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PubMed2006

Tetraploid somatic hybrids of potato (Solanum tuberosum L.) obtained from diploid breeding lines.

Intraspecific somatic hybrids between 16 different diploid breeding lines of Solanum tuberosum L. were produced by PEG-induced fusion. Manually selected heterokaryons were cultured in a Millicells-CM using a post-fusion protoplast mixture. Plants were regenerated from calli derived from heterokaryons obtained from 10 out of 38 combinations of diploid lines. Of the tested putative somatic hybrids, 14.2% were diploid, 72.8% were tetraploid and 13% pentaploid. The DNA amplification pattern obtained with RAPD or semi-random primers confirmed that 6 fusion combinations were hybrids. In most cases, the morphological traits were intermediate to those of the diploid fusion partners. About 23.0% of the tested somatic hybrids showed variation in their morphology. Of the tested somatic hybrids, 78.0% flowered and 86.0% tuberized. The cytoplasm of 9 diploid lines and 6 somatic hybrid combinations was analysed. Two of the diploid lines had W/S chloroplasts and alpha or epsilon mitochondria; the remainder contained T chloroplasts and beta mitochondria. All the analysed somatic hybrids carried T chloroplasts and beta mitochondria.

Breeding↗

PubMedDate not supplied

In vitro response to hyperthermia or X-irradiation of diploid and tetraploid RIF-1 cells separated by centrifugal elutriation.

The RIF-1 cell line at the University of Utah is comprised of approximately 65 per cent diploid and 35 per cent tetraploid cells. Because sensitivity to heat cell killing has been shown to be ploidy dependent (Lucke-Huhle 1978), the responses of these subpopulations were examined independently. Diploid and tetraploid cells were separated from stock in vitro cultures by centrifugal elutriation and maintained entirely in vitro. No influence of ploidy on Do or Dq of heat dose survival curves could be detected. Neither did ploidy affect sensitivity to X-irradiation. However, separation of the diploid and tetraploid subpopulations was imperfect. The ratio of diploid to tetraploid cells in the separated and parent (mixed ploidy) lines was therefore monitored at each passage. Tetraploid contamination of the diploid cell subline was undetectable at the time of separation but regrew to 35 per cent by 40 days after separation. Diploid contamination of the tetraploid subline was initially less than 5 per cent and remained quite low until it became undetectable at 74 days. Differences in regrowth of the contaminating subline could not be accounted for by differences in plating efficiency or doubling time, but might result from subpopulation interactions. If so, such phenomena should be considered when using ploidy-dependent cytotoxic treatments.

Cell Line↗

PubMed2000

Multiparameter flow cytometry for simultaneous assessment of p53 protein expression and cellular DNA content in oral squamous cell carcinomas: evidence for the development of aneuploid clones from p53-deficient diploid progenitor cells.

Diploid tumour cells regularly continue to progress after the development of aneuploid cell populations in head and neck squamous cell carcinomas. The coexistence of aneuploid clones with their diploid progenitor cells provides a unique opportunity to study the order of appearance of p53 mutation and aneuploidy in the same tumour. Multiparameter flow cytometry was therefore applied to 22 oral squamous cell carcinomas to simultaneously assess cellular DNA content and p53 protein expression on a single-cell basis. Concurrent measurements of cytokeratin expression served to identify tumour cells of epithelial origin. One of 5 diploid and 2 of 17 aneuploid carcinomas were p53-negative. For 15 p53-positive aneuploid tumours, overexpression of p53 protein was identified for the aneuploid clones as well as for coexisting diploid tumour cell populations in 14 cases. On the understanding that coexisting diploid and aneuploid tumour cell populations have a common clonal origin, these results provide evidence that aneuploid tumour clones typically develop from p53-deficient diploid progenitor cells. Loss of wild-type p53 function may therefore contribute to the development of aneuploidy in head and neck cancer.

Aneuploidy↗

PubMed2001

Heterofertilization exhibited by trifluralin-induced bicellular pollen on diploid and tetraploid maize crosses.

