Delayed termination of nuclear histone doubling after premeiotic DNA synthesis inTriturus vulgaris male meiosis

It has been shown by means of double wavelength cytophotometry of DNA (Feulgen reaction) and histone (fast green, pH 8.2) inTriturus vulgaris spermatocytes that the doubling of DNA content in nuclei terminates at the end of preleptotene to beginning of leptotene whereas the doubling of histone content begun at premeiotic interphase is delayed and proceeds till the end of leptotene to beginning of zygotene. As a result preleptotene spermatocytes contain approximately 4C DNA and only 3C histone. Histone content in leptotene amounts to 93% of 4C, and in zygotene, pachytene and metaphase I both DNA and histone contents equal 4C. Thus, the temporal pattern of nucleo-histone doubling in meiotic chromosomes ofT. vulgaris differs from the synchronous DNA and histone doubling in mitotic chromosomes of all previously studied species. The delay of histone doubling inT. vulgaris meiocytes is less pronounced than in the previously studied insectsAcheta domestica andPyrrhocoris apterus where the histone content amounts to 3C in leptotene—zygotene and the equal histone/DNA ratio is restored only in pachytene.—Responsibilities for this phenomenon and its biolgoical sinnificance are discussed in connection with recent hypotheses concerning mechanisms of homologous chromosome pairing.     

THE SYNTHESES OF DEOXYRIBONUCLEIC ACID AND HISTONE IN THE ONION ROOT MERISTEM

A comparison of the times necessary to incorporate tritium-labeled lysine and arginine into histones and tritium-labeled thymidine into DNA indicates that the periods of DNA and histone synthesis prior to division closely coincide. (The comparison was made by determining the times necessary, after pulse labeling, for cells with marked chromosomes to enter and then leave the division stages.) An additional period of chromosomal protein synthesis, of short duration, occurs late in interphase. Most of the chromosomal proteins appear either to be synthesized in the nucleus or to migrate there shortly after synthesis. Much of this protein is conserved from one division to the next. Studies of the effects of puromycin and fluorodeoxyuridine on the syntheses of DNA and histone suggest that continuation of DNA synthesis is dependent on a concurrent protein synthesis. Histone synthesis, on the other hand, can proceed at a normal rate under conditions in which DNA synthesis is inhibited.     

INCORPORATION OF TRITIUM-LABELED THYMIDINE AND LYSINE INTO CHROMOSOMES OF CULTURED HUMAN LEUKOCYTES

The incorporation of thymidine-H³ and lysine-H³ into human leukocyte chromosomes was studied in order to determine the temporal relationships between the syntheses of chromosomal deoxyribonucleic acid and chromosomal protein. The labeled compounds were incorporated into nuclei of interphase cells. Label from both precursors became apparent over the chromosomes of dividing cells. Incorporation of thymidine-H³ occurred during a restricted period of midinterphase (S) which was preceded by a nonsynthetic period (G₁) and followed by a nonsynthetic period (G₂). Incorporation of lysine-H³ into chromosomal protein occurred throughout interphase. Grain counts made over chromosomes of dividing cells revealed that the rate of incorporation of lysine-H³ into chromosomal protein differed during various periods of interphase. The rate of incorporation was diminished during G₁. During early S period the rate of incorporation increased, reaching a peak in late S. The high rate continued into G₂. Thymidine-H³ incorporated into DNA was distributed to mitotic chromosomes of daughter cells in a manner which has been referred to as a "semi-conservative segregation." No such semi-conservative mechanism was found to affect the distribution of lysine-H³ to the mitotic chromosomes of daughter cells. Therefore, it is concluded that synthesis of chromosomal protein and its distribution to chromosomes of daughter cells are not directly influenced by synthesis and distribution of the chromosomal DNA with which the protein is associated.     

