A Cytophotometric analysis of the DNA in the nucleus of the giant cell,R-2, in Aplysia

DNA was cytophotometrically measured in Feulgen stained nuclei of R-2, the giant neuron of the abdominal ganglion in Aplysia. The data indicate that the nucleus hag a volume of more than 7 X 10^{6 3} in large animals, and contains as much as 75000 times the haploid amount of DNA. To our knowledge, this is the most highly polyploid nucleus yet described. Furthermore, the amount of DNA increases with growth, going from approximately 2000 times the haploid amount in small animals to over 75000 times in large animals. The data suggest that the increase in DNA occurs in increments, each increment having approximately twice the DNA as the one before. Thus we suggest that the increase in DNA in the nucleus of R-2 results from the entire genome replicating without accompanying cell division.This study was supported by USPHS Grants no. 5R01NS07711 and 5R01-NS08109 and NSF grant no. GB7284. Richard E. Coggeshall is a recipient of Career Development Award 5-K3-GM-31754 and Frank J. Swartz is a recipient of a travelling fellowship GM05079.     

DNA amounts in the nuclei of Paramecium bursaria

DNA amounts in the macronuclei and micronuclei of syngen 4 of Paramecium bursaria were determined. There was a wide distribution of values for the amount of DNA in the macronucleus. This distribution remained wide, but moved to lower values as the animals were starved. The micro-nuclear DNA values were constant under all culture conditions, at a value of 7.5 × 10^–12 g per nucleus. This gave an estimate of the haploid genome size, 2.25 × 10¹² daltons, which is approximately ten times that obtained by DNA renaturation studies.This work was supported by a Commonwealth Scholarship to the author. Thanks are due to the Medical Research Council for funds to purchase the Vickers densitometer.     

Variable transmission rates of a B-chromosome in <Emphasis Type="Italic">Myrmeleotettix maculatus</Emphasis> (Thunb.) (<Emphasis Type="Italic">Acrididae: Orthoptera</Emphasis>)

Amounts of Feulgen staining in individual spermatid and primary spermatocyte nuclei ofTricholioproctia impatiens were measured by the two wavelength method of cytospectrophotometry and compared with Feulgen-DNA values found for bull sperm, taken as a presumed reference standard of 3.24×10^–12 g DNA per nucleus. The amount of DNA estimated for the haploid male genome ofTricholioproctia was 0.39×10^–12 g DNA. This value was used to determine the DNA content and degree of polyteny of Malpighian tubule nuclei sampled from the larval stages of development.     

DNA content of mitotically-active condensed chromosomes of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

Two-wavelength Feulgen microspectrophotometry was used to determine the DNA content of mitotically-active ganglionic cells of first-and thirdinstar larvae of Drosophila melanogaster. The measurements revealed that the DNA values differ, on the average, by a factor of approximately two, with the metaphase cells of the first-instar larvae having about four times the haploid amount of the spermatozoon, and the metaphase cells of the third-instar larvae having about eight times the haploid amount. The increase from 4C to 8C in the course of development without any pronounced modification of the heterochromatic—euchromatic ratio is interpreted as evidence of an increase in the number of chromosomal strands. It is suggested, accordingly, that these mitotically-active chromosomes are multistranded or polynemie.     

Determination of the nuclear DNA content of Saccharomyces cerevisiae and implications for the organization of DNA in yeast chromosomes.

