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.     

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.     

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.     

A Cytophotometric analysis of the DNA in the nucleus of the giant cell,R-2, in <Emphasis Type="Italic">Aplysia</Emphasis>

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 10⁶ μ³ 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.     

CYTOLOGICAL CONSEQUENCES OF DNA AMPLIFICATION IN AN ANTHER CULTURE-DERIVED DOUBLED HAPLOID LINE OF NICOTIANA TABACUM

An increase in nuclear DNA, without an increase in chromosome number, has been found to occur as a result of anther culture in Nicotiana tabacum L. The objective of this study was to determine the cytological consequences of this DNA amplification in F₁ hybrids between a doubled haploid that had undergone a substantial increase in DNA and the cultivar from which that doubled haploid was derived. Mitotic and meiotic analyses were performed on plants obtained from reciprocal crosses of N. tabacum cv. NC95 and NC95 SCDHL 12, a doubled haploid line that has 41% more DNA than the parental cultivar. While no cytological abnormalities were observed in either parental line, numerous abnormalities were seen in both somatic and meiotic tissues of the F₁ hybrid. Chromosome losses, which appeared to result from spindle errors, were observed in these tissues. It is speculated that the spindle errors may be the result of a genetic unbalance caused by combining genomes with widely differing amounts of DNA. In addition to the spindle errors, a quadrivalent with an atypical morphology was observed in meiotic diplotene and metaphase I cells of the hybrid. The quadrivalent configuration was interpreted to represent pairing between amplified homologous regions in homeologous chromosomes. Further investigations of additional doubled haploid × cultivar lines is required before the significance of these findings to the anther culture process in N. tabacum can be fully assessed.     

The ovulation and activation of primary and secondary oocytes in LT/Sv strain mice

Eggs were isolated from the oviducts or ovaries of LT/Sv strain mice in order to investigate the pathways taken by them following spontaneous or induced parthenogenetic activation. The chromosome preparations from the ovarian oocytes that matured in vitro to metaphase I were all morphologically normal. Of 42 recently ovulated eggs that failed to activate parthenogenetically in culture, 57% on nuclear densitometric analysis were found to have the normal 2C amount of DNA, and 1N (haploid) number of chromosomes present, and were arrested at metaphase II. Somewhat unexpectedly, 43% had a 4C amount of DNA, and 2N (diploid) number of chromosomes present, had been arrested at metaphase I, and were evidently ovulated as primary oocytes. Following parthenogenetic activation, the majority of oocytes extruded a polar body and developed a single pronucleus. The activated eggs could be divided into two sub-populations according to the diameter (and therefore volume) of the pronucleus—in one group this was about one-third greater than in the other. The chromosome constitution of the two groups was determined separately at the first cleavage mitosis. Those with a normal-sized pronucleus were invariably haploid, while those with an enlarged pronuclear volume were invariably found to be diploid. The chromosomes in the diploid spreads often appeared to be associated in homologous pairs. We conclude that almost uniquely in LT/Sv strain females eggs may be activated parthenogenetically at either stage of meiotic maturation giving rise to diploid or haploid embryos, respectively.     

Cytophotometric and autoradiographic evidence for functional apomixis in a gynogenetic fish,Poecilia formosa and its related,triploid unisexuals

Summary Amounts of DNA in individual Feulgen-stained nuclei from squash preparations of ovaries and testes from wild-caught and laboratory-reared stocks of Poecilia spp. were determined with an integrating microdensitometer. The DNA content of primary spermatocytes (4C) at zygotene, pachytene, or at metaphase I (3.3–3.4 pg) was approximately twice that found in secondary spermatocytes (2C) and four times that found for young spermatids (1C). Rarely, mature sperm were found with 2C DNA amounts. Nuclei from follicular epithelium and oogonia from both bisexual and diploid unisexual fish contained about 1.6–1.7 pg DNA; whereas, the DNA content of primary oocyte nuclei was about 3.5–3.7 pg DNA, indicating that just one cycle of chromosomal replication had occurred in these cells during the period of DNA synthesis before the visible onset of meiotic prophase. Similar results were obtained for triploid unisexuals whose 6C primary oocyte nuclei contained 5.0–5.1 pg DNA, which was twice the DNA content of 3C oogonia and follicular epithelial cells (2.4–2.5 pg DNA). Autoradiographic studies, designed to monitor the incorporation of ³H-thymidine by oogonia and primary oocytes in vivo and in vitro, also showed that there is no additional synthesis of DNA during the course of meiotic prophase in these unisexual fish. Therefore, we conclude that apomixis, not endoreduplication, is the cytological basis of reproduction in Poecilia formosa and its related, triploid biotypes.     