The heterofertilization rates and fertility of trifluralin-induced bicellular pollen were investigated in maize (Zea mays L.). A diploid inbred line, Oh43 (r1/r1), and a tetraploid line, Q28-1 (r1/r1/r1/r1), were pollinated with a trifluralin treated diploid stock heterozygous for R1-scm2. The gene R1-scm2 conditions purple pigmentation in both the embryo and the aleurone layer. Heterofertilized kernels were detected as discordant kernels, i.e., yellow kernel with purple embryo or purple kernel with white embryo. The diploid-diploid crosses treated with 0.2-0.3% Trefanocide solution (0.09-0.13% trifluralin) resulted in incidences of discordant kernels (3.7-4.8%) that were significantly higher than the control (2.3%). Most of the seedlings (86%) of the discordant kernels in the 0.3% treatment were triploids or triploid-class aneuploids. In tetraploid-diploid crosses, trifluralin treatments increased the number of plump kernels on the tetraploid ears. In the 0.3% treatment, 5.2% of ovaries produced plump kernels on the ears and most of the seedlings (92%) were tetraploids or tetraploid-class aneuploids, whereas in the control, only 1.5% ovaries produced plump kernels and most of the seedlings (98%) were triploids or triploid-class aneuploids. A high rate of discordance was observed among the plump kernels both in the treated plots (36.1-48.0%) and in the control (33.3%). Consequently, almost all of the plump kernels from the tetraploid-diploid crosses were considered to be the results of heterofertilization.

Crosses, Genetic↗

PubMedDate not supplied

[Comparative study of the protein makeup in diploid and haploid forms of Saccharomyces and Pichia].

The rates of growth, biomass accumulation, and electrophoretic spectra of mobile cytoplasmic proteins were studied with nonisogenous haploid and diploid cultures of Saccharomyces cerevisiae and Pichia guilliermondii as well as with isogenous haploid-diploid pairs of Saccharomyces cerevisiae and Pichia pinus. On a mineral medium with glucose, differences in these parameters in various yeast strains were found to be due to the genotype of a strain rather than to ploidy: nonisogenous haploid and diploid cultures displayed considerable and random variability of these properties while no differences were found in isogenous haploid-diploid pairs. Studies on solubility of protein fractions in various solvents made it possible to reveal differences connected with ploidy, namely: both in nonisogenous and isogenous haploid-diploid systems, the content of the water-soluble fraction decreased in diploid cultures.

Ascomycota↗

PubMed1981

[Mechanism of mutant induction in the ade2 gene of diploid Saccharomyces cerevisiae yeasts by ultraviolet rays].

Ultraviolet light (UV) at 3000 ergs/mm-2 induces ade2 mutants with a frequency about 10(-4) in wild-type haploid strains of yeast and about 10(-5) in diploid wild-type strains. UV irradiation effectively induced mitotic segregation of ade2 in the heterozygous diploid (the frequency of segregation is 6%). Interallelic complementation and localization spectra are similar for mutations induced both in haploids and diploids. The occurrence of ade2 mutants in diploids correlated with mitotic segregation of the marker his8 which is situated in the same arm of XY chromosome as ade2 is, distal to the centromere. Our data about the frequency of ade2 mutants in diploids and haploids, the frequency of ade2 mitotic segregation, mitotic segregation of other markers and genetic characteristics of ade2 mutations confirm the suggestion that the major mechanism of diploid ade2 mutants appearance is mutation in one of the two ADE2 alleles and consequent mitotic homozygotisation of mutation as a result of mitotic crossingover between ade2 and the centromere.

Diploidy↗

PubMed1993

Cellular and nuclear volume of primitive red blood cells in digynic and diandric triploid and control diploid mouse embryos.

