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PubMed1995

Growth and gene expression in diploid epithelial cell lines derived from normal human parotid gland.

Secretions of salivary glands are essential for the maintenance of oral health. Due to the lack of suitable in vitro models, studies to examine biochemical and molecular mechanisms of the cellular secretions have been difficult. Furthermore, adequate quantities of human epithelial cells could not be obtained, because normal diploid cells are believed to exhibit a limited lifespan of two to three passages (40-50 population doublings). This report describes for the first time the development of two diploid epithelial acinar cell lines, HPAM1 and HPAF1, derived from the normal human parotid gland. The cell lines are propagated in serum-free medium comprised of keratinocyte basal medium supplemented with insulin (5 micrograms/ml), hydrocortisone (0.5 micrograms/ml), epidermal growth factor (EGF, 10 ng/ml), bovine pituitary extract (25 micrograms/ml), and antibiotics. The HPAM1 cell line has been passaged more than 50 times (> 189 population doublings) and HPAF1 more than 40 times (> 185 population doublings). Both cell lines exhibit normal diploid karyotypes, lack transformed phenotypes and are non-tumorigenic in nude mice. Both cell lines produce tissue-specific proteins, i.e. alpha-amylase 1, basic proline-rich protein, and cystatins; and express the corresponding genes as determined by RT-PCR analyses. These results demonstrate that normal diploid human cells do not inherently exhibit limited life-span in vitro and can, under optimum conditions, be propagated indefinitely.

Animals↗

PubMed2001

Microsatellite-stable diploid carcinoma: a biologically distinct and aggressive subset of sporadic colorectal cancer.

Chromosomal instability and microsatellite instability represent the major pathways for colorectal cancer (CRC) progression. However, a significant percentage of CRC shows neither pattern of instability, and thus represents a potentially distinctive form of the disease. Flow cytometry was used to determine the degree of DNA aneuploidy in 46 consecutive sporadic colorectal cancers. Microsatellite status was determined by PCR amplification using standard markers, while immunostaining was used to examine the expression of p53. K- ras status was determined by restriction-mediated PCR assay. Twenty-five (54%) tumours were aneuploid, 14 (30%) were diploid and microsatellite-stable and seven (15%) were diploid and microsatellite-unstable. Tumours with microsatellite instability were more likely to be right sided, to occur in women and to be associated with an improved survival. Aneuploid tumours were significantly more common in men and were likely to be left sided. The diploid microsatellite-stable (MSS) tumours did not show a sex or site predilection, but were strongly associated with the presence of metastatic disease at the time of diagnosis. Our data suggests that diploid, MSS tumours represent a biologically and phenotypically distinct subset of colorectal carcinoma, and one that is associated with the early development of metastases. We suggest that the genetic stability that characterizes these tumours may favour the maintenance of an invasive phenotype, and thus facilitate disease progression. These findings may have important implications for treatment options in this disease subset.

Aged↗

PubMed1972

Parasexual recombination in Dictyostelium discoideum: selection of stable diploid heterozygotes and stable haploid segregants (clones-temperature sensitive-ploidy-fruiting bodies-spore-slime mold).

Haploid strains of Dictyostelium discoideum bearing temperature-sensitive mutations have been used to select stable diploid, heterozygotic clones, which arise at low frequency (about 10(-5)). Segregants arise from such diploids at low frequency (about 10(-3)). The diploids were heterozygous for resistance to cycloheximide and were phenotypically sensitive to the drug. Growth of the diploid cells in the presence of cycloheximide automatically selected those segregants bearing the resistant allele, and facilitated examination of the assortment of unselected markers. The combination of the two selective methods provides a workable system of genetic analysis in this species. We have used this method to locate six markers on three different linkage groups.

Clone Cells↗

PubMed1977

Microsurgically produced homozygous-diploid uniparental mice.

