Molecular and cytological characteristics of nuclear DNA and chromatin for angiosperm systematics: DNA diversification in the evolution of four orchids

The species- and genus-specific DNA content, average base composition of nuclear DNA, presence or absence of satellite DNA, the percentage of heterochromatin and other characteristics of nuclear DNA and nuclear structure allow to deduce the molecular changes which accompanied, or more probably caused, cladogenesis in the orchids studied. It is suggested that saltatory replication (generative amplification) of certain DNA sequenes, diversification of reiterated DNA sequences, and loss of DNA play an important role in the evolution of orchids.—The relationship between changes of genome composition and of nuclear structure and ultrastructure is discussed on the basis of cot curves, heterochromatin staining with Giemsa (C banding), electron microscopy of nuclei, and molecular hybridization in situ.Some aspects of this paper have been presented at the Helsinki Chromosome Conference, August 1977 (Nagl & Capesius 1977).     

Cell cycle activity during seed priming is not essential for germination advancement in tomato

Seed priming is a technique of controlled hydration and drying that results in more rapid gemination when the seeds are reimbibed. Advancement of radicle meristem cells into the S and G2 phases of the cell cycle, increasing the percentage of nuclei having a 4C DNA content, has been reported to occur during priming. It has been suggested that the efficiency of priming is related to the accumulation of 4C nuclei in the radicle meristem, but the extent of cell cycle activity varied among different treatments and seed lots. A wide range of priming treatments across temperatures, water potentials and durations can be compared on a common basis using the hydrothermal priming time model. Flow cytometry was used to monitor cell cycle activity in a number of tomato (Lycopersicon esculentum Mill.) seed lots during priming in relation to the accumulation of hydrothermal priming time and the subsequent germination rate response. In some seed lots, the percentage of 4C nuclei in the radicle meristems prior to emergence increased in proportion to accumulated hydrothermal priming time, while in other lots, no increase in nuclear DNA content was detected. All lots, however, demonstrated rapid radicle emergence following priming. Thus, replicative DNA synthesis in radicle meristem nuclei often occurred during seed priming, but an increase in the percentage of 4C nuclei was not essential for germination advancement.     

The interphase distribution of satellite DNA-containing heterochromatin in mouse nuclei

The distribution of sites capable of binding mouse satellite-complementary RNA in the cytological hybridization reaction has been examined in mouse liver and testis interphase nuclei. The approach taken has been to combine hybridization with semi-thin sectioning and autoradiography in order to obtain a clear picture of the relationship of satellite DNA-containing structures to the rest of the interphase nucleus. In liver nuclei, hybridization occurs primarily with blocks of heterochromatin associated with the nuclear envelope. The most prominent of these, in terms both of size and intensity of hybridization, is the nucleolar stalk and the rest of the nucleolus-associated heterochromatin. The nucleolar body itself is not labeled, nor is much of the peripheral condensed chromatin ; in fact, a polarized distribution of satellite DNA is evident. In Sertoli and spematid nuclei, satellite DNA is found in a small number of large heterochromatin blocks with which the nucleolus is associated; some of this material bears a relationship to the nuclear envelope in these cells also.     

Extra DNA synthesis in the dedifferentiating cells ofVicia faba roots

Summary Cell dedifferentiation was induced inVicia faba root tissues by removing the whole root meristem (decapitation) and the behaviour of the nuclear DNA in the dedifferentiating cells was studied by means of cytophotometric and autoradiographic analyses. Cytophotometric determination after Feulgen-staining showed that: 1. the vast majority of nuclei in differentiated cells were in the DNA postsynthetic phase, but their Feulgen absorption was lower than that of DNA postsynthetic nuclei (G₂, 4 C) in the meristem; 2. such a Feulgen absorption was detected in certain nuclei after root decapitation; 3. all the mitoses in the dedifferentiating tissues were diploid, fully matching the Feulgen absorption of mitoses in the meristem.After³H-thymidine (³H-T) feeding of the decapitated roots and autoradiography, the following results were obtained: 1. two populations of labeled nuclei, characterized by two different levels of scattered labeling occurred in dedifferentiating tissues, slightly labeled nuclei being much more numerous than heavily labeled nuclei; 2. the percentage of labeled nuclei was much greater than that of DNA presynthetic nuclei in the root tissues; 3. almost all the mitoses were labeled after a 16-hour³H-T feeding; 4. the percentage of slightly labeled nuclei paralleled that of dedifferentiating cells; 5. the duration of the DNA synthesis phase and that of the gap between completion of DNA synthesis and mitosis differed in heavily and slightly labeled nuclei; 6. all nuclei which entered DNA synthesis also entered mitosis.These results are interpreted to mean that: 1. after decapitation, two different DNA syntheses occur in the dedifferentiating root tissues ofV. faba: DNA reduplication in cells which dedifferentiate starting from a DNA presynthetic nuclear condition (heavily labeled nuclei) and extra DNA synthesis in cells which dedifferentiate starting from a DNA postsynthetic nuclear condition (slightly labeled nuclei); 2. extra DNA synthesis is required in these dedifferentiating cells for entry into mitosis.     