Synchronous synthesis of DNA in coleoptiles and the initial leaf of developing etiolated wheat shoots: the nature and correlation of nuclear and mitochondrial DNA syntheses

In etiolated coleoptiles and initial leaf of developing wheat shoots the DNA synthesis is periodical and synchronous. In the initial leaf each step of DNA synthesis results in a stepwise increase of DNA content and is doubled at the first three steps. During the leaf plane formation the synthesis of nuclear DNA (nDNA) is decreased, while that of mitochondrial DNA (mitDNA) continues in synchronous cycles. This is the cause of relative stabilization of DNA content per unit of leaf plane length. The DNA increase in this organ occurs due to synchronous synthesis of nDNA and mitDNA in intercalary meristem cells. In coleoptiles a marked replication of nDNA is observed at the first three steps of the synthesis; in each cycle nDNA synthesis precedes that of mitDNA. With completion of coleoptile formation the nDNA synthesis in it practically ceases, whereas that of mitDNA continues in synchronous cycles. MitDNA is non-methylated and its composition (56 mol.% GC) differs significantly from that of the newly synthesized nDNA (44 mol.% GC; 100 X m5C/(C + m5C) = 16-17%). It may be concluded that in various organs of wheat shoots the composition and methylation of newly synthesized DNA depend on the age of the shoot and on the ratio of nDNA/mitDNA syntheses.     

DIVISION IN THE DINOFLAGELLATE GYRODINIUM COHNII (SCHILLER) : A New Type of Nuclear Reproduction

Dinoflagellates are of interest because their chromosomes resemble the nucleoplasm of prokaryotes both chemically and ultrastructurally. We have studied nuclear division in the dinoflagellate Gyrodinium cohnii (Schiller), using cells obtained from cultures undergoing phasic growth. Electron micrographs of serial sections were used to prepare three-dimensional reconstructions of nuclei and chromosomes at various stages of nuclear division. During division, a complex process of invagination of the intact nuclear envelope takes place at one side of the nucleus and results in the formation of parallel cylindrical cytoplasmic channels through the nucleus. These invaginations contain bundles of microtubules, and each of the bundles comes to lie in the cytoplasm of a cylindrical channel. Nuclear constriction occurs perpendicular to these channels without displacement of the microtubules. There are no associations between chromosomes and the cytoplasmic microtubules. In dividing cells most chromosomes become V-shaped, and the apices of the V's make contact with the membrane surrounding cytoplasmic channels. It is proposed that the membrane surrounding cytoplasmic channels in the dividing nucleus may be involved in the separation of daughter chromosomes. Thus, dinoflagellates may resemble prokaryotes in the manner of genophore separation as well as in genophore chemistry and ultrastructure.     

VITAMIN B12 AND THE MACROMOLECULAR COMPOSITION OF EUGLENA : II. Recovery from Unbalanced Growth Induced by Vitamin B12 Deficiency

When vitamin B₁₂ is added to B₁₂-deficient cultures of Euglena gracilis, the cells undergo two relatively synchronous cell divisions within a shorter than usual period of time, apparently as a result of a transitory shortening of the cell cycle. The first cell division pulse, occurring 4.5 h after addition of B₁₂, is preceded by the completion of DNA duplication, but appears to involve no net synthesis of RNA or protein. Before the second round of cell division at about 11 h, a significant amount of DNA synthesis is observed. This time it is accompanied by a minor increase in the RNA and protein content of the culture. The cellular contents of RNA and protein were observed to decrease steadily after the resumption of cell division in B₁₂-depleted cultures receiving the vitamin. Ultimately all three macromolecules returned to their nondeficient, plateau stage levels; by this time, cell division had ceased.     

The cytology of cotyledon cells and the induction of giant polytene chromosomes inPisum sativum

Summary The cotyledon cells ofPisum sativum have high DNA contents. By appropriate culture techniques, some of these cells can be triggered into division. Two types of dividing nuclei were seen. Firstly those that were polyploid with metaphases containing chromosome numbers ranging in value from 4 x to 32 x. Included among these were unexpected numbers equivalent to 12 x and 14 x. Secondly there were cells containing giant polytene chromosomes and these progressed from prophase to a metaphase where the polytene chromosomes separated into constituent single chromosomes.     

