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PubMed1979

DNA reassociation kinetics in diploid and phylogenetically tetraploid cyprinidae.

Four diploid and three phylogenetically tetraploid Cyprinidae (Ostariophysi) have been characterized as for nuclear DNA content, modal chromosome number and DNA reassociation kinetics (hydroxyapatite chromatography). Among the diploid species nuclear DNA content (10(-12) g DNA/2C) was 1.62 for Tinca tinca, 1.87 for Scardinius erythrophthalmus, 2.53 for Leuciscus cephalus and 2.75 for Alburnus alburnus, while the phylogenetically tetraploid species Carassius auratus, Barbus barbus and Cyprinus carpio attained 3.40, 3.66 and 3.80 respectively. Modal chromosome number was 2n = 48-50 for diploid individuals and 2n = 100-104 for phylogenetically tetraploid ones. In all the species 5--8% of the genome is represented by highly repetitive and foldback DNA. In DNA reassociation kinetics of phylogenetically tetraploid Cyprinidae a distinct plateau separates an intermediate reassociating sequence fraction (about 22% of the genome; with average repetition frequencies between 1,000 and 1,400) from a slow reassociating one (unique DNA; about 72% of the genome). These two genome fractions are not clearly distinguishable from each other in Cot curves of the diploid Cyprinidae, where a similar plateau is not evident. Since simple ploidy changes are not expected to affect DNA reassociation kinetics we suggest a different evolution in the genome organization of the two ploidy groups. Some possible hypotheses are discussed.

Animals↗

PubMed1990

Differential limb regeneration in diploid and triploid Rana pipiens larvae with reference to spinal motor neuron development.

Developmental manipulations that can alter nerve-limb relationships can assist in understanding the neural control of limb regeneration. Pressure-induced triploidy in Rana pipiens tadpoles results in alterations of the quantitative characteristics of the spinal motor neurons that innervate the limbs, whereas the limbs appear unaltered. Unilateral midthigh amputations at larval stages IX, XI, and XIII of diploid and triploid animals resulted in complete regeneration for only stage IX animals regardless of ploidy. Nevertheless, triploid limbs regenerated much faster than did diploids, an event that can be related to the differential dynamics of nerve fiber extension and/or the altered numbers and sizes of triploid spinal motor neurons. Although normal limb development from stage IX to the endpoint at stage XVIII was the same in diploids and triploids, the rate of regeneration in triploids was nearly twice that of diploids. The data of this noninvasive means of altering the quantitative relationship of nerve-to-peripheral target suggest a unique means of studying nerve-dependent limb regeneration in an animal that progressively loses its regenerative capability during development.

Animals↗

PubMed2005

Mouse zygotes with one diploid pronucleus formed as a result of ICSI can develop normally beyond birth.

A mouse spermatozoon was injected into mouse secondary oocytes (ICSI) in the vicinity of the metaphase spindle. In 22% of oocytes injected successfully, the maternal chromatin (the haploid chromatids formed after the second meiotic division) and paternal chromatin (from the sperm nucleus) were surrounded by a common nuclear envelope to form one diploid bi-parental pronucleus. However, the use of spermatozoa in which BrdU had been incorporated into DNA during spermatogenesis revealed, that maternal and paternal chromatin occupied two separate compartments within the one pronucleus. In the living state, the diploid pronucleus could be distinguished from a haploid one by its distinctly larger size and by a greater number of "nucleolus-like bodies"-criteria confirmed karylogically at the 1st cleavage division. Such zygotes with one diploid pronucleus were able to develop in vitro into blastocysts as often as those with two haploid pronuclei [11/29 (38%) vs. 14/35 (40%)]. Seventy nine 2-cell embryos developing in vitro from zygotes with one diploid pronucleus were transplanted to the oviducts of pseudopregnant recipients: two females had six foetuses when killed on the 17th day, and two females gave birth to nine young, eight of which survived and developed into normal fertile animals.

Animals↗

PubMed2005

Experimentally produced diploid-triploid mouse chimaeras develop up to adulthood.