The DNA content of the nucleus of the yeast Saccharomyces cerevisiae has been determined by both renaturation kinetics and DNA per cell measurements. Renaturation kinetics experiments were performed by following the decrease of optical hyperchromicity at 260 nm and by hydroxyapatite chromatography. DNA per cell measurements were made by the diaminobenzoic acid method and by the ethidium bromide method of Klotz &; Zimm (1972b). The conclusion from the above experiments is that the S. cerevisiae nucleus contains 9 × 10⁹ ± 2 × 10⁹ daltons of DNA. Previously we (Lauer &; Klotz, 1975) had measured the molecular weight of the largest piece of DNA in the yeast nucleus to be 2 × 10⁹ ± 0.2 × 10⁹. Here we extend this work by using a more highly protein-denaturing buffer system and conclude that the largest piece of DNA in the S. cerevisiae nucleus contains 1.5 × 10⁹ to 2.2 × 10⁹ daltons of DNA in both haploid and diploid cell lysates. From genetics, the largest yeast chromosome should contain 13% of the genome, or 0.9 × 10⁹ to 1.5 × 10⁹ daltons of DNA (using our DNA per cell range). Thus, the large DNA we measure contains from one to two times the amount of the DNA predicted from genetics to be in the largest chromosome. In light of these new data, viscoelastic measurements on yeast DNA are now consistent with the idea that each chromosome contains one piece of DNA.     

Evidence for dosage compensation in parthenogenetic Hymenoptera

Amount of DNA-Feulgen staining in individual somatic nuclei and mature sperm of the parthenogenetic wasps, Habrobracon juglandis, H. serinopae, and Mormoniella vitripennis, were determined with a scanning microdensitometer. The haploid genome for both species of Habrobracon was estimated to be 0.15–0.16×10^–12 g DNA, corresponding to a molecular weight of roughly 10×10¹⁰ daltons. The haploid genome of M. vitripennis is approximately twice this value, 0.33–0.34×10^–12 g, or about 20×10¹⁰ daltons. Measurements made on dividing nuclei from syncytial preblastoderm embryos of H. juglandis and M. vitripennis showed that the chromosomes of impaternate males were present in the haploid number and contained the C amount of DNA; whereas nuclei from female preblastoderm embryos contained the diploid number of chromosomes and the 2C amount of DNA. However, hemocyte and brain cell nuclei from either male or female adult wasps contained 2C and 4C amounts of DNA. Both sexes also showed equivalent levels of polyploidy (8C, 16C, or 32C) in Malpighian tubule nuclei. Therefore, in these parthenogenetic species, a mechanism must exist that compensates during later development for the initial two-fold difference in the chromatin content of somatic nuclei in haploid male and diploid female embryos. Hemocytes from impaternate Mormoniella diploid males and triploid females contain the 2C and 3C amounts of DNA, respectively. Therefore dosage compensation involves an additional cycle of DNA replication only in haploid cells, and it insures that a certain minimum quantity of DNA is received by each somatic cell.     

DNA content of mitotically-active condensed chromosomes of Drosophila melanogaster

Two-wavelength Feulgen microspectrophotometry was used to determine the DNA content of mitotically-active ganglionic cells of first-and thirdinstar larvae of Drosophila melanogaster. The measurements revealed that the DNA values differ, on the average, by a factor of approximately two, with the metaphase cells of the first-instar larvae having about four times the haploid amount of the spermatozoon, and the metaphase cells of the third-instar larvae having about eight times the haploid amount. The increase from 4C to 8C in the course of development without any pronounced modification of the heterochromatic—euchromatic ratio is interpreted as evidence of an increase in the number of chromosomal strands. It is suggested, accordingly, that these mitotically-active chromosomes are multistranded or polynemie.This study was supported by a Research Grant (GM 10499) from the National Institutes of Health, U. S. Public Health Service.     

The Role of Cytokinin in the Regulation of Growth, DNA Synthesis and Cell Proliferation in Cultured Soybean Tissues (Glycine max var. Biloxi)