It has long been known that a relationship exists between ploidy and cellular and nuclear volume. This has been observed in amphibia and plants, and more recently in postimplantation tetraploid mouse embryos. We wished to establish whether a similar relationship exists in digynic and diandric triploid mouse embryos, as well as establishing whether the cellular or nuclear volume of primitive nucleated red blood cells of these two classes of triploids were the same. Spontaneously occurring digynic triploid embryos isolated from LT/Sv strain female mice result from the fertilisation of primary oocytes. These embryos develop to the forelimb-bud stage but invariably possess neural tube and cardiac abnormalities. Diandric triploid embryos were also analysed, and were produced experimentally by standard micromanipulatory techniques. They have a relatively normal morphology, and can survive up to the forelimb--bud stage. Primitive red blood cellular and nuclear volume was analysed in serially sectioned digynic and diandric triploid and in developmentally matched diploid control embryos isolated both as littermates of triploid embryos from LT/Sv strain mice, and from other genetically dissimilar diploid controls. The triploid and diploid embryos were analysed between 8-8.5 and 10-10.5 days of gestation, respectively. The cellular and nuclear volumes of the primitive red blood cells in the digynic and diandric triploid embryos were not significantly different, though they were significantly greater than comparable measurements for diploid embryos. We have therefore confirmed that a predictable relationship exists between cellular and nuclear volume and ploidy in the material analysed. The red blood cellular volume in triploid and diploid embryos increased in a predictable way over time, while their nuclear volume decreased in a predictable way over the same time period. A similar relationship has previously been observed when the nucleated red blood cells of diploid and tetraploid embryos were analysed morphometrically.

Animals↗

PubMed1996

The oocyte of triploid fluke receiving intrusion of sperm from a diploid fluke--evidence for the origin of tetraploids in Paragonimus westermani.

An experiment was conducted to elucidate the origin of tetraploids (2n = 4x = 44) of Paragonimus westermani that occur together with diploid (2n = 2x = 22) and triploid (2n = 3x = 33) types in Liaoning Province, the People's Republic of China. Metacercariae of the diploid type, obtained from Hyogo Prefecture, Japan, and those of the triploid type from Tsushima, Nagasaki Prefecture, Japan, were mixed and inoculated into dogs and cats. The following results were obtained. The flukes were found in pairs within cysts in random combinations of 2x + 2x, 2x + 3x, and 3x + 3x (7:15:7). Oocytes in the oviduct were at stages from diplotene to metaphase. In a triploid fluke encysted with a diploid fluke, the primary oocytes were intruded by sperms from the diploid fluke. In the primary oocytes of diploid as well as triploid flukes, from diplotene to diakinesis, the homologues of the nucleolar chromosomes were heteromorphic as far as the size of the short arm was concerned. This implies that the triploid is an autotriploid generated in an ancestral diploid population that was polymorphic for the nucleolar chromosome.

Animals↗

PubMed2001

Analysis of Ki-ras gene mutations associated with DNA diploid, aneuploid, and multiploid colorectal carcinomas using a crypt isolation technique.

BACKGROUND AND AIM: Current evidence suggests a possible relationship between DNA ploidy status and Ki-ras gene mutations in human cancers. However, the conventional method does not enable accurate determination of DNA ploidy status of a tumor cell. The present study attempts to clarify whether Ki-ras gene mutations are associated with DNA ploidy status in sporadic colorectal carcinomas using a crypt isolation technique coupled with DNA cytometric sorting. METHODS: Polymerase chain reaction and single-strand conformation polymorphism and direct sequencing were used to analyze Ki-ras gene mutations in 82 sporadic colorectal carcinomas: 21 diploid, 12 aneuploid, and 49 multiploid. In addition, microsatellite instability (MSI) was assessed using seven microsatellite markers to study the relationship to Ki-ras mutations. RESULTS: Ki-ras mutations were found in 12 of 21 diploid carcinomas and in 8 of 12 aneuploid carcinomas. In contrast, Ki-ras gene mutations were detected infrequently in the 34 multiploid carcinomas examined, 8 of which were seen in diploid populations and 10 in aneuploid populations. On the other hand, Ki-ras gene mutations were inversely correlated with MSI, which was found in diploid carcinomas only. CONCLUSIONS: The low frequency of Ki-ras gene mutations that we observed in multiclonal colorectal carcinomas suggests that development of multiclonal colorectal carcinoma may involve a mechanism different from that involved in the development of diploid or aneuploid colorectal carcinomas.

Adult↗

PubMed1992

Image cytometric DNA analysis in human breast cancer analysis may add prognostic information in diploid cases with low S-phase fraction by flow cytometry.