Spontaneous diploid-triploid chimaeras occur sporadically in various mammalian species including man, but so far have never been produced experimentally. In order to get a deeper insight into the developmental consequences of this anomaly, we have developed two procedures that enabled for the first time to produce routinely diploid-triploid embryos, foetuses, and animals in the mouse. These procedures are: (1) aggregation of cleaving diploid embryos with triploid embryos produced by suppression of the second polar body in zygotes, and (2) fusion of a haploid karyoplast with one blastomere of the two-cell diploid embryos. The first procedure yielded 23 living and 6 dead postimplantation embryos and foetuses (age: 8th-19th day) out of which 22 were chimaeric. In addition, three chimaeric neonates reached adulthood. Two animals were fertile, and one--an overt chimaera--was an infertile male. The rate of postimplantation development of aggregation chimaeras was normal or only slightly retarded, and with one exception the foetuses were morphologically normal. Generally, the highest contribution of the 3n component in extra-embryonic structures was noted in the yolk sac, and usually it was higher than its contribution to the organs of the body. Chimaerism was most often noted in the liver, the heart, the intestine, and the lungs. Participation of triploid cells to all tissues studied, both in the body and in extra-embryonic structures, appeared to decrease slightly as development progressed. The second procedure yielded 10 foetuses and 6 adults. Three foetuses were chimaeric. Six fertile adults were probably non-chimaeras: the triploid component was absent in the coat and in the blood.

Animals↗

PubMed1995

Restricted distribution of tetraploid cells in mouse tetraploid<==>diploid chimaeras.

Tetraploid mouse embryos were produced by electrofusion at the 2-cell stage, cultured overnight, and aggregated with normal diploid embryos to produce tetraploid<==>diploid (4n<==>2n) chimaeric conceptuses. At 7 1/2 days the 4n<==>2n chimaeras were usually smaller and developmentally retarded compared to control diploid<==>diploid chimaeras. At 12 1/2 days the 4n<==>2n chimaeras had heavier placentas but there was no significant difference in fetal size. Tetraploid cells showed a restricted tissue distribution at both developmental stages studied: 4n cells were commonly present in both the primitive endoderm and the trophectoderm lineages but they rarely contributed to the primitive ectoderm lineage. The overall similarity in the distribution of tetraploid cells at 7 1/2 and 12 1/2 days implies that whatever causes the restricted tissue distribution operates largely before 7 1/2 days. There was no evidence for excessive embryonic losses of 4n<==>2n chimaeras. So, if the restricted distribution of 4n cells was a result of cell selection, the mechanism is more likely to involve loss of 4n cells from the primitive ectoderm early in development rather than selective death of conceptuses with tetraploid cells in this lineage. Alternatively, 4n cells may be preferentially allocated to the trophectoderm and primitive endoderm rather than the primitive ectoderm layer at the blastocyst stage.

Animals↗

PubMed1977

Replication of chromosomal DNA in diploid Drosophila melanogaster cells cultured in vitro.

Replication rate and replicon sizes in chromosomal DNA of in vitro cultured diploid D. melanogaster cells were determined using autoradiography of 3H-thymidine labeled DNA. Synthesis of DNA in euchromatic and heterochromatic regions of Drosophila diploid cells occurs at different periods of the S phase which last 10 h. During the first 4 h the synthesis is observed only in euchromatic regions. The heterochromatic synthesis starts shortly before the synthesis in euchromatic regions is completed and lasts for 6 h until the end of the S phase. The cells were synchronized by 5-fluorodeoxyuridine which blocked the diploid cell DNA synthesis. Synthesis was found to start simultaneously in most euchromatic replicons. In the majority of the replicons the synthesis started at a single point and proceeded bidirectionally. The average rate of DNA synthesis per fork was 12.5 mum/h (38kb). The mean distance between the middle points of adjacent labeled regions was 70 mgm (210 kb). The size of most replicons ranged from 40 to 120 mum. - These estimates do not apply to the heterochromatic portions of the D. melanogaster genome since the measurements have been carried out on DNA preparations obtained during the first 2 h of the S phase. - On the average, a replicon can consist of 7 chromomeres since the size of a replicon in diploid cell chromosomal DNA and DNA length of a polytene chromomere average 210 and 30 kb, respectively.

Animals↗

PubMed1979

Difference between diploid and aneuploid Chinese hamster cells in replication at mid-S-phase.

Following partial synchronization of the heteroploid Chinese hamster cell line V-79 and of normal diploid lung fibroblasts of the Chinese hamster in culture, their DNA replication during S-phase aws compared by means of a BrdU-incorporation/thymidine pulse technique and Hoechst-Giemsa differential staining of metaphase chromosomes. This comparison indirectly shows the S-phase of the heteroploid cells of V-79 to be 2 h shorter than the diploid cell S-phase. When the thymidine pulse is applied to diploid lung fibroblasts at mid-S-phase, differential staining colours metaphase chromosomes a pale blue. Performing the corresponding experiment with V-79 cells, neither a pale blue nor dark red staining is obtained, but rather an intermediate shade, showing prominently dark staining regions in parts. The pause in DNA synthesis observed at mid-S-phase of the diploid Chinese hamster lung fibroblasts seems to be omitted at mid-S-phase of the V-79 cells.

