starvation arrest point, ts-559 block, isoleucine deprivation arrest point, ts-550C block, hydroxyurea or excess-thymidine arrest segment.     

Induction of differentiation in a human promyelocytic leukemic cell line (HL-60). Production of granule proteins

Serum fibronectin inhibits the adhesion of neutrophil granulocytes (PMNs) to clean glass, HSA-coated glass, and gelatin-coated glass. It does not affect adhesion to collagen-coated glass which itself provides a substratum of low adhesiveness for PMNs. Cell-cell adhesion is not affected. During the acute inflammatory response in vivo, PMNs must migrate through the fibronectin and collagen containing extracellular matrix: reducing cell-substratum adhesion in these circumstances might facilitate locomotion towards inflammatory foci.     

DNA synthesis catalysed by endogenous templates and DNA-dependent DNA polymerases in spermatogenic cells from rat

The activity levels of DNA polymerases α and β have been measured by autoradiography in squash preparations from rat testis of sexually mature animals. Similar results were obtained with ‘fixed’ samples (dipped in acetone: ethanol for 5 min at 25 °C) or ‘unfixed’ samples (frozen in liquid nitrogen and freeze-dried). The activities of DNA polymerases α and β in situ were distinguished by differential assay conditions and by selective inhibition with compounds such as N-ethylmaleimide and aphidicolin. Using the endogenous chromatin as template, maximal activity for both enzymes was obtained in the presence of all four deoxyribonucleoside triphosphates, MgCl₂ and ethylene glycol. When DNA polymerase activities in several predominant testicular cell types (pre-leptotene primary spermatocytes, pachytene primary spermatocytes, round spermatids and elongated spermatids) were quantitatively compared, on a per cell basis, the following percentage distribution was observed:

     

10.

Effects of 3-aminobenzamide on DNA synthesis and cell cycle progression in Chinese hamster ovary cells     

Jeffrey L. Schwartz  William F. Morgan  Leon N. Kapp  Sheldon Wolff 《Experimental cell research》1983,143(2):377-382

3-Aminobenzamide (3AB), an inhibitor of poly(ADP-ribose) polymerase, is a potent inducer of sister chromatid exchanges (SCEs). Because of the possible relation between SCEs and DNA synthesis, the effects of 3AB on DNA synthesis and cell cycle progression in Chinese hamster ovary (CHO) cells were examined. Unlike all other SCE-inducing agents whose effects on DNA synthesis have been studied, short term exposures (30–120 min) of 3AB did not inhibit the overall rate of DNA synthesis and this result was independent of the amount of bromodeoxyuridine (BrdU) in the DNA. Longer exposure times (>24 h) did result in an extended S phase, but this was not due to an effect on the rate of DNA chain elongation. 3AB also delayed the entry of cells into S phase. The overall cell cycle delay was dose dependent, approaching 9 h after a 54 h exposure to 10 mM 3AB. Earlier reports that 3AB is neither mutagenic nor cytotoxic were confirmed. Thus 3AB acts to increase SCE frequency by a mechanism distinct from that which causes cytotoxicity and mutagenicity, and does not involve any inhibition in the rate of DNA chain growth.     

11.

Survival of apurinic SV40 DNA in the d-complementation group of xeroderma pigmentosum.     

R D Kudrna  J Smith  S Linn  E E Penhoet 《Mutation research》1979,62(1):173-181

The survival of depurinated Form I SV40 DNA was studied in normal human fibroblasts and in D-complementation Xeroderma pigmentosum (XP) fibroblasts. Survival was measured with an infective center assay. Heat-acid and methyl methanesulfonate (MMS) were used as depurinating agents. After 3 hrs of depurination by heat--acid treatment, infectivity in normal cells was less than 15% of the controls compared to more than 50% for the XP D cell strains. Similar results were obtained with MMS-treated DNA. These results are contrary to expectation since apurinic endonuclease activity, which is presumed to be involved in the repair of apurinic sites, is much lower in XP D cell strains than in normal cell strains. Our results indicate that another mechanism for the repair of apurinic sites could exist.     

12.