Measurements of DNA ploidy can be performed either with image cytometry (ICM) or flow cytometry (FCM); both methods provide independent prognostic information in primary breast cancer. The aim of the present investigation was to compare the two methods and to relate the findings to prognosis (median follow-up 42 months). Concordance in ploidy status (diploid, tetraploid, aneuploid) was obtained in 76% of the samples (168/222). When the fraction of S-phase cells (SPF) from FCM analysis was also taken into consideration, four different groups of samples were obtained (Flow I-IV), which were considered to correspond to the Auer classification (Auer I-IV) of DNA histograms obtained from image cytometry. Complete concordance between the two techniques now was 70% (155/222). Samples classified as Flow I (diploid or near-diploid with low SPF) and Auer I had a distant metastasis rate of 3/60 (5%), as compared to 62/154 (40%) for all other combinations of the Flow and Auer classifications taken together. Thus, the only findings of prognostic importance were that some samples were Flow I but not Auer I, or vice versa. These two groups represent 17 (7.7%) and 14 (6.3%), respectively, of the total number of samples, and had frequencies of distant metastasis similar to those of the other high-risk groups, namely, 7/17 and 5/14, respectively. In a multivariate analysis, flow cytometric S-phase value was a stronger prognostic factor than either the Flow and Auer classification. We conclude that when routine FCM DNA analysis is used, diploid or near-diploid samples with a low S-phase value should be reanalyzed with ICM.

Breast Neoplasms↗

PubMed1995

S-phase cells of the lymphoplasmocytic compartment in hyperdiploid multiple myeloma are diploid cells.

In vivo S-phase cell labeling with iododexoyuridine (IdUrd) was performed in six multiple myeloma (MM) patients. Myeloma cells from four patients were hyperploid. In three out of four patients, DNA/IdUrd flow cytometry revealed that most of the labeled cells, which had divided during the period, elapsed between flash labeling and sampling, had returned to the diploid G0/G1 compartment and not to the hyperdiploid peak. To eliminate contaminating cells belonging to the normal hematopoiesis, plasmocytic and lymphocytic cells were fractionated and analyzed separately. Cell enrichment was performed with use of murine monoclonal antibodies (MoAbs) against plasmocytic and lymphocytic cell markers and subsequent magnetic activated cell sorting with immunobeads, i.e., polysterene magnetic particles coated with sheep anti-mouse IgG. The purified plasmocytic cells were mainly hyperdiploid and largely unlabeled. The lymphocytic cells appeared to belong chiefly to the diploid cell population. The IdUrd-labeled cells were predominantly lymphocytic cells, returning after mitosis to the diploid G0/G1 peak. Although this pattern of S-phase cells in hyperdiploid MM, belonging to the diploid cell compartment, was observed in three out of four hyperploid cases and although the number of observations is small, S-phase cells may demonstrate an aspect of tumor cell kinetics in hyperploid MM, which has been debated for many years and which indicates the existence of a non-plasmocytic stem cell compartment that feeds the plasmocytoma. The behavior of the labeled cells as observed in a few cases of MM provides another, hitherto undescribed, argument that, at least in some MM patients, a part of the proliferating tumor cells may be diploid lymphocytic (precursor) cells. These findings should be considered when targeting and monitoring treatment of MM and also in purging procedures of bone marrow in patients to be treated by ablative cytotoxic therapy and autologous bone marrow transplantation.

Aged↗

PubMed2003

Proteome analysis of diploid, tetraploid and hexaploid wheat: towards understanding genome interaction in protein expression.

Hexaploid wheat (Triticum aestivum L.) is derived from a complex hybridization procedure involving three diploid species carrying the A, B and D genomes. The proteome patterns of diploid, tetraploid and hexaploid wheat were analyzed to explore the genome interaction in protein expression. At least two species from each of the diploid and tetraploid were used to compare their proteome maps with a hexaploid wheat cv. Chinese Spring. The ancestral cultivars were selected based on their history of closeness with the cultivated wheat. Proteins were extracted from seed flour and separated by two-dimensional electrophoresis (2-DE) with isoelectric focusing of pH range from 4-10. 2-DE maps of cultivated and ancestral species were analyzed by computer assisted image analyzer. The region of high molecular weight glutenin subunits of hexaploid wheat showed similarity with those of the diploid donors, BB and DD genomes. The omega gliadin, which is controlled by B genome in common wheat, was assumed to have evolved as a result of interaction between AA and BB genomes. The low molecular weight glutenins and alpha and beta gliadin regions were contributed by the three genomes. This result suggests that the function of donor genomes particularly in the expression of proteins in hexaploid wheat is not totally independent; rather it is the product of interactions among the diploid genomes in the hexaploid nuclear constitutions. The expression of nonstorage proteins was affected substantially due to the removal of the D genome from hexaploid constitution. Location of the structural gene controlling one of the alpha amylase inhibitor proteins in the nonstorage protein region was identified in the short arm of chromosome 3D.