Repetitious DNA in some Anemone Species

The DNA from several Anemone species, which contain different amounts of heterochromatin as revealed by Giemsa staining, was analysed by ultra-centrifugation and renaturation. No satellite band was observed in any of the samples centrifuged in cesium chloride gradients. Renaturation studies showed the presence of repetitive sequences. The proportion of repetitive DNA per genome varied from 53% to 67% and did not correlate with either the DNA content per cell or the relative amount of heterochromatin.     

DNA, RNA, protein and heterochromatin changes during embryo development and germination of soybean (Glycine max L.)

DNA, RNA, protein and heterochromatin were measured cytophotometrically in developing soybean (Glycine max) seeds. The average 2C DNA content for the soybean genome was 2.64 pg. The amounts of nuclear DNA in embryo axes showed no significant change during embryo development, whereas the DNA content in cotyledon nuclei increased significantly from 3.58 pg to 5.49 pg. The number of endopolyploid nuclei increased from 26% to 48% and the DNA content from 4.45 to 5.49 pg after cessation of cell division. The changes in RNA and protein content during embryo development were in general similar to those in DNA content. This can be interpreted that increased DNA levels in soybean cotyledons generated during embryogeny increase the protein synthesizing capacity. During the first 15 days of germination, the number of endopolyploid nuclei in cotyledons declined from 46% to 4%, and this decline is interpreted as DNA degradation providing a ready source of nucleosides and phosphates during early embryo growth. A later decline, however, between 15 and 20 days after germination, was age related similar to leaf senescence, because the percentage of endopolyploid nuclei remained unchanged while the number of non-viable cells increased. In senescing cotyledons, 73% and 80% of RNA and protein but only 20% of DNA were lost, as compared to dormant cotyledons. The heterochromatin (condensed chromatin) measurements indicated that nuclei of metabolically inactive dormant and senescent cotyledon nuclei contained an average of 33% more heterochromatin than nuclei from the green cotyledons of seedlings.     

Satellite DNA in the kangaroo Macropus rufogriseus

Two distinct satellite DNAs, amounting to 25% of the total DNA, were isolated from the nuclei of the red-necked wallaby, Macropus rufogriseus. The physical properties of native, single-stranded and reassociated molecules were studied in buoyant-density gradient centrifugation. The homogeneity of each satellite fraction was examined using melting characteristics of native and reassociated DNA, and renaturation kinetics. These data suggest that sequence heterogeneity exists in both fractions. Each satellite fraction was found by in situ hybridization to be localized in heterochromatin of interphase nuclei and in the centromeric regions of metaphase chromosomes. The chromosomal distributions of the two satellite DNAs differentiate the sex chromosomes, which have sequences of only one satellite, from the autosomes which have sequences of both satellites in the centromeric heterochromatin. Giemsa C-banding techniques also showed a differentiation of the centromeric regions of sex chromosomes from those of the autosomes.     

Mouse centromeric heterochromatin: Isolation and some characteristics

A method is suggested for isolation of highly purified mouse centromeric heterochromatin. Treatment of mouse liver nuclei with decreasing concentrations of Ca2+ resulted in the gradual unraveling of chromatin in the nucleus and at 0.1 mM Ca2+ electron microscopy revealed several dense particles per nucleus, surrounded by decondensed chromatin. These particles, assumed to represent centromere regions of interphase chromosomes by in situ hybridization with radioactive mouse satellite DNA and by differential staining for centromere heterochromatin, were isolated in preparative amounts and their DNA and protein composition was analyzed. The preparation represented practically pure mouse centromere heterochromatin, since more than 90% of its DNA was satellite DNA.     