Changes in protein content and cell proliferative activity were followed after a cytokinin-requiring strain of cultured Glycine max tissue was transferred to freshly prepared media which either contained or lacked cytokinin. Cell numbers doubled within the first two days after transfer, both in the presence and absence of cytokinin. However, after the second day no further increase in cell number was observed in the absence of cytokinin, while cell numbers continued to increase logarithmically in the presence of cytokinin. The size of the cell population attained after the first six days of growth was a function of the cytokinin concentration of the culture medium. However, the amount of ³H-thymidine incorporated into nuclear DNA bore no relation to the rate of cell proliferation. Tissues cultured on medium lacking cytokinin incorporated the greatest amount of ³H-thymidine per microgram of DNA, while the actively dividing tissues incorporated somewhat less. Using autoradiography and isopycnic CsCl gradient centrifugation, it was shown that the radioactivity derived from ³H-thymidine was associated with nuclear DNA in the cytokinin-deprived cells. Biochemical measurements demonstrated that cells cultured for six days without cytokinin had approximately twice the DNA content of the actively proliferating cells cultured on cytokinin-containing medium. Furthermore, in autoradiographs labeled cells were found to average nearly three times as many silver grains per nucleus in tissues cultured without cytokinin as the cytokinin-grown tissues. This suggests that the ³H-thymidine incorporation in the non-proliferating soybean cells results from nuclear DNA synthesis and that some of the cells became polypoid in the absence of cytokinin. These findings would be consistent with the idea that cytokinin acts as a specific trigger for cytokinesis.     

Genome Size, Complexity, and Ploidy of the Pathogenic Fungus Histoplasma capsulatum

The genome size, complexity, and ploidy of the dimorphic pathogenic fungus Histoplasma capsulatum was determined by using DNA renaturation kinetics, genomic reconstruction, and flow cytometry. Nuclear DNA was isolated from two strains, G186AS and Downs, and analyzed by renaturation kinetics and genomic reconstruction with three putative single-copy genes (calmodulin, α-tubulin, and β-tubulin). G186AS was found to have a genome of approximately 2.3 × 10⁷ bp with less than 0.5% repetitive sequences. The Downs strain, however, was found to have a genome approximately 40% larger with more than 16 times more repetitive DNA. The Downs genome was determined to be 3.2 × 10⁷ bp with approximately 8% repetitive DNA. To determine ploidy, the DNA mass per cell measured by flow cytometry was compared with the 1n genome estimate to yield a DNA index (DNA per cell/1n genome size). Strain G186AS was found to have a DNA index of 0.96, and Downs had a DNA index of 0.94, indicating that both strains are haploid. Genomic reconstruction and Southern blot data obtained with α- and β-tubulin probes indicated that some genetic duplication has occurred in the Downs strain, which may be aneuploid or partially diploid.     

Cell-cycle dependency of radiosensitivity and mutagenesis in fertilized egg cells of rice,Oryza sativa L.

Summary To determine the time and duration of the first and second DNA synthetic phases in fertilized egg cells and central cells of rice, a total of 753 ovules were sampled at 2 h intervals during the first 30 h after pollination and exposed to ³H-thymidine for 2 h at 25 °C. Autoradiographic observation of labeled nuclei was made for fertilized egg cells, as well as for central and antipodal cells. The first and second DNA synthetic phases in fertilized egg cells were found 8–12 h and 21–25 h after pollination, respectively. The durations of each cell-cycle phase in the egg cell were estimated to be 4–6 h for G₁, 4 h vor S and for G₂, and 2 h for M. In the central cell, the first DNA synthesis took place at 3–4 h after pollination, i.e., immediately after fertilization, followed by the formation of the primary endosperm nucleus. Antipodal cells also showed labeled nuclei in the early stages after fertilization. The first divisions of fertilized egg cell and primary endosperm nucleus were observed at 16–18h and at 4–6 h after pollination, respectively. The present observations suggest that sperm and egg nuclei participate in fertilization with haploid amount (1C) of DNA and fertilized egg cell originates thus in 2C state.     