Shortly after fertilization, either the male or the female pronucleus was microsurgically removed from 202 F(1) hybrid eggs derived from crosses of two inbred strains. Subsequent incubation of these haploid eggs in medium containing cytochalasin B, which inhibits cytokinesis but not nuclear division, enabled the remaining pronucleus to become diploid. After nuclear diploidization and transfer to regular culture medium, cleavage commenced normally, and a total of 135 successfully manipulated eggs continued in development and yielded 93 morulae and blastocysts. These embryos were surgically transferred to the uteri of pseudopregnant foster mothers who gave birth to seven live female offspring. Five of the females were derived from the maternal genome (gynogenesis) and the remaining two mice inherited only the paternal genes (androgenesis), depending on whether the female or male pronucleus had been retained in the egg, respectively. Homozygosity for a number of genetic loci positioned on different chromosomes and effecting the coat color phenotype and strain-specific allelic variants of several enzymes, urinary and plasma proteins, and hemoglobins could be demonstrated unequivocally in all instances. Chromosomal analysis revealed a normal diploid karyotype including two X chromosomes. Thus far, six of the seven homozygous-diploid (isogeneic) females have proved to be fertile and have given birth to progeny corresponding only to the pronuclear genotype of the mother.

Animals↗

PubMed1991

Genetic diversity and adaptedness in tetraploid Avena barbata and its diploid ancestors Avena hirtula and Avena wiestii.

Avena barbata, a tetraploid grass, is much more widely adapted and successful in forming dense stands than its diploid ancestors. The success of such polyploids has often been attributed to heterosis associated with ability to breed true for a highly heterozygous state in which allelic differences between the parents are fixed in the polyploid by chromosome doubling. We have examined the relationship between genetic diversity and adaptedness for 14 allozyme loci in A. barbata and its diploid ancestors in samples collected from diverse habitats in Israel and Spain. The relationship varied from locus to locus: superior adaptedness was associated with genetic uniformity for five loci, in part with genetic uniformity and in part with genetic diversity (monomorphism for a single heteroallelic quadriplex) for one locus, and with allelic diversity in the form of heteroallelic quadriplexes combined with genotypic diversity in the form of complex polymorphisms among different homoallelic and/or heteroallelic quadriplexes for the eight remaining loci. These results indicate that allelic diversity fixed in nonsegregating form through chromosome doubling was an important factor in the evolution of adaptedness in A. barbata. However, it is unlikely that heterosis associated with heterozygosity contributed significantly to superior adaptedness in either the diploids or the tetraploid because virtually all loci (approximately 99%) were homozygous in the Avena diploids and tetraploid.

Adaptation, Physiological↗

PubMed1976

Variation in aryl hydrocarbon (benzo(a)pyrene) hydroxylase activity in heteroploid and predominantly diploid rat liver cells in culture.

Aryl hydrocarbon (benzo(a)pyrene) hydroxylase is present and inducible in Buffalo rat liver cells in culture. There is substantial variation in both basal and inducible hydroxylase activities among heteroploid subclones isolated from a heteroploid parent population, and among diploid subclones isolated from a diploid parent population. This variation is not related to differences in the growth characteristics of the subclones, or to differences in their chromosome number. The results indicate that substantial heterogeneity in both basal and induced hydroxylase activity develops during the growth of both heteroploid and diploid cell strains in culture. These findings indicate that diploid cell populations are not necessarily homogeneous with respect to aryl hydrocarbon hydroxylas activity. This observation may complicate the interpretation of experiments involving somatic cell hybridization or polycyclic hydrocarbon-induced transformation and/or cytotoxicity. This heterogeneity in hydroxylase activity develops rather rapidly (2-3 mo of culture), in the absence of any apparent mutational stress.

Benz(a)Anthracenes↗

PubMed2005

Generation of fertile and diploid fish, medaka (Oryzias latipes), from nuclear transplantation of blastula and four-somite-stage embryonic cells into nonenucleated unfertilized eggs.

In two experimental series of transplantation of embryonic cell nuclei into nonenucleated unfertilized eggs in medaka (Oryzias latipes), fertile and diploid nuclear transplants were successfully generated