Genetic studies on heterochromatin in Drosophila melanogaster and their implications for the functions of satellite DNA

In Drosophila melanogaster the centromeric heterochromatin of all chromosomes consists almost entirely of several different satellite DNA sequences. In view of this we have examined by genetic means the meiotic consequences of X chromosomes with partial deletions of their heterochromatin, and have found that the amount and position of recombination on each heterochromatically deleted X is substantially different from that of a normal X. It appears that the amount of heterochromatin is important in modifying the centromere effect on recombination. — In all the deleted Xs tested, chromosome segregation is not appreciably altered from that of a nondeleted control chromosome. Thus satellite DNA does not appear to be an important factor in determining the regular segregation of sex chromosomes in Drosophila. Additionally, since X chromosomes with massive satellite DNA deficiencies are able to participate in a chromocenter within salivary gland nuclei, a major role of satellite DNA in chromocenter formation in this tissue is also quite unlikely. — In order to examine the mechanisms by which the amount of satellite DNA is increased or decreased in vivo, we have measured cytologically the frequency of spontaneous sister chromatid exchanges in a ring Y chromosome which is entirely heterochromatic and consists almost exclusively of satellite DNA. In larval neuroblast cells the frequency of spontaneous SCE in this Y is approximately 0.3% per cell division. Since there is no meiotic recombination in D. melanogaster males and since meiotic recombination in the female does not occur in heterochromatin, our results provide a minimum estimate of the in vivo frequency of SCE in C-banded heterochromatin (which is predominantly simple sequence DNA), without the usual complications of substituted base analogs, incorporated radioactive label or substantial genetic content. — We emphasise that: (a) satellite DNA is not implicated in any major way in recognition processes such as meiotic homologue recognition or chromocenter formation in salivaries, (b) there is likely to be continuous variation in the amount of satellite DNA between individuals of a species; and (c) the amount of satellite DNA can have a crucial functional role in the meiotic recombination system.     

Nuclear ultrastructure: Condensed chromatin in plants is species-specific (karyotypical), but not tissue-specific (functional)

Summary In contrast to mammalian cell nuclei those of plants display nearly an identical ultrastructure in all developmental stages and tissues. This indicates that the gross organization of chromatin is species-specific, but not tissue-specific and function-dependent. The species-specific nuclear ultrastructure is determined by the basic nuclear DNA content (2 C value). The higher the DNA content, the more the euchromatin remains in the condensed state during interphase, but to a lower coiling order than the heterochromatin.Some difficulties in the interpretation of electron micrographs of cell nuclei, and the possible role of repetitive DNA sequences in the karyotypical condensation of euchromatin in plants are discussed.     

Anomalous electrophoretic mobility of Drosophila phosphorylated H1 histone: is it related to the compaction of satellite DNA into heterochromatin?

In embryonic nuclei of Drosophila virilis, 45% of the DNA is satellite, and congruent to 50% of the H1 histone is phosphorylated. In polytene salivary gland nuclei, less than 1% of the DNA is satellite, and less than 10tion. The phosphorylated H1's migrate 4% slower than the unphosphorylated H1's on SDS-acrylamide gels. The mobility difference may arise because the phosphorylated and unphosphorylated H1's have different conformations in SDS. This putative conformational difference could be essential to the compaction of satellite DNA into heterochromatin.     

Behaviour of sex chromosome associated satellite DNAs in somatic and germ cells in snakes

Sex chromosome associated satellite DNAs isolated from the snakes Elaphe radiata (sat III) (Singh et al., 1976) and Bungarus fasciatus (Elapidae) (minor satellite) are evolutionarily conserved throughout the suborder Ophidia. An autosome limited satellite DNA (B. fasciatus major satellite) is not similarly conserved. Both types of satellites have been studied by in situ hybridisation in various somatic tissues and germ cells where it has been observed that the W sex chromosome remains condensed in interphase nuclei. In growing oocytes however, the W chromosome satellite rich heterochromatin decondenses completely whilst the autosomal satellite rich regions remain condensed. Later, the cycle is reversed and the W chromosome condenses whilst the autosomal satellite regions decondense. In a primitive snake (Eryx johni johni) where the sex chromosomes are not differentiated and where there is no satellite DNA specific to them, these phenomena are absent. — The differential behaviour of autosomal and sex chromosome associated satellite DNAs is discussed in the light of gene regulation.     