The replicative organization of DNA in polytene chromosomes ofDrosophila hydei

Salivary-gland nuclei ofDrosophila hydei were pulse-labeledin vitro with³H-thymidine and studied autoradiographically in squash preparations. The distribution of radioactive label over the length of the polytene chromosomes was discontinuous in most of the labeled nuclei; in some nuclei the pattern of incorporation was continuous. Comparison of the various labeling patterns of homologous chromosome regions in different nuclei showed that specific replicating units are replicated in a specific order. By combining autoradiography with cytophotometry of Feulgen-stained chromosomes, it was possible to correlate thymidine labeling of specific bands with their DNA content. The resulting data indicate that during the S-period many or perhaps all of the replicating units in a salivary-gland nucleus start DNA synthesis simultaneously but complete it at different times. Furthermore, the data support the hypothesis that the chromomere is a unit of replication or replicon. The DNA content of haploid chromomeres was found to be about 5×10^-4 pg for the largest bands inDrosophila hydei. From the results of H³-thymidine autoradiography and Feulgen-cytophotometry on neuroblast and anlage nuclei it was concluded that during growth of the polytenic nucleus heterochromatin is for the most part excluded from duplication. The results of DNA measurements in interbands of polytene chromosomes do not agree with a multistrand structure for the haploid chromatid. A chromosome model is proposed which is in accordance with the reported results and with current views concerning the replicative organization of chromosomes.     

Characterization of postreplication repair in Saccharomyces cerevisiae and effects of rad6, rad18, rev3 and rad52 mutations

Summary Postreplication repair of nuclear DNA was examined in an excision defective haploid strain of yeast lacking mitochondrial DNA (ral ⁰). The size of the DNA synthesized in cells exposed to various fluences of ultraviolet light (UV) corresponds approximately to the average interdimer distance in the parental DNA. Upon further incubation of cells following exposure to 2.5 J/m², the DNA increases in size; by 4 h, it corresponds to DNA from uniformly labeled cells. The alkaline sucrose sedimentation pattern of DNA pulse labeled at various times after UV irradiation, for up to 4 h, does not change substantially, indicating that dimers continue to block DNA replication. A significant amount of postreplication repair requires de novo protein synthesis, as determined by its inhibition by cycloheximide. The rad6 mutant does not carry out postreplication repair, the rad18 and rad52 mutants show great inhibition while the rev3 mutation does not affect postreplication repair. Both recombinational and nonrecombinational repair mechanisms may function in postreplication repair and most of postreplication repair is error free.     

DNA sequence organization in the genome of Cycas revoluta

The pattern of DNA sequence organization in the genome of Cycas revoluta was analyzed by DNA/DNA reassociation. Reassociation of 400 base pair (bp) fragments to various C₀t values indicates the presence of at least four kinetic classes: the foldback plus very highly repetitive sequences (15%), the fast repeats (24%), the slow repeats (44%), and the single copy (17%). The latter component reassociates with a rate constant 1×10^–4 M^–1S^–1 corresponding to a complexity of 1.6× 10⁶ kb per haploid genome. A haploid C. revoluta nucleus contains approximately 10.3 pg DNA. The single-copy sequences account for about 28% of the DNA, but only 17% reassociate with single-copy kinetics because of interspersion with repetitive sequences. — The interspersion of repetitive and single-copy sequences was examined by reassociation of DNA fragments of varying length to C₀t values of 70 and 500. A major (65%) and homogeneous class of single-copy sequences averaging 1,100 bp in length is interspersed in a short period pattern with repeated sequences. A minor (35%) heterogeneous single-copy component is interspersed in a long-period pattern. The majority of repetitive sequences have a length distribution of 100–350 bp with subclasses averaging 150 and 300 bp in length. Repeat sequences with a wide range in sizes exceeding 2 kilobase pair (kb) are also present in this genome. — The size and distribution of inverted repeat (ir) sequences in the DNA of C. revoluta were studied by electron microscopy. It is estimated that there are approximately 4 × 10⁶ ir pairs (one per 2.33 kb) that form almost equal numbers of looped and unlooped palindromes. This high value is 2.5 times that found in wheat DNA. These palindromes are in general randomly distributed in the genome with an average interpalindrome distance of 1.6 kb. The majority (about 85%) of ir sequences of both types of palindromes belong to a main-size class, with an average length of 210 bp in the unlooped and and 163 bp in the looped type. These values are comparable to those reported for some other plant and animal genomes. Distribution of length of single stranded loops showed a main-size class (75%) with an average length of 220 bp.     