AN ANALYSIS OF HETEROCHROMATIN IN MAIZE ROOT TIPS

The B chromosomes of maize are condensed in appearance during interphase and are relatively inert genetically; therefore they fulfill the definition of heterochromatin. This heterochromatin was studied in root meristem cells by radioautography following administration of tritiated thymidine and cytidine, and was found to behave in a characteristic way, i.e. it showed asynchronous DNA synthesis and very low, if any, RNA synthesis. A cytochemical comparison of normal maize nuclei with nuclei from isogenic maize stock containing approximately 15–20 B-chromosomes in addition to the normal complement has revealed the following: (a) the DNA and histone contents are greater in nuclei with B chromosomes; (b) the proportion of DNA to histone is identical with that of nuclei containing only normal chromosomes; (c) the amount of nonhistone protein in proportion to DNA in interphase is less in nuclei with B chromosomes than in normal nuclei. In condensed B chromosomes the ratio of nonhistone protein to DNA is similar to that in other condensed chromatin, such as metaphase chromosomes and degenerating nuclei. The B chromosomes appear to have no effect on nucleolar RNA and protein. Replication of B chromosomes is precisely controlled and is comparable to that of the ordinary chromosomes not only in synthesis for mitosis but also in formation of polyploid nuclei of root cap and protoxylem cells.     

Control of DNA replication and spatial distribution of defined DNA sequences in salivary gland cells of Drosophila melanogaster

In dividing cells, each sequence replicates exactly once in each S-phase, but in cells with polytene chromosomes, some sequences may replicate more than once or fail to replicate during S-phase. Because of this differential replication, the control of replication in polytene cells must have some unusual features. Dennhöfer (1982a) has recently concluded that the total DNA content of the polytene cells of Drosophila salivary glands exactly doubles in each S-phase. This observation, along with previous studies demonstrating satellite underreplication in salivary gland cells, led us to consider the hypothesis that there is a doubling of DNA mechanism for the control of DNA replication in polytene cells. With this mechanism, a doubling of DNA content, rather than the replication of each sequence, would signal the end of a cycle of DNA replication. To test this hypothesis, we have reinvestigated the replication of several sequences (satellite, ribosomal, histone and telomere) in salivary gland cells using quantitative in situ hybridization. We find that underreplication of some sequences does occur. In addition we have repeated Dennhöfer's cytophotometric and labeling studies. In contrast to Dennhöfer, we find that the total DNA contents of nonreplicating nuclei do reflect this partial replication, in accord with Rudkin's (1969) result. We conclude that DNA replication in polytene cells is controlled by modifications of the mechanism operating in dividing cells, where control is sequence autonomous, and not by a doubling of DNA mechanism. — In situ hybridization to unbroken salivary gland nuclei reveals the distribution of specific sequences. As expected, satellite, histone and 5S sequences are usually in a single cluster. This rules out the possibility that sequences known to be underreplicated in chromosomal DNA exist as extrachromosomal copies. Telomere sequences are grouped into two to six clusters, as if the chromosome ends are partially but not completely paired in salivary gland nuclei.     

Cytological studies on degenerative nuclei at pollen development ofCarex ciliato-marginata

Chromosomes in degenerative and functional nuclei ofCarex ciliato-marginata Nakai were investigated during meiotic and primary pollen nuclear division. The nuclear DNA content of these nuclei was also measured using Feulgen microspectrophotometry. At metaphase of the primary pollen nuclear division, the chromosomes of degenerative nuclei were the same length as those of the functional nucleus, but only half their width. The functional nucleus divided into two, each of which moved to a pole, but the degenerative nuclei did not divide. The nuclear DNA content of the degenerative nucleus was half that of the functional nucleus and equal to that of one of the tetrads of a meiotic division. It is concluded that DNA replication was carried out in only one nucleus of the tetrad and that the other three nuclei were composed of unreplicated chromosomes at metaphase of the primary pollen nuclear division.     

Macromolecular synthesis in synchronized cultures of Anacystis nidulans.

Synchronous culture of Anacystis nidulans has been induced by the light-dark-light regimen. At various time intervals during synchronous growth, samples were pulsed with radioactive labels to determine phospholipid, protein, ribonucleic acid (RNA), and deoxyribonucleic acid (DNA) syntheses within the cell division cycle. A temporal order of protein, RNA, and DNA syntheses occurred within the cell division cycle, whereas phospholipid was characteristically synthesized during midcycle (during cell enlargement) and during the time of cell division. Chemically determined protein, RNA, and DNA syntheses were found to support the schedule of these macromolecules in cultures growing at an 8-h doubling time.     