Interactions of mercury and copper with constitutive heterochromatin and euchromatin in vivo and in vitro.

Mouse liver nuclei were fractionated into (condensed) heterochromatin and (noncondensed) euchromatin by differential centrifugation of sonicated nuclei. The fractions were subsequently characterized as unique nuclear species by thermal denaturation derivative profile analysis, which revealed the heterochromatin fraction enriched in satellite DNA and by endogenous metal content, which displayed partitioning of mercury in euchromatin over heterochromatin by a 10:1 ratio, with a comparatively uniform distribution of copper in both fractions. Fractionation of nuclei following in vivo challenge with copper showed enrichment of copper in heterochromatin, relative to euchromatin, while in vivo exposure to mercury resulted in a 20-fold accumulation of mercury in euchromatin, relative to heterochromatin. Using gel filtration and equilibrium dialysis to measure in vitro binding under relatively physiologic conditions of pH (6.0-7.0) and ionic strength (standard saline citrate or saline), the condensed and noncondensed chromatin fractions exhibited binding specificities toward mercury and copper similar to that observed in the in vivo metal challenge experiments. The level of mercury which binds to euchromatin in vitro, when measured either in physiologic [standard saline citrate (SSC)] or in dilute (1:100 SSC) salt solutions, was comparable (approximately 3 mug of Hg/mg of DNA) to that of in vivo euchromatin-bound mercury after 1 month of challenge with dietary metal. In contrast, copper showed little or no preference for the nuclear fractions in dilute salt solutions and displayed patterns which mimic in vivo binding only at higher ionic strengths (saline). Removal of proteins from the chromatin fractions resulted in a loss of binding specificity toward both metals. Therefore, the binding selectivity of condensed and noncondensed chromatin toward both mercury and copper appears to arise from protein or from protein-DNA associations. The state of chromatin condensation is especially critical in the case of copper.     

DNA CONTENT AND HETEROCHROMATIN VARIATIONS IN VARIOUS TISSUES OF PEANUT (ARACHIS HYPOGAEA)

The average 2C DNA amount for the peanut (Arachis hypogaea L.) genome is 4.21 pg, and 73% of the dormant peanut cotyledon nuclei displayed 8C DNA amounts or higher, as compared to 0 to 4% in root-shoot apices and leaf tissue. Thermal melt profiles and heterochromatin values indicated replication of the whole genome. Cotyledon nuclear DNA declined in the percent of polyploid nuclei as well as DNA amounts within ploidy classes during germination. The presence of high DNA C levels in cotyledons generated during embryogeny is interpreted to increase the protein-synthesizing capacity and subsequently supplies a ready source of nucleosides and phosphates during early embryo growth as a result of DNA degradation. However, the later DNA decline at the onset of cotyledon senescence was age related similarly to leaf senescence. The change in proportion of heterochromatin was related to the metabolic state of the tissue and not to the DNA content, as dormant and senescing nuclei contained a higher proportion of heterochromatin as compared to nuclei from metabolically active tissue such as germinating roots. The shift in heterochromatin is interpreted to be involved in gene expression.     

Percent satellite DNA as a function of tissue and age of mice.

A selective loss of satellite DNA was found to occur to different extents as a function of tissue and age of mice using several common DNA extraction and purification procedures. This result emphasizes a serious problem that may be encountered in comparative studies of DNA structure and composition if selective loss of specific DNA sequences occurs. We have developed a DNA extraction and purification procedure that is simple and reliable and gives a high percent DNA yield, which substantially reduces the selective loss of heterochromatin DNA sequences. The method features a centrifugation step of a proteolytic digest of chromatin in 2.4 M CsCl. Percent DNA yield of 82-98% are routinely obtained with no apparent loss of satellite DNA sequences from different tissues or ages of mice. Utilizing this method, percent satellite DNA was found to remain essentially constant at 11 +/- 1% for spleen, kidney, and brain tissues obtained from mice of 10-780 days of age. However, for liver, percent satellite DNA remained at about 7-8% from 10 to 300 days of age and then increased to about 12-13% from 300 to 600 days of age. During this latter time interval (300-600 days), an increase of DNA per nucleus of about 3-fold occurred, due to the formation of tetra- and octaploid cell types. A steady loss in the total number of nuclei per gram of liver as a function of age was also found. These two opposing effects resulted in a nearly constant amount of DNA per gram and per organ for liver throughout the lifespan of the mouse.     