Chromosome Aberrations↗

PubMed2001

The kinetics of the hypoferraemic response and changes in levels of alternative complement activity in diploid and triploid Atlantic salmon, following injection of lipopolysaccharide.

To study any possible effects of triploidy on the kinetics of the response of two non-specific disease factors, full sibling diploid and triploid Atlantic salmon were injected intraperitoneally with either lipopolysaccharide (1 mg kg(-1) body weight) or saline. Individually marked fish were repetitively blood sampled for up to 19 days. Total serum protein concentrations remained constant throughout the experiment indicating that the sampling regime did not cause haemodilution. The alternative complement pathway activity (measured by the titre of haemolytic activity against rabbit erythrocytes) in the serum of saline injected fish remained constant but in LPS-injected fish it fell to barely detectable levels 2 days after injection, but recovered to pre-treatment levels by about day 5. Triploid fish took slightly longer to reach full recovery levels than diploids. All groups of fish showed a hypoferraemic response, suggesting that the sampling regime was at least partially responsible. However, the response was more rapid and pronounced in the LPS-injected fish. In the latter, serum iron concentrations decreased to very low levels by day 2 post-injection in the diploid fish and by day 3 in the triploid fish. Pre-treatment iron levels were re-established by about 15 days post-injection in all groups. The data show only slight differences between the diploid and triploid fish, but the longer time taken for the triploids to recover complement activity and the slower onset of the hypoferraemic response following injection of LPS, suggest that they may be at a disadvantage compared with their diploid siblings in their defence against bacterial infections.

Animals↗

PubMed1989

Methylation of ribosomal cistrons in diploid and tetraploid Odontophrynus americanus (Amphibia, Anura).

Odontophyrynus americanus (Amphibia, Anura) genomic DNA from diploid and tetraploid specimens was treated with restriction enzymes sensitive to cytosine and adenine methylation (5 meC and 6 meA). In both diploids and tetraploids a high proportion of the total DNA was not cleaved by 5 meC-sensitive enzymes as observed on agarose gels stained with ethidium bromide. The DNAs were transferred to nitrocellulose filters and hybridized with cloned fragments containing sequences of Xenopus laevis 28S and 18S ribosomal DNA (rDNA). A high level of methylation of the ribosomal repeat units was revealed by 5 meC-sensitive enzymes in blood, liver, kidney and testis tissues. Adenine was methylated to a lesser degree and similarly in the rDNA from both germinative and somatic tissues. Comparison of the results obtained with DNA of diploids and tetraploids showed that methylation of ribosomal genes was increased in tetraploid genomes of adult frogs, but exact quantitative determinations could not be performed by this methodology. Cloning of the 28S region of the rDNA repeat unit was performed in the lambda gtWES lambda C vector. Restriction patterns obtained with methylation-sensitive enzymes using diploid and tetraploid derived clones confirmed the high level of methylation of the corresponding region of the ribosomal repeat unit in genomic DNAs. The implications of these results in the regulation of expression of the ribosomal genes in diploids and tetraploids are discussed.

Adenine↗

PubMed1975

The effects of three rad genes on UV induced mutation rates in haploid and diploid Saccharomyces cells.