Nucleotide sequence of a highly repeated DNA sequence and its chromosomal localization in Allium fistulosum

A highly repeated DNA sequence with a repeating unit of approximately 380bp was found in EcoRV digests of the total genomic DNA of Allium fistulosum. Three independent clones containing this unit were isolated, and their repeating units sequenced. These units showed more than 94% sequence homology, and the copy number was estimated to be about 2.8×10⁶ per haploid genome. In situ hybridization, with the repeating unit as a probe, and C-banding analyses indicated that the repeated DNA sequence of A. fistulosum is closely associated with the major C-heterochromatin in the terminal regions of all 16 chromosomes at mitotic metaphase. The characters of the repeating unit are similar to those of the A. cepa unit, which is taxonomically closely related to A. fistulosum.     

Intraspecific variation in nuclear DNA content among world populations of a mosquito,Aedes albopictus (Skuse)

Summary Aedes albopictus is commonly distributed in most parts of the Oriental region and on many islands in the Indian and the Pacific Oceans. The species was recently introduced into the United States and Brazil. Feulgen cytophotometric quantitation of haploid nuclear DNA content was carried out for 37 populations of Ae. albopictus to determine the extent of intraspecific variation in nuclear DNA content and whether the range expansion of the species has coincided with an increase in DNA content. The haploid nuclear DNA content varied nearly three-fold. The minimum DNA content was 0.62 pg in Koh Samui from Thailand, and the maximum DNA content was 1.66 pg in Houston-61 from the United States. Statistical comparisons of populations revealed significant differences in DNA contents. No geographic clustering of populations was noted with respect to DNA content. In general, populations from the United States and Brazil had higher DNA contents, but there was no indication that the range expansion had occurred hand in hand with an increase in DNA content. Each population had a specific amount of DNA that is probably imposed by the microenvironment.     

PLOIDY ANALYSIS OF THE TWO MOTILE FORMS OF CHRYSOCHROMULINA POLYLEPIS (PRYMNESIOPHYCEAE)1

In some cultures of the flagellate Chrysochromulina polylepis Manton et Parke, established from cells isolated from the massive bloom in Skagerrak and Kattegat in 1988, we observed, two motile cell types. They were termed authentic and alternate cells and differed with respect to scale morphology. To investigate whether or not the two cell forms were joined in a sexual life cycle, the relative DNA content per cell and relative size of cells of several clonal cultures of C. polylepis were determined by flow cytometry. Percentages of authentic and alternate cells in the cultures were estimated by transmission electron microscopy. Pure authentic cultures (α) contained cells with the lowest level of DNA and were termed haploid. Two pure alternate cultures (β) contained cells with double the DNA content of authentic cells and were termed diploid. Other pure alternate cultures contained haploid cells only, or both haploid and diploid cells. Three cell types were observed, each capable of vegetative propagation: authentic haploid, alternate haploid, and alternate diploid cells. Both the haploid and diploid alternate cells were larger than the haploid authentic cells. Cultures containing diploid cells appeared unstable: cell type ratio and ploidy ratio changed during the experiment where this cell type was present, particularly when grown in continuous light. In contrast, cultures with only haploid cells remained unchanged at all growth conditions tested. Light condition may influence cell type ratio and ploidy ratio. Our attempt to induce syngamy by mixing different authentic haploid clones did not result in mating. Assuming that the authentic and alternate cell types are of the same species, the life cycle of C. polylepis includes three flagellated scale-covered cell forms. Two of the cell types are haploid and may function as gametes, and the third is diploid, possibly being the result of syngamy.     

Mitotic polyploidization of mouse heart myocytes during the first postnatal week

Summary Polyploidization of myocytes in the cardiac ventricle of mice occurs predominantly during the first week of the postnatal life. Using isolated cells it was shown that about 70 % of the cardiomyocytes become binuclear at this time (2c×2), while 10 % are mononuclear but contain 4c of DNA, where c was the haploid level of DNA. About 2 % of the cell population were represented by 4c×2 or 8c cells.Cytophotometry of Feulgen-stained DNA in ¹⁴C-thymidine-labeled nuclei has shown that the cells that enter the mitotic cycle are mainly diploid. After mitosis (30 h or more after thymidine application) the label was found predominantly in 2c×2 and 4c cell types. Comparison of the curves presenting dynamics of labeled mitoses and the accumulation of labeled binuclear cells reveals that binuclear 2c×2 cells are formed by acytokinetic mitosis. The formation of 4c mononuclear cells is accomplished via other types of mitotic arrest; this may be due, for example, to a block in the pro-or metaphase.Only very rare cases of cytotomy were detected and the number of newly formed 2c cells was very low. It is concluded that cell multiplication is practically arrested at this period of life, and growth of the ventricular mass is due to polyploidization of virtually all cycling cells, while their number remains unchanged. Mechanisms and functional significance of cardiomyocyte polyploidization are discussed.     