FtsK-dependent dimer resolution on multiple chromosomes in the pathogen Vibrio cholerae

Unlike most bacteria, Vibrio cholerae harbors two distinct, nonhomologous circular chromosomes (chromosome I and II). Many features of chromosome II are plasmid-like, which raised questions concerning its chromosomal nature. Plasmid replication and segregation are generally not coordinated with the bacterial cell cycle, further calling into question the mechanisms ensuring the synchronous management of chromosome I and II. Maintenance of circular replicons requires the resolution of dimers created by homologous recombination events. In Escherichia coli, chromosome dimers are resolved by the addition of a crossover at a specific site, dif, by two tyrosine recombinases, XerC and XerD. The process is coordinated with cell division through the activity of a DNA translocase, FtsK. Many E. coli plasmids also use XerCD for dimer resolution. However, the process is FtsK-independent. The two chromosomes of the V. cholerae N16961 strain carry divergent dimer resolution sites, dif1 and dif2. Here, we show that V. cholerae FtsK controls the addition of a crossover at dif1 and dif2 by a common pair of Xer recombinases. In addition, we show that specific DNA motifs dictate its orientation of translocation, the distribution of these motifs on chromosome I and chromosome II supporting the idea that FtsK translocation serves to bring together the resolution sites carried by a dimer at the time of cell division. Taken together, these results suggest that the same FtsK-dependent mechanism coordinates dimer resolution with cell division for each of the two V. cholerae chromosomes. Chromosome II dimer resolution thus stands as a bona fide chromosomal process.     

contribution to the study of nuclear total proteins and DNA during the mitotic cycle in fibroblasts cultivated in vitro and in Ehrlich ascites cells

Summary The dry weight and total protein content of nuclei has been measured by interferometry in living or fixed cells cultivated in vitro (freshly prepared chick, mouse or rat embryo fibroblasts) and in fixed Ehrlich ascites tumor cells of the mouse growing in vivo. The DNA content was estimated by cytophotometry after Feulgen reaction in the same nuclei. The dry weight of nucleoli in fibroblasts and the dry weight and DNA content of chromosomes in dividing fibroblasts and Ehrlich tumor cells have also been measured.During the interphase in fibroblasts, the dry weight of the living nucleus and the nuclear total protein content as measured in fixed cells doubles during the preparation for mitosis, as the DNA content does. In chick and mammal fibroblasts and within the limits of accuracy of our measurements, the synthesis curves for nuclear proteins and DNA do not seem to be necessarily identical.In our fibroblasts, the nucleolar total dry weight per nucleus doubles during the interphase (nucleolar preparation for mitosis); it increases in proportion to the nuclear total protein content, even in polyploid nuclei.During the mitosis, the chromosomes contain all the DNA of the nucleus but some nuclear proteins (non chromosomal proteins) seem to move into the cytoplasm during the mitosis and return into the nucleus at the post-telophase.According to our observations, Ehrlich ascites mouse tumor cells are near-tetraploid as far as the number of chromosomes, nuclear total protein content and DNA content are concerned. During the preparation for mitosis, these amounts double but no necessary close time relation seems to link these premitotic syntheses. Prom this point of view, our results show no clear-cut differences between these tumor cells and the fibroblasts. Except the polyploidy, the behaviour of nuclear proteins and DNA during mitosis in the tumor cells is the same as that observed in our fibroblasts.The effects of various antimitotic agents on rat fibroblasts cultivated in vitro have also been studied with our cytochemical methods. Our measurements of nuclear protein, DNA and nucleolar material content have been made in cells in which mitosis was prevented by alkylating agents, beryllium sulphate, RNase or neutral DNase. The effects of colchicine on these cellular parameters have also been studied.     

X-Ray and Ultraviolet Sensitivity of Synchronously Dividing Escherichia coli

A paper pile filtration technique was used to obtain synchronously dividing populations of E. coli strains B and B/r from cultures in the exponential growth phase. Three generations of highly phased cell division were obtained by rapid pressure filtration which selected approximately 1 per cent of the exponentially growing culture. The sensitivity of E. coli strain B to x-ray and UV inactivation as a function of the cell division cycle was determined on synchronous populations. E. coli strain B showed a sharp decrease in sensitivity to inactivation by both radiations in the middle of the division cycle, and a further decrease near the end of the cycle. The sensitivity of E. coli strain B/r to x-irradiation was also investigated. Only the mid-cycle decrease in sensitivity was found during the division cycle of this strain. It was concluded that the repetition of the observed sensitivity patterns in both strains through the first three cycles after synchronization indicates that the same basic sensitivity patterns are probably also present in the individual cells of an exponential phase culture.     