Aneuploidy↗

PubMedDate not supplied

Diploid yeast cells yield homozygous spontaneous mutations.

A leucine-requiring hybrid of Saccharomyces cerevisiae, homoallelic at the LEU1 locus (leu1-12/leu1-12) and heterozygous for three chromosome-VII genetic markers distal to the LEU1 locus, was employed to inquire: (1) whether spontaneous gene mutation and mitotic segregation of heterozygous markers occur in positive nonrandom association and (2) whether homozygous LEU1/LEU1 mutant diploids are generated. The results demonstrate that gene mutation of leu1-12 to LEU1 and mitotic segregation of heterozygous chromosome-VII markers occur in strong positive nonrandom association, suggesting that the stimulatory DNA lesion is both mutagenic and recombinogenic. In addition, genetic analysis of diploid Leu+ revertants revealed that approximately 3% of mutations of leu1-12 to LEU1 result in LEU1/LEU1 homozygotes. Red-white sectored Leu+ colonies exhibit genotypes that implicate post-replicational chromatid breakage and exchange near the site of leu1-12 reversion, chromosome loss, and subsequent restitution of diploidy, in the sequence of events leading to mutational homozygosis. By analogy, diploid cell populations can yield variants homozygous for novel recessive gene mutations at biologically significant rates. Mutational homozygosis may be relevant to both carcinogenesis and the evolution of asexual diploid organisms.

Diploidy↗

PubMed1976

Isolation and characterization of compatible diploids of Schizophyllum commune.

Common-AB diploids with several heterozygous biochemical markers were mated with appropriately marked haploid strains of S. commune in an effort to obtain compatible, common-A, and common-B diploid progeny with biochemical markers identical to those of the common-AB parent. The spores from these crosses were germinated on minimal medium. Five compatible diploids, but no common-A or common-B diploids, marked as desired, were isolated by this method. Two possessed some dikaryotic cells and two had many dikaryotic cells. One of the latter was shown to have peculiar behaviour associated with one of its B mating-type factors.

Agaricales↗

PubMed1983

Ribosomal RNA gene number and sequence divergence in the diploid-tetraploid species pair of North American hylid tree frogs.

Hyla chrysoscelis (2n = 24) and H. versicolor (2n = 48) are a diploid-tetraploid species pair of tree frogs. Hybridization saturation of isolated 125I-labeled ribosomal RNAs (rRNAs) with filter-immobilized DNA shows that there are twice as many rRNA genes in the tetraploid as in the diploid. For the diploid, saturation occurs at 0.037%, from which it is calculated that there are about 618 copies of the (18 S + 28 S) rRNA genes per haploid genome. Analysis of the extent of hybridization and also the thermal stability of homologous and heterologous hybrids shows that considerably more base substitutions have occurred in the tetraploid rDNA genes than in the diploid since their divergence. This is interpreted to reflect either a relaxation of the gene regulatory "correction" mechanism hypothesized to be responsible for the maintenance of identical tandem rRNA genes in the tetraploid or a release of one gene set from the normal selective constraints.

Animals↗

PubMed1987

Paragonimus westermani: a comparative study on the migration route of the diploid and triploid types in the final hosts.

Recent studies on chromosomes have shown the presence of diploid and triploid types of Paragonimus westermani. To determine any possible biological differences between them, the migration route and development of the diploid type in the final hosts were compared with those of the triploid type. In the cat, the definitive host, larvae of the diploid type migrated to the abdominal wall, remained there for two weeks, and then migrated to the lung. In the rat, the abnormal host, some orally administered metacercariae of the diploid type made cysts in the lung and laid eggs, but the triploid type did not lay any eggs. Neither type of P. westermani migrated to the liver of the rat, in contrast to the previously reported fact that P. miyazakii and P. ohirai migrate to the liver and develop there. From these results, it seems likely that the two types of P. westermani are closely related.

Abdomen↗

PubMed1980

Genetic analysis of tumorigenesis: VI. Chromosome rearrangements in tumors derived from diploid premalignant Chinese hamster cells in nude mice.