Cytological evidence of spontaneous androgenesis in the freshwater clam Corbicula leana Prime

 Cytological observations and DNA microfluorometry of the hermaphrodite freshwater triploid clam Corbicula leana revealed unusual androgenetic development as follows: (1) the maternal genome of zygotes was extruded as two polar bodies just after karyokinesis of the first meiosis, (2) only chromosomes derived from one male pronucleus constituted the metaphase of the first cleavage of zygotes, (3) DNA content of 7-day-old veliger larvae was identical to the somatic cells of the parent. This spontaneous androgenetic process in C. leana zygotes is the first case in the phylum Mollusca and may be related to the specialized mode of reproduction; i.e. hermaphroditism and self-fertilization. Received: 22 September 1997/Accepted: 7 December 1997     

Endomitosis and the effect of gibberellic acid in different Pisum sativum L. cultivars

The evolution of the total amount of DNA in epicotyls and of the amount of DNA per cell nucleus in epicotyl cortex cells during germination was followed in two closely related pea varieties, Pisum sativum cv. Finale and Pisum sativum cv. Rondo. Under etiolating conditions, growth of the cv. Rondo occurs only by cell elongation which is preceded by endomitotic DNA synthesis, while in the cv. Finale growth is the result of cell elongation accompanied by endomitotic DNA synthesis and cell division. The maximum C-level attained in both cultivars under etiolating conditions is 8 C (C=haploid amount of DNA in a gamete cell). Both the maximum C-level reached and the percentage of cells reaching this C-level seem to be under strict genetic control. In both cultivars, light inhibits the endomitotic DNA replication.Neither gibberellic acid (GA₃), nor AMO 1618 alter the maximum C-level or the percentage distribution of the C-classes. Both growth regulators are effective, although in an opposite way, only in tissues where cell division occurs or where endomitotic DNA synthesis is blocked, as in light-grown pea epicotyls.     

Chromosome-sized DNA molecules from Drosophila

Measurements of viscoelastic retardation times of detergent-Pronase lysates of Drosophila cells demonstrated the presence of large numbers of DNA molecules of a size commensurate with that of the chromosomes. The values estimated from the retardation times for the molecular weights of the largest molecules ranged from about 20×10⁹ to 80×10⁹ daltons depending on the species of Drosophila. The molecular weights of the DNA molecules were independent of the metaphase shapes (i.e., metacentric or submetacentric), but were proportional to the DNA contents of the chromosomes in the case of translocations or deletions. It was concluded, therefore, that the DNA molecules must run the length of the chromosome and cannot be discontinuous at the centromere. When compared with the values of the DNA contents of Drosophila chromosomes determined by other methods, the results were consistent with the model of one, or possibly two, DNA molecules per chromosome; the simplest conclusion, that there is only one DNA molecule per chromosome (for simple chromosomes), rests on a long extrapolation of an empirical relation between retardation time and molecular weight, but is also favored by indirect evidence. Further possibilities which could not be excluded were that the large DNA molecules contained Pronase-resistant, non-DNA links, or that a fraction of smaller DNA molecules might also have been present in the chromosomes. Chromosome-sized DNA molecules were obtained almost quantitatively from unsynchronized cultured cells, suggesting that the size of the chromosomal DNA is conserved throughout much of the cell cycle. The molecules were stable for periods of up to several days at 50° C in solutions containing detergent, Pronase, and EDTA.     

Repair of DNA double-strand breaks requires two homologous DNA duplexes

DNA repair and cell survival in haploid and its diploid derivative strains ofSaccharomyces cerevisiae were studied after 100 krad X-ray irradiation. The cells were in theG ₁ stage of the cell cycle, where haploid cells had only one copy of genetic material per genome and diploid had two copies. It was found that diploid could repair double-strand breaks in its DNA after 48 hr of liquid holding which was accompanied by a four-fold rise in survival. In contrast a haploid strain failed to repair its DNA and showed no increase in survival after liquid holding. It is concluded that (1) repair of DNA double-strand breaks requires the availability of two homologous DNA duplexes, (2) restoration of cell viability during liquid holding is connected with repair of DNA double-strand breaks and (3) this repair is a slow process possibly associated with slow finding and conjugation of homologous chromosomes.     