Histone metabolism during amphibian metamorphosis. Isolation, characterization, and biosynthesis.

The amino acid incorporation rates of several classes of liver protein from Rana catesbeiana tadpoles were examined at different stages of spontaneous and thyroxine-induced metamorphosis, particular attention being given to histones. Incorporation data were corrected for the specific radioactivity of the free amino acid pools in tadpole liver. Little change was observed in the overall incorporation rates for the crude mitochondrial and total liver proteins during thyroxine treatment or at selected stages of spontaneous metamorphosis, except that the incorporation rates for these proteins were approximately twofold greater for the newly metamorphosed froglet than for the other stages. However, an increase in the ratio of the specific radioactivities of the total and crude mitochondrial liver protein within each set of animals was observed during late stages of spontaneous metamorphosis, as well as during the second through sixth days of thyroxine treatment. The amino acid incorporation rates of the histones for the late metamorphic and froglet stages of spontaneous metamorphosis were three- to fourfold higher than those of premetamorphic animals, but no significant changes were observed during thyroxine treatment. Thyroxine treatment also produced no detectable changes in the relative amounts or incorporation rates of the histone fractions or subfractions. Apparently the developmental changes induced by thyroxine do not involve a reorganization of the histone complement of chromatin at this level of analysis. Furthermore, since histone and DNA syntheses are tightly coupled, our results show that the extensive metabolic changes induced in tadpole liver by thyroxine occur in the absence of significant levels of cell division.     

Histone acetylation: A possible mechanism for the inheritance of cell memory at mitosis

Immunofluorescent labelling demonstrates that human metaphase chromosomes contain hyperacetylated histone H4. With the exception of the inactive X chromosome in female cells, where the bulk of histone H4 is under-acetylated, H4 hyperacetylation is non-uniformly distributed along the chromosomes and clustered in cytologically resolvable chromatin domains that correspond, in general, with the R-bands of conventional staining. The strongest immunolabelling is often found in T-bands, the subset of intense R-bands having the highest GC content. The majority of mapped genes also occurs in R-band regions, with the highest gene density in T-bands. These observations are consistent with a model in which hyperacetylation of histone H4 marks the position of potentially active gene sequences on metaphase chromosomes. Since acetylation is maintained during mitosis, progeny cells receive an imprint of the histone H4 acetylation pattern that was present on the parental chromosomes before cell division. Histone acetylation could provide a mechanism for propagating cell memory, defined as the maintenance of committed states of gene expression through cell lineages.     

New observations of the nuclear division in Entamoeba histolytica. The chromosomes

The morphologic organization of the nucleus and DNA during the nuclear division of Entamoeba histolytica was examined. The DNA of dividing amebic trophozoites was visualized with the fluorescent probe, Hoechst 33258 for light microscopy, and a DNA-specific antibody and phosphotungstic acid for electron microscopy. These techniques demonstrated features of the dividing amebic nuclei and the presence of spherical DNA-containing bodies corresponding to the condensed chromosomes. Based on light microscopy observations the number of chromosomes in E histolytica is five. Microtubules (MT) radiating from the microtubule organizing center (MTOC) were observed attached to the putative chromosomes.     

THERMAL DEPOLARIZATION OF FLUORESCENCE FROM POLYTENE CHROMOSOMES STAINED WITH ACRIDINE ORANGE

The degree of polarization of fluorescence from stretched Chironomus thummi polytene chromosomes, stained with low concentrations of acridine orange (AO), decreases with increasing temperature. The "half temperature" of this decrease (T_½R) is lower than the expected DNA thermal denaturation temperature (T_m) by about 20°C. T_½R is lowered as histone is removed from chromosomes. Balbiani ring regions of the fourth chromosome have T_½R's much lower than other regions, and nearly as low as chromosomes which had been extensively pretreated with trypsin to remove histone and other proteins. Measurements of the thermal change in the rotational diffusion rate of AO in solution with DNA indicate that the temperature at which the DNA-AO bonding changes from a "rigid" to a "loose" mode varies with the GC percentage of the DNA, and in the same fashion as T_m, although 20°C lower.