Comparative study of the cellular replication kinetics of rat mammary epithelial cells in vivo and in vitro     

S Eberhard  L Tait  L K Tay  J Russo 《Experimental cell research》1982,141(1):31-38

Ts-131b, one of the temperature-sensitive (ts) mutants isolated from mouse FM3A cells, was found to be defective in DNA replication at a non-permissive temperature. After the cells were transferred to 39.5 °C, the cell number increased by only 10% and the rate of incorporation of precursors into cellular DNA decreased rapidly. Cell cycle analysis by a flow cytometric method with the cells incubated at 39.5 °C revealed that progression of the cells through the S phase was inhibited and most of the cells were arrested in the S phase. To study the defect in DNA replication of this ts-mutant at 39.5 °C, DNA-fiber autoradiography was performed to measure the rate of DNA-chain elongation. The results showed that the rate of DNA-chain elongation was decreased at 6 h after the temperature shift. However, since the decrease in the rate of DNA-chain elongation was not sufficient to account for the decrease in the rate of incorporation of the precursors, it was suggested that there was also a decrease in the rate of initiation of DNA replication at some of the replicon origins.     

13.

Adenosine receptor activation in quiescent Swiss 3T3 cells. Enhancement of cAMP levels, DNA synthesis and cell division   总被引：6，自引：0，他引：6  

E Rozengurt 《Experimental cell research》1982,139(1):71-78

Anti-tubulin immunofluorescence microscopy is used here to demonstrate that eggs of Lytechinus variegatus are induced to assemble cytoplasmic microtubules upon artificial activation. These microtubules progress through three distinct configurations followed by cycles of abortive division. The first of these is a configuration in which microtubules are found in a disordered network near the egg cortex; the progressive thickening of the microtubule-containing layer appears to be responsible for the centripetal movement of the egg nucleus that occurs shortly after activation. These microtubules are replaced at about 40 min by a population of long, radially arrayed microtubules, which are restructured by about 70 min to form the apolar mitotic apparatus. Each of the microtubule configurations characteristic of activated eggs becomes more prominent when eggs are treated at the appropriate times after activation with the microtubule-stabilizing drug taxol. Any microtubule organizing centers within the activated egg must have very limited authority, since aster-like structures are not seen, and microtubules are not observed to be closely associated with the nucleus or egg cortex. Activation of eggs with ammonia in Ca²⁺-free sea water (a treatment that bypasses the cortical reaction and the Ca²⁺ transient) induces the appearance of microtubules as readily and in the same patterns as does treatment with ionophore A23187 or butyric acid, both of which activate by inducing an intracellular calcium release and the cortical reaction.     

14.

Density-dependent regulation of growth in somatic hybrids between normal Chinese hamster fibroblasts and V79-8 (G1) cells   总被引：1，自引：0，他引：1  

G. Marin  D. Wynford-Thomas  A. Lamontagne  D. M. Prescott 《Experimental cell research》1984,155(2):575-582

The purpose of this work was to determine the relationship between the presence of a G1 period in the mitotic cycle and a cell's ability to respond to density-dependent regulation of growth (DDR). Somatic hybrids were obtained between normal fibroblasts from newborn Chinese hamsters, which show a strong response to DDR, and V79-8 Chinese hamster cells, which are insensitive to DDR. Two variant V79-8 sublines were used, one reported to lack a G1 period (G1-) and the other with a G1 period (G1+). Fourteen hybrid clones were isolated in selective medium and analysed for growth properties and cell cycle parameters; their hybrid nature was supported by chromosome counts. All hybrid clones, irrespective of whether a V79-8 G1- or G1+ cell was one of the parents, showed pronounced DDR and had G1 periods of various lengths. Previous experiments had shown the absence of G1 to be dominant in somatic hybrids between V79-8 G1- and G1+ cell lines. Our results may mean that the G1- property provided by V79-8 is unable to overcome the very long G1 of normal fibroblasts, or in cells that can be arrested in G1 in response to DDR, some function prevents the dominant effect of the G1- cell on at least part of the G1 period.     

15.