Relative ribosomal RNA cistron multiplicity in oocytes and postembryonic stages of the eutelic nematodePanagrellus silusiae

Summary The number of ribosomal RNA cistrons has been measured in the total DNA extracted from L2 juvenile and adult stages of the free-living nematodePanagrellus silusiae. Saturation hybridization studies with homologous rRNA indicate that both stages have about 275 ribosomal genes per haploid equivalent. Using homologous¹²⁵I-labelled rRNA for in situ hybridization, the mean number of silver grains per DNA content for oocyte, hypodermis and gut nuclei was similar. The mean DNA contents of maturing oocyte, hypodermis and gut nuclei are about 20C, 2C, and 10C respectively. We conclude that rDNA amplification alone is insufficient to account for the variation in DNA content of oocytes and that postembryonic development in this eutelic organism occurs without a significant differential increase in the number of ribosomal cistrons per worm.Supported by the National Research Council of Canada     

Haploid and diploid cell cultures from a haplo-diploid insect

Summary

This is the first report of haploid and diploid cell culture from the haplo-diploid parasitoid wasp, Mormoniella vitripennis. Cells were cultured from haploid and diploid wasps by collecting populations of eggs from virgin females (unfertilized, haploid, parthenogenetic eggs) and mated females (mostly fertilized, diploid eggs). Eggs were surface sterilized in 70% ethanol, followed by 50% Chlorox, and rinsed in phosphate buffered saline; larvae were allowed to hatch in culture. Larval cells were dissociated and cultured at 28°C in the presence of Grace's medium supplemented with fetal bovine serum. Most cells in the HMV (predominantly haploid) and DMV (predominantly diploid) cell cultures grew in suspension in the first week, formed monolayers of fibroblasts and epithelial cells by the second week in culture, and continued to grow in monolayers and vesicle-like structures for up to three months. Chromosome analysis of HMV. cells demonstrated over 70% haploid cells, with five chromosomes (N=5). The remainder were aneuploid. No diploid cells (2N= 10) were found in the HMV cell culture. Chromosome analysis of DMV cultures revealed 62% diploid, with ten chromosomes; 13% were haploid, with five chromosomes; the remainder were aneuploid. These data confirm that haploid and diploid cells can be cultured from a haplo-diploid insect species. The HMV cells which are predominantly haploid, and DMV cells which are predominantly diploid may be valuable models for the study of cellular and gene activity in haploid and diploid genetic milieux.     

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.     

MEIOSIS WITH TELOMERIC PAIRING IN A HAPLOID OF TRITICUM MONOCOCCUM (x = 7)

In the microsporocytes of a haploid of Trilicum monococcum (x = 7), foldback and other nonhomologous pairing was observed at pachytene. At the diplotene equivalent stage of meiosis, nonhomologous chromosomes were connected by their telomeres in associations involving two to seven chromosomes. Telomeric connections were Feulgen-positive for DNA and were disjoined by metaphase I. These connections may have resulted from earlier base-pairing of repeated sequences of guanine-rich telomere overhangs of nonhomologous chromosomes. Recent molecular studies of several widely divergent organisms have shown that all telomeres of nonhomologous chromosomes in a genome are identical, and telomere structure is conserved among widely divergent eukaryotes. Chromosome distribution at anaphase I fitted theoretical expectations of random movement of each of the seven chromosomes to one or the other of the two poles as did pollen fertility (stainability) resulting from such distribution. A single bivalent in 3.78% of the metaphase I cells provided evidence for a duplication in the genome of Triticum monococcum.     

Genomic organization in the flesh fly Sarcophaga bullata

The genome of the flesh fly Sarcophaga bullata has been characterized both cytologically and biochemically. S. bullata has a haploid DNA level of 0.61 picograms which is five times larger than the haploid genome size of Drosophila melanogaster. Reassociation kinetics of Sarcophaga DNA shows that its sequence organization is very similar to that of D. melanogaster in having a very large proportion of single copy DNA (81%) and only small amounts of highly and moderately repetitive DNA (9% and 6%, respectively). cRNAs from all three sequence classes were prepared and their cytological distributions on diploid and polytene cells determined by in situ hybridization. The cytological distribution of the highly repetitive probe was found to be restricted to the centromeric heterochromatin of two of the five autosomes and this sequence class was also found to be markedly underreplicated in polytene foot-pad cells. No highly repetitive DNA was localized on either of the sex chromosomes, but only on the two large centromeric regions of chromosomes C and E. Moderately repetitive DNA was found uniformly distributed on all of the autosomes in both testis and polytene foot-pad squashes. As in the case of the highly repetitive sequence probe, no moderately repetitive DNA was detected on either the X or Y chromosomes. Moderately repetitive DNA in Sarcophaga was also shown to have the Drosophila type pattern of sequence interspersion with a moderately repetitive element of 5,000 nucleotides adjacent to a unique element of greater than 10,000 nucleotides. The Sarcophaga genome is the largest for which this type of interspersion has so far been demonstrated.