The chromosome constitution of CHEF/16 clones recovered from methylcellulose and of uncloned, tumor-derived CHEF/16 populations are compared. Nine of 11 clones recovered from methycellulose were initiated by diploid cells. Moreover, chromosomally diploid cells were still present in most CHEF/16 clones even after growth in anchorage-independent conditions. In contrast, none of the CHEF/16 cells recovered from tumors were diploid. Nonrandom chromosome changes were observed, but no specific chromosome alterations were consistently found in tumor-derived CHEF cells. Although CHEF/16 cells are uniformly tumorigenic in nude mice, each of 10 uncloned tumor-derived populations from inocula of 10(2), 10(4), and 10(6) CHEF/16 cells consisted of only 1-3 stemlines. Our results show that diploid CHEF/16 cells are premalignant and undergo karyotypic changes leading to successful and usually clonal establishment of tumors in nude mice.

Animals↗

PubMed1980

Genetic analysis of tumorigenesis: V. Chromosomal analysis of tumorigenic and nontumorigenic diploid chinese hamster cell lines.

The chromosomal constitution of four established diploid Chinese hamster embryo fibroblast (CHEF) cell lines is described. CHEF/18 exhibits anchorage-dependent growth and is not tumorigenic in nude mice. CHEF/16 has a high plating efficiency in methylcellulose and is highly tumorigenic in nude mice. Both CHEF/8 and CHEF/16 have a normal Giemsa banding pattern and constitutive heterochromatin distribution characteristic of normal diploid Chinese hamster cells and exhibit relatively little chromosomal variation within their cell populations. These results suggest that the nuclear changes responsible for tumorigenicity of CHEF/16 involve alterations below the level detectable by Giemsa banding analysis. Chromosome rearrangements were detected in two other CHEF cell lines; CHEF/205-30, a diploid, thioguanine-resistant derivative of CHEF/18, and CHEF/204-Bu 50, a diploid, 5-bromodeoxyuridine-resistant derivative of CHEF/16, which are being used as genetic markers in intraspecific somatic cell hybrids.

Animals↗

PubMed1996

S-phase fraction identifies high-risk subgroups among DNA-diploid breast cancers.

The prognostic value of DNA content measured by means of flow cytometry was analyzed in formalin-fixed, paraffin-embedded samples from 231 breast cancer patients treated between 1984 and 1988, with a mean follow-up period of 55 months. We followed the guidelines of a Consensus Meeting held on this issue in Maine, USA, in 1992. DNA-diploid and -aneuploid tumors were evaluated separately for the fraction of cells in S-phase (SPF) contained in them, this being divided into three groups ('high', 'intermediate', and 'low'), defined by the 25th and 75th centile of the SPF-distribution corresponding to either DNA-diploid or DNA-aneuploid tumors. Unequivocally readable histograms were obtained from 174 samples (75.3%). A high SPF in diploid tumors was significantly associated with a higher recurrence rate (p = 0.015), a shorter disease-free survival (p = 0.014), advanced (IIIB) clinical stage (p = 0.034), and almost significantly with total survival (p = 0.055). In a multivariate Cox regression analysis, a high SPF in diploid tumors retained its independent prognostic power, being significantly associated with a shorter disease-free survival (p = 0.00049) and total survival (p = 0.0077). It also allowed to identify a subgroup with an ominous prognosis among patients < or = 50 years of age with early stage tumors. Our results fully validate the recommendations of the 1992 Maine Consensus Meeting.

Adult↗

PubMed1975

Establishment of normal diploid and malignant heteroploid cell lines from non-treated and benzo(a)pyrene treated hamster embryo cell cultures.

Two normal diploid control cell lines and a heteroploid malignant transformed cell line from B(a)P treated hamster embryo cell cultures were established. The 14-month-old B(a)P transformed cell line grew 8-times faster than the 20-month-old control cell line. The control cell line showed normal diploid chromosome complement in 93% cells and heteroploidy in 7% cells while B(a)P treated line showed 83% heteroploid cells and only 17% diploid cells. This is the first report on the establishment of diploid hamster cell cultures grown for extended period.

Animals↗

PubMed1996

Distribution of 5S and 18S-28S rDNA loci in a tetraploid cotton (Gossypium hirsutum L.) and its putative diploid ancestors.