Extent of histone modifications and H1 content during cell cycle progression in the presence of butyrate     

J. A. D''Anna  L. R. Gurley  R. A. Tobey 《Experimental cell research》1983,147(2):407-417

The effects of butyrate upon the extents of phosphorylation of histones H1 and H1(0) during cell-cycle progression have been investigated. Chinese hamster (line CHO) cells were synchronized in early S phase and released into medium containing 0 or 15 mM butyrate to resume cell-cycle traverse into G1 of the next cell cycle. Cells were also mechanically selected from monolayer cultures grown in the presence of colcemid and 0 or 15 mM butyrate to obtain greater than 98% pure populations of metaphase cells. Although cell cycle progression is altered by butyrate, electrophoretic patterns of histones H1, H1(0), H3, and H4 indicate that butyrate has little, if any, effect on the extents of H1 and H1(0) phosphorylation during the cell cycle or the mitotic-specific phosphorylation of histone H3. Butyrate does, however, inhibit removal of extraordinary levels of histone H4 acetylation (hyperacetylation) during metaphase, and it appears to cause an increase in the content of H1(0) in chromatin during the S or G2 phases of the cell cycle.     

16.

Size maturation process of nascent DNA intermediates into chromosomal-sized DNA in Tetrahymena pyriformis macronuclear DNA replication     

T Kozu  T Yagura  T Seno 《Experimental cell research》1983,149(1):189-200

An analysis was made of the size maturation process of nascent DNA intermediates in macronuclear DNA replication of Tetrahymena pyriformis. The first discrete size class of nascent intermediates larger than Okazaki fragments were replicon-sized DNA (about 2 X 10(7) D single-stranded (ss) DNA) and accumulated in cells treated with cycloheximide. On removal of cycloheximide, the replicon-sized intermediates were converted to middle-sized intermediates (about 10 X 10(7) D ssDNA) and then merged into chromosomal-sized DNA. As indicated by either aphidicolin inhibition or the technique of the photolysis of bromodeoxyuridine (BrdU)-substituted DNA with long-wave ultraviolet light, four to eight replicon-sized intermediates were joined together to form a middle-sized intermediate after rapid sealing by DNA synthesis of the late-replicating regions located between adjacent replicon-sized intermediates. The late-replicating regions may represent the short gaps or terminal regions where DNA synthesis was retarded by cycloheximide, since the size of late-replicating regions was suggested to be shorter than the replicon size by DNA fiber autoradiography. Therefore, it is probable that four to eight completed replicons are joined as a group such as a replicon cluster, as has been reported in DNA replication of other eukaryotic cells.     

17.

Doubling of cell mass is not necessary in order to achieve cell division in cultured human cells     

Ø.W. Rønning  E.O. Pettersen 《Experimental cell research》1984,155(1):267-272

When exponentially growing NHIK 3025 cells were shifted from medium containing 30% serum to medium containing 0.03% serum the rate of net protein accumulation was reduced due to both a reduction in the rate of protein synthesis and an increase in the rate of protein degradation. This change in growth conditions increased the protein doubling time from 18 to 140 h. The cell cycle duration of cells synchronized by mitotic selection was, however, only increased from 17 to 26 h by this treatment. Therefore, when the cells divide by the end of the first cell cycle following synchronization, the cells shifted to 0.03% serum contained far less protein than those growing continuously in 30% serum. Hence, the attainment of a critical cell mass is probably not controlling cell division for cells growing in a balanced state.     

18.

Biochemical and immunochemical characterization of two simian virus 40 (SV40)-specific glycoproteins in nuclear and surface membranes of SV40-transformed cells.     

R Schmidt-Ullrich  W S Thompson  D F Wallach 《Biochemical and biophysical research communications》1979,88(3):887-894

Plasma membranes of several simian virus 40-transformed cells contain virus-specific proteins with molecular masses of ～ 100,000D and ～ 60,000D and isoelectric points of ～ 4.7 and ～ 4.5, respectively. Triton X-100 extracts of purified nuclei from simian virus 40-transformed hamster lymphocytes contain the same proteins but in different proportions, the high molecular mass component being enriched six-fold in terms of the lower molecular mass one. Both proteins can be labeled metabolically with [¹⁴C]glucosamine and their isoelectric points altered by neuraminidase treatment, showing that they are sialoglycoproteins.     