Effects of the rad 2-20, rad 9-4, r1s, and the corresponding wild type RAD alleles in haploid and homozygous diploid Saccharomyces strains on UV induced mutation rates from adenine, lysine and histidine dependence to independence are reported. The UV induced mutation rates were similar for the RAD, r1s, and rad 9-4 haploids, whereas the rad 2-20 mutation causes a marked increase in the UV induced mutation rates. The diploid rad 2-20 strain also exhibits a marked increase in the UV induced mutation rates, whereas the rad 9-4 diploid has reduced mutation rates when compared to the wildtype. The UV induced mutation rates of haploid and diploid RAD strains are almost identical. For the rad 2-20 and rad 9-4 diploids, however, these rates are smaller than in the corresponding haploid strains. Differential effects of the rad genes on the ratio of locus to suppressor mutations were found. The implications of these findings on possible repair processes in yeasts are discussed.

Adenine↗

PubMed1997

6-N-hydroxylaminopurine (HAP)-induced accumulation of variability in haploid and diploid strains of Aspergillus nidulans.

Haploid and diploid strains of Aspergillus nidulans have been repeatedly treated with the strong mutagen 6-N-hydroxylaminopurine (HAP) which causes only base substitutions. An enormous amount of variability may be rapidly accumulated in haploid or diploid strains of A. nidulans. In particular, in the diploids the analysis of the results shows that after 12 cycles of treatment the conidia differ from each other for about ten recessive lethals and therefore probably for several hundreds of mutations. The viability of the heterozygous multiply mutant diploids is not appreciably different from that of untreated controls. In the diploid strains the accumulated variability was very high. The treatment of a haploid strain during vegetative growth also caused a strong accumulation of mutations, even though deleterious, because they can be maintained in the heterokaryotic condition.

Adenine↗

PubMed1991

Plasma growth hormone levels during sexual maturation in diploid and triploid rainbow trout (Oncorhynchus mykiss).

Plasma growth hormone concentrations were determined in diploid and triploid rainbow trout of both sexes during sexual maturation. Diploid females grow large ovaries, whereas triploid female trout show no ovarian development. The plasma growth hormone concentration in triploid female trout remained low and unchanged throughout the study, whereas it rose slightly, but significantly, in the diploid females that matured, but not in those that remained immature. On the other hand, triploid males do develop tests like their diploid counterparts. In both groups spermiation was accompanied by a steady rise in the plasma growth hormone concentration. The results suggest that the elevated growth hormone concentration in mature male trout was a consequence not of reproduction per se, but of the loss of condition that accompanied spawning. This hypothesis was supported by the results from the females. Apart from a temporary loss in the mature diploids caused by stripping of the eggs, female trout did not lose condition, neither did they show any change in the plasma growth hormone concentration during the period when they ovulated. These results suggest that it was the nutritional insufficiency accompanying reproduction in male fish that caused the elevation in growth hormone concentration. In fact, a strong negative correlation between the plasma growth hormone concentration and the condition factor of the fish was observed.

Animal Nutritional Physiological Phenomena↗

PubMed1989

Longterm survival of normal, diploid Syrian hamster-cells in tumors induced by transfection with v-Ha-ras and v-myc oncogenes.

Tumors were induced following transfection of normal, diploid hamster embryo cells with plasmids containing the viral Harvey ras and viral myc oncogenes. Direct cytogenetic studies of the tumors performed at 3-7 weeks after injection of the transfected hamster cells into nude mice revealed that 100% of the hamster cells were aneuploid and no detectable diploid cells in mitosis were observed. However, when tumor explants were cultured in vitro, diploid Syrian hamster cells were frequently detected at early passages. The percentage of diploid hamster cells in the cultures varied from 2 to 94% at the first passage. After several passages in vitro, only aneuploid hamster cells were observed. The diploid hamster cells in culture had a flat morphology and senesced. The aneuploid cells in the cultures were readily cloned and formed tumors after reinjection in nude mice. Reconstruction experiments consisting of injecting cloned, aneuploid tumor cells mixed with 6 X 10(6) normal Syrian hamster embryo cells were performed. Cell cultures derived from these mixed tumors contained both normal and aneuploid hamster cells indicating that normal cells survived in vivo during the growth of the tumor cells for up to 4 weeks. During this period, some normal cells transplanted in vivo did not die or terminally differentiate. Since normal cells can persist in vivo, tumor-derived cell cultures are mixed populations and caution should be exercised in interpreting quantitative molecular or cellular studies of these uncloned populations.

Aneuploidy↗