The most widely cultivated species of cotton, Gossypium hirsutum, is a disomic tetraploid (2n=4x=52). It has been proposed previously that extant A- and D-genome species are most closely related to the diploid progenitors of the tetraploid. We used fluorescent in situ hybridization (FISH) to determine the distribution of 5S and 18S-28S rDNA loci in the A-genome species G. herbaceum and G. arboreum, the D-genome species G. raimondii and G. thurberi, and the AD tetraploid G. hirsutum. High signal-to-noise, single-label FISH was used to enumerate rDNA loci, and simultaneous, dual-label FISH was used to determine the syntenic relationships of 5S rDNA loci relative to 18S-28S rDNA loci. These techniques provided greater sensitivity than our previous methods and permitted detection of six new G. hirsutum 18S-28S rDNA loci, bringing the total number of observed loci to 11. Differences in the intensity of the hybridization signal at these loci allowed us to designate them as major, intermediate, or minor 18S-28S loci. Using genomic painting with labeled A-genome DNA, five 18S-28S loci were localized to the G. hirsutum A-subgenome and six to the D-subgenome. Four of the 11 18S-28S rDNA loci in G. hirsutum could not be accounted for in its presumed diploid progenitors, as both A-genome species had three loci and both D-genome species had four. G. hirsutum has two 5S rDNA loci, both of which are syntenic to major 18S-28S rDNA loci. All four of the diploid genomes we examined contained a single 5S locus. In g. herbaceum (A1) and G. thurberi (D1), the 5S locus is syntenic to a major 18S-28S locus, but in G. arboreum (A2) and G. raimondii (D5), the proposed D-genome progenitor of G. hirsutum, the 5S loci are syntenic to minor and intermediate 18S-28S loci, respectively. The multiplicity, variation in size and site number, and lack of additivity between the tetraploid species and its putative diploid ancestors indicate that the behavior of rDNA loci in cotton is nondogmatic, and considerably more complex and dynamic than previously envisioned. The relative variability of 18S-28S rDNA loci versus 5S rDNA loci suggests that the behavior of tandem repeats can differ widely.

Chromosomes↗

PubMed1985

Isolation of tetraploid clones with high efficiency from diploid 3Y1 rat fibroblasts treated with sodium butyrate.

Sodium butyrate causes proliferation arrest with a G2 (4C) DNA content and induces formation of tetraploid cells upon removal of the inhibitor, in rat 3Y1 diploid fibroblasts. We isolated tetraploid clones from the butyrate-treated 3Y1 cells with high efficiency; among 21 clones randomly isolated, 5 were pure diploid, 7 were mainly tetraploid with a small contaminating diploid population, and 7 were pure tetraploid. Among the pure tetraploid clones, two showed doubled chromosome numbers with slightly broader distributions than that seen in parental 3Y1 cells. Butyrate further induced polyploid formation in the tetraploid cells thus produced, but octaploid cells that resulted could not be maintained for prolonged cultivation. We found no difference between the tetraploid and the (parental and parallel isolated) diploid clones in terms of colony-forming ability, proliferation rate, and sensitivity to density-dependent inhibition of proliferation. These results suggest that doubling of chromosome number by itself does not cause a change in proliferation property. The tetraploid clones had lower average saturation densities possibly due to enlargement of cell size represented by higher cellular protein content.

Animals↗

PubMed2002

Dmc1 fluorescent foci in prophase I nuclei of diploid, triploid and hybrid lilies.

We examined the distribution of meiotic epitopes for the Dmc1 protein of lilies in a normal diploid, a triploid, and in a diploid species-hybrid. The triploid has an extra chromosome set; all three sets align, but only two of the three axes intimately pair at a given location. Our findings with the triploid support the idea that retention of the foci until the pachytene stage requires a successful homology check and synaptonemal complex (SC) initiation; the number of foci in the triploid diminishes by approximately 30% from early zygotene to pachytene, and the triploid pachytene values are similar to the pachytene values of the diploid. The species-hybrid lacks chromosome homology, has reduced SC formation and few reciprocal genetic exchanges. In this species-hybrid the number of foci at early zygotene is similar to that in the normal diploid but is dramatically reduced by mid-zygotene. The extent to which the number of Dmc1 foci is reduced is similar to the extent that SC formation is reduced. In contrast the extent of the reduction in reciprocal genetic exchange in the species-hybrid is much greater than the reduction in the number of foci. We conclude that Dmc1 protein is involved in homology checking, but the impact of failure to find homology affects SC formation and reciprocal genetic exchange differentially.

Blotting, Western↗