19.

Mitochondrial DNA synthesis in overmatured eggs of the starfish     

H Nagano  K Okano  S Ikegami 《Experimental cell research》1983,146(1):207-211

DNA synthesis was studied during the overmaturational stage of starfish eggs. Some DNA synthesis took place in the overmature eggs of the starfish. The DNA synthesis was resistant to aphidicolin. Judging from its circularity and small molecular size, the DNA synthesized is of mitochondrial origin.     

20.

Resolution of mitochondrial DNA structures in the large yeast Wickerhamia fluorescens   总被引：1，自引：0，他引：1  

B C Hyman  B Bainbridge  T W James 《Experimental cell research》1982,141(1):221-230

DAPI (4′6 Diamidino-2-phenylindole) staining of the large yeast Wickerhamia fluorescens revealed extensive cytoplasmic staining material attributable to mitochondrial DNA (mtDNA), often present in large network-like structures. The maintenance of this mtDNA appears to be insensitive to a variety of mitochondrial specific mutagens, suggesting that W. fluorescens may be classified as a petite negative yeast. Restriction enzyme analysis generated a unit genome size of 42(10⁶) D for this mtDNA which, together with determinations of the average mtDNA per cell, allowed an estimate of the cellular copy number of mitochondrial genomes. A physical map of this mtDNA was also derived. These experiments suggested models which might reflect the cytological structures resolved by DAPI staining in W. fluorescens relative to other yeasts.     

	Pre-leptotene primary spermatocyte %	Pachytene primary spermatocyte %	Round spermatid %	Elongated spermatid %
DNA polymerase α	25	42	30	3
DNA polymerase β	29	34	36	1

Effects of 3-aminobenzamide on DNA synthesis and cell cycle progression in Chinese hamster ovary cells

3-Aminobenzamide (3AB), an inhibitor of poly(ADP-ribose) polymerase, is a potent inducer of sister chromatid exchanges (SCEs). Because of the possible relation between SCEs and DNA synthesis, the effects of 3AB on DNA synthesis and cell cycle progression in Chinese hamster ovary (CHO) cells were examined. Unlike all other SCE-inducing agents whose effects on DNA synthesis have been studied, short term exposures (30–120 min) of 3AB did not inhibit the overall rate of DNA synthesis and this result was independent of the amount of bromodeoxyuridine (BrdU) in the DNA. Longer exposure times (>24 h) did result in an extended S phase, but this was not due to an effect on the rate of DNA chain elongation. 3AB also delayed the entry of cells into S phase. The overall cell cycle delay was dose dependent, approaching 9 h after a 54 h exposure to 10 mM 3AB. Earlier reports that 3AB is neither mutagenic nor cytotoxic were confirmed. Thus 3AB acts to increase SCE frequency by a mechanism distinct from that which causes cytotoxicity and mutagenicity, and does not involve any inhibition in the rate of DNA chain growth.     

Survival of apurinic SV40 DNA in the d-complementation group of xeroderma pigmentosum.

The survival of depurinated Form I SV40 DNA was studied in normal human fibroblasts and in D-complementation Xeroderma pigmentosum (XP) fibroblasts. Survival was measured with an infective center assay. Heat-acid and methyl methanesulfonate (MMS) were used as depurinating agents. After 3 hrs of depurination by heat--acid treatment, infectivity in normal cells was less than 15% of the controls compared to more than 50% for the XP D cell strains. Similar results were obtained with MMS-treated DNA. These results are contrary to expectation since apurinic endonuclease activity, which is presumed to be involved in the repair of apurinic sites, is much lower in XP D cell strains than in normal cell strains. Our results indicate that another mechanism for the repair of apurinic sites could exist.     