Percent satellite DNA as a function of tissue and age of mice

A selective loss of satellite DNA was found to occur to different extents as a function of tissue and age of mice using several common DNA extraction and purification procedures. This result emphasizes a serious problem that may be encountered in comparative studies of DNA structure and composition if selective loss of specific DNA sequences occurs. We have developed a DNA extraction and purification procedure that is simple and reliable and gives a high percent DNA yield, which substantially reduces the selective loss of heterochromatin DNA sequences. The method features a centrifugation step of a proteolytic digest of chromatin in 2.4 M CsCl. Percent DNA yield of 82–98% are routinely obtained with no apparent loss of satellite DNA sequences from different tissues or ages of mice. Utilizing this method, percent satellite DNA was found to remain essentially constant at 11 ± 1% for spleen, kidney, and brain tissues obtained from mice of 10–780 days of age. However, for liver, percent satellite DNA remained at about 7–8% from 10 to 300 days of age and then increased to about 12–13% from 300 to 600 days of age. During this latter time interval (300–600 days), an increase of DNA per nucleus of about 3-fold occurred, due to the formation of tetra- and octaploid cell types. A steady loss in the total number of nuclei per gram of liver as a function of age was also found. These two opposing effects resulted in a nearly constant amount of DNA per gram and per organ for liver throughout the lifespan of the mouse.     

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.     

VARIATION OF NUCLEAR DNA CONTENT IN HELIANTHUS ANNUUS (ASTERACEAE)

Nuclear 2C DNA content was determined by laser flow cytometry for 13 diploid (2n = 34) lines (cultivated varieties and inbred lines) of Helianthus annuus. Mean DNA amount of second leaf nuclei varied from 6.01 to 7.95 pg (32%) among lines. Mean DNA content varied up to 19% within lines. Variability in mean DNA content exceeding 27% and 48% was detected among leaves from different nodes of plants of the open-pollinated variety, Californicus, and the inbred line, RHA 299, respectively. The root tip and shoot tip nuclei of H. annuus have been reported to consist of a mixture of aneuploid (17 to 33 chromosomes) and diploid (34 chromosomes) cells, a condition called aneusomaty. Chromosome counts of root tips and an analysis of the distribution of DNA content of large numbers of nuclei from leaves indicate that aneusomaty either does not occur, or is not common, among the lines investigated. The intraspecific, intraline, and intraplant variation in DNA content in H. annuus support the concept that a sizable portion of a plant genome is unstable and subject to rapid changes in DNA amount.     

The chromosomal localization of a heavy satellite DNA in the testis of Plethodon c. cinereus

When DNA from blood or liver of Plethodon c. cinereus is centrifuged to equilibrium in cesium chloride it separates out into 2 components. The smaller or satellite component is relatively rich in G + C and is therefore heavy, and it amounts to about 2% of the total DNA. The heavy satellite does not include the ribosomal cistrons, and it is unrelated to the nucleolar organizer. When squash preparations of cells from the testis of P. c. cinereus are incubated in synthetic E³RNA complementary to the satellite DNA, the RNA anneals specifically to the centromeric heterochromatin of spermatogonia, spermatocytes, and spermatids, and to the centromeric regions of all discernible chromosomes. RNA/DNA hybrids were located by autoradiography. H³RNA complementary to the major component of the DNA anneals to all nuclei and to all parts of the chromosomes. H³RNA complementary to nucleolar DNA from Xenopus laevis anneals specifically to the chromatin associated with nucleoli in nuclei at various stages of the meiotic divisions. The nature of the centromeric heterochromatin and its role in the meiotic divisions are discussed.     

Chromocenters of interphase nuclei in the mouse liver contain satellite DNA]

In isolated interphase mouse liver nuclei after hypotonic treatment only the chromocenters belonging to the pericentromeric heterochromatin remain in dense form while the main mass of a chromatin is completely decondensed. The centromeric nature of these chromocenters is demonstrated by their capability for C-banding and for hybridization with a satellite mouse DNA.