The DNA of Arabidopsis thaliana

Summary Arabidopsis thaliana is a small flowering plant of the mustard family. It has a four to five week generation time, can be self- or cross-pollinated and bears as many as 10⁴ seeds per plant. Many visible and biochemical mutations exist and have been mapped by recombination to one of the five chromosomes that comprise the haploid karyotype. With the experiments reported here we demonstrate that Arabidopsis has an extraordinarily small haploid genome size (approximately 7×10⁷ nucleotide pairs) and a low level of cytosine methylation for an angiosperm. In addition, it appears to have little repetitive DNA in its nuclear DNA, in contrast to other higher plants.     

Die Verteilung der Chromosomen auf die Tochterzellkerne multipolarer Mitosen in euploiden Gewebekulturen von Microtus agrestis

In kidney epithelial cultures from female Microtus agrestis, 3,55% of all mitoses are multipolar, 94% of them tripolar. Feulgen photometric measurements of 21 tripolar mitoses reveal a total DNA amount corresponding to the mitotic diploid value (4c) in 5 cases, and to the tetraploid value (8c) in 16 cases, Diploid tripolar mitoses divide into one daughter nucleus with a diploid DNA value (2c) and two nuclei each with a haploid DNA value (1c). Most tetraploid tripolar mitoses divide into one daughter nucleus with a diploid DNA value (2c) and two nuclei with a triploid DNA value (3c). Also the sex chromosomes are distributed to the daughter nuclei in the relation of 2∶3∶3. This can be seen in anaphase figures as well as in interphase nuclei presumably derived from tripolar mitoses, showing chromocenters according to the number of X-chromosomes. In two cases of tripolar tetraploid mitoses the resulting nuclei have a haploid, a triploid and a tetraploid DNA value. The DNA replication pattern is always identical in the daughter nuclei of diploid and tetraploid tripolar mitoses. — Our observations suggest segregation and distribution of haploid chromosome sets or multiples of haploid sets to the daughter nuclei of multipolar mitoses. They also show a possible way of formation of haploid and triploid cells in a basically diploid tissue. Presumably triploid nuclei (with 3 chromocenters) are capable of DNA synthesis.     

Apogamic induction of haploid plasmodia in a myxomycete,Didymium iridis

Interisolate crosses between haploid (mean DNA = 0.32) CR 5-5 (A²) myxamoebae and polyploid (mean DNA = 1.80) CR 2–25 (A⁵) myxamoebae of the myxomycete Didymium iridis result in plasmodia that have the haploid (mean DNA = 0.32) DNA content rather than the predicted polyploid value. F₁ clones possess the mating type allele of the CR 5-5 clone only, and they also have the same mean DNA content as CR 5-5 myxamoebae. Crosses between these F₁ clones and CR 2–25 myxamoebae again resulted in the production of haploid plasmodia. Hence, the polyploid CR 2–25 clone appears to induce the CR 5-5 clone to produce plasmodia without involving itself in nuclear fusion.     

Induction of host nuclear DNA synthesis in coccidia-infected chicken intestinal cells