Comparative study of the cellular replication kinetics of rat mammary epithelial cells in vivo and in vitro

Ts-131b, one of the temperature-sensitive (ts) mutants isolated from mouse FM3A cells, was found to be defective in DNA replication at a non-permissive temperature. After the cells were transferred to 39.5 °C, the cell number increased by only 10% and the rate of incorporation of precursors into cellular DNA decreased rapidly. Cell cycle analysis by a flow cytometric method with the cells incubated at 39.5 °C revealed that progression of the cells through the S phase was inhibited and most of the cells were arrested in the S phase. To study the defect in DNA replication of this ts-mutant at 39.5 °C, DNA-fiber autoradiography was performed to measure the rate of DNA-chain elongation. The results showed that the rate of DNA-chain elongation was decreased at 6 h after the temperature shift. However, since the decrease in the rate of DNA-chain elongation was not sufficient to account for the decrease in the rate of incorporation of the precursors, it was suggested that there was also a decrease in the rate of initiation of DNA replication at some of the replicon origins.     

Adenosine receptor activation in quiescent Swiss 3T3 cells. Enhancement of cAMP levels, DNA synthesis and cell division

Anti-tubulin immunofluorescence microscopy is used here to demonstrate that eggs of Lytechinus variegatus are induced to assemble cytoplasmic microtubules upon artificial activation. These microtubules progress through three distinct configurations followed by cycles of abortive division. The first of these is a configuration in which microtubules are found in a disordered network near the egg cortex; the progressive thickening of the microtubule-containing layer appears to be responsible for the centripetal movement of the egg nucleus that occurs shortly after activation. These microtubules are replaced at about 40 min by a population of long, radially arrayed microtubules, which are restructured by about 70 min to form the apolar mitotic apparatus. Each of the microtubule configurations characteristic of activated eggs becomes more prominent when eggs are treated at the appropriate times after activation with the microtubule-stabilizing drug taxol. Any microtubule organizing centers within the activated egg must have very limited authority, since aster-like structures are not seen, and microtubules are not observed to be closely associated with the nucleus or egg cortex. Activation of eggs with ammonia in Ca²⁺-free sea water (a treatment that bypasses the cortical reaction and the Ca²⁺ transient) induces the appearance of microtubules as readily and in the same patterns as does treatment with ionophore A23187 or butyric acid, both of which activate by inducing an intracellular calcium release and the cortical reaction.     

Density-dependent regulation of growth in somatic hybrids between normal Chinese hamster fibroblasts and V79-8 (G1) cells

The purpose of this work was to determine the relationship between the presence of a G1 period in the mitotic cycle and a cell's ability to respond to density-dependent regulation of growth (DDR). Somatic hybrids were obtained between normal fibroblasts from newborn Chinese hamsters, which show a strong response to DDR, and V79-8 Chinese hamster cells, which are insensitive to DDR. Two variant V79-8 sublines were used, one reported to lack a G1 period (G1-) and the other with a G1 period (G1+). Fourteen hybrid clones were isolated in selective medium and analysed for growth properties and cell cycle parameters; their hybrid nature was supported by chromosome counts. All hybrid clones, irrespective of whether a V79-8 G1- or G1+ cell was one of the parents, showed pronounced DDR and had G1 periods of various lengths. Previous experiments had shown the absence of G1 to be dominant in somatic hybrids between V79-8 G1- and G1+ cell lines. Our results may mean that the G1- property provided by V79-8 is unable to overcome the very long G1 of normal fibroblasts, or in cells that can be arrested in G1 in response to DDR, some function prevents the dominant effect of the G1- cell on at least part of the G1 period.     

Extent of histone modifications and H1 content during cell cycle progression in the presence of butyrate

The effects of butyrate upon the extents of phosphorylation of histones H1 and H1(0) during cell-cycle progression have been investigated. Chinese hamster (line CHO) cells were synchronized in early S phase and released into medium containing 0 or 15 mM butyrate to resume cell-cycle traverse into G1 of the next cell cycle. Cells were also mechanically selected from monolayer cultures grown in the presence of colcemid and 0 or 15 mM butyrate to obtain greater than 98% pure populations of metaphase cells. Although cell cycle progression is altered by butyrate, electrophoretic patterns of histones H1, H1(0), H3, and H4 indicate that butyrate has little, if any, effect on the extents of H1 and H1(0) phosphorylation during the cell cycle or the mitotic-specific phosphorylation of histone H3. Butyrate does, however, inhibit removal of extraordinary levels of histone H4 acetylation (hyperacetylation) during metaphase, and it appears to cause an increase in the content of H1(0) in chromatin during the S or G2 phases of the cell cycle.     

Size maturation process of nascent DNA intermediates into chromosomal-sized DNA in Tetrahymena pyriformis macronuclear DNA replication

An analysis was made of the size maturation process of nascent DNA intermediates in macronuclear DNA replication of Tetrahymena pyriformis. The first discrete size class of nascent intermediates larger than Okazaki fragments were replicon-sized DNA (about 2 X 10(7) D single-stranded (ss) DNA) and accumulated in cells treated with cycloheximide. On removal of cycloheximide, the replicon-sized intermediates were converted to middle-sized intermediates (about 10 X 10(7) D ssDNA) and then merged into chromosomal-sized DNA. As indicated by either aphidicolin inhibition or the technique of the photolysis of bromodeoxyuridine (BrdU)-substituted DNA with long-wave ultraviolet light, four to eight replicon-sized intermediates were joined together to form a middle-sized intermediate after rapid sealing by DNA synthesis of the late-replicating regions located between adjacent replicon-sized intermediates. The late-replicating regions may represent the short gaps or terminal regions where DNA synthesis was retarded by cycloheximide, since the size of late-replicating regions was suggested to be shorter than the replicon size by DNA fiber autoradiography. Therefore, it is probable that four to eight completed replicons are joined as a group such as a replicon cluster, as has been reported in DNA replication of other eukaryotic cells.     

Doubling of cell mass is not necessary in order to achieve cell division in cultured human cells

When exponentially growing NHIK 3025 cells were shifted from medium containing 30% serum to medium containing 0.03% serum the rate of net protein accumulation was reduced due to both a reduction in the rate of protein synthesis and an increase in the rate of protein degradation. This change in growth conditions increased the protein doubling time from 18 to 140 h. The cell cycle duration of cells synchronized by mitotic selection was, however, only increased from 17 to 26 h by this treatment. Therefore, when the cells divide by the end of the first cell cycle following synchronization, the cells shifted to 0.03% serum contained far less protein than those growing continuously in 30% serum. Hence, the attainment of a critical cell mass is probably not controlling cell division for cells growing in a balanced state.     

Biochemical and immunochemical characterization of two simian virus 40 (SV40)-specific glycoproteins in nuclear and surface membranes of SV40-transformed cells.

Plasma membranes of several simian virus 40-transformed cells contain virus-specific proteins with molecular masses of ～ 100,000D and ～ 60,000D and isoelectric points of ～ 4.7 and ～ 4.5, respectively. Triton X-100 extracts of purified nuclei from simian virus 40-transformed hamster lymphocytes contain the same proteins but in different proportions, the high molecular mass component being enriched six-fold in terms of the lower molecular mass one. Both proteins can be labeled metabolically with [¹⁴C]glucosamine and their isoelectric points altered by neuraminidase treatment, showing that they are sialoglycoproteins.     

Mitochondrial DNA synthesis in overmatured eggs of the starfish

DNA synthesis was studied during the overmaturational stage of starfish eggs. Some DNA synthesis took place in the overmature eggs of the starfish. The DNA synthesis was resistant to aphidicolin. Judging from its circularity and small molecular size, the DNA synthesized is of mitochondrial origin.     

Resolution of mitochondrial DNA structures in the large yeast Wickerhamia fluorescens

DAPI (4′6 Diamidino-2-phenylindole) staining of the large yeast Wickerhamia fluorescens revealed extensive cytoplasmic staining material attributable to mitochondrial DNA (mtDNA), often present in large network-like structures. The maintenance of this mtDNA appears to be insensitive to a variety of mitochondrial specific mutagens, suggesting that W. fluorescens may be classified as a petite negative yeast. Restriction enzyme analysis generated a unit genome size of 42(10⁶) D for this mtDNA which, together with determinations of the average mtDNA per cell, allowed an estimate of the cellular copy number of mitochondrial genomes. A physical map of this mtDNA was also derived. These experiments suggested models which might reflect the cytological structures resolved by DAPI staining in W. fluorescens relative to other yeasts.