When Eimeria maxima (gamonts) infects villus epithelial cells of the chicken duodenum there is extensive cellular enlargement with no alteration in nuclear size. Feulgen DNA microspectrophotometric measurements indicated that the infected host-cell nucleus contains the same amount of DNA as an uninfected cell nucleus. Evidence is presented to indicate that second generation schizonts of E. necatrix develop in crypt epithelial cells that are displaced/migrate into the lamina propria. The developing parasite causes cellular and nuclear hypertrophy in these cells as does E. tenella in cecal cells of the chicken. In these two cases nuclear enlargement is accompanied by induced rounds of DNA synthesis in the host-cell. Analyses indicated that the DNA content of enlarged nuclei does not fall into classes that correspond to a geometric series 2:4:6:8:16: etc. times the DNA content of a 2C equivalent, and that nuclear size and DNA content in infected cells are not significantly correlated. Autoradiographic studies on E. necatrix infected chicks administered ³H-thymidine show that DNA synthesis takes place in the nuclei of cells containing all developing stages but not mature schizonts, and that this synthesis is not a continuous process. The data suggest that intestinal cells that are capable of undergoing cell division and therefore additional rounds of DNA synthesis, can be induced by coccidial infection in the absence of concomitant cell division.     

Mechanism of Accumulation of DNA in Giant Cells of Mouse Trophoblast

IN rodents, the foetal part of the placenta contains giant trophoblast cells which are unique among mammalian cell types in that each nucleus contains several hundred times the haploid amount of DNA^1–4. We have investigated the mechanism by which this DNA is accumulated, in order to understand its relation to trophoblast function. Galassi⁵ suggested that engulfment of maternal cells might be responsible for the formation of the giant nuclei, while Avery and Hunt⁶ raised the possibility that diploid trophoblast cells fused. Recent studies^4,7 make both these possibilities seem unlikely. On the basis mainly of cytological observations, Zybina¹ has proposed that giant trophoblast nuclei arise in the rat by a series of endoreduplications, that is replication of the genome without subsequent mitosis and cell division. Her claim⁸ that polytene chromosomes⁹ could be seen in these nuclei was not supported by our studies on mouse trophoblast⁴.     

Differentiation trees,a junk DNA molecular clock,and the evolution of neoteny in salamanders

Obligate neotenic salamanders die if forced to metamorphose. We suggest that this can be explained by assuming: 1) their “excess” DNA is “junk” DNA; 2) the “adult” specifying portion of the DNA becomes junk DNA and is available for repeated duplication. This suggests a “new” junk DNA molecular clock. We obtain remarkable agreement in “predicting” the amount of DNA per nucleus in present day non-obligate neotene salamanders from this molecular clock. These observatons are consistent with the idea that the development of these animals is describable in terms of differentiation trees whose branches (gene cascades) corresponding to adult somatic tissues accumulate deleterious mutations over evolutionary time. We show that the amount of DNA per nucleus increases linearly with the phylogenetic age of salamander families. The lack of constraints by natural selection, on unused adult branches, may account for the large amount of so-called “junk DNA” in obligate neotenic salamanders. The effects of this excess DNA, via increased cell size, suggest a positive feedback, ecophysiological explanation for such junk DNA: adaptation to cool water environments is enhanced by the lower metabolism associated with more DNA, larger cells and slower developmental time.     

H-thymidine incorporation into nuclear DNA of leaf cells

The incorporation of ³H-thymidine into nuclear DNA of leaf cells of Nanthium pennsylvanicum was studied as a function of concentration and specific activity of the radioisotope. From the assessment of the average number of grains per nucleus and the percent of labeled nuclei, it was concluded that the incorporation was a linear function of concentration of the exogenous radioisotopic solution and a logarithmic function of the incubation time. Ten microcuries per milliliter on the average yielded 20% of labeled nuclei with 18 grains per nucleus. Seven-fold increase in concentration only doubled the amount of ³H-thymidine incorporated. The lamina regions near the vein incorporated a significantly greater amount of the radioisotope than the lamina region at some distance from the vein. The specific activities of 2, 3.35, 6.7 and 15.3 c/mmole had no effect upon the amount of ³H-thymidine incorporated, if the amount of microcuries of the incubation solution was the same in each activity. Considering the total number of molecules, the estimated rates of incorporation indicated that at the activity of 2 c/mmole, the system operated with about 7 times higher rates as compared with the activity of 15.3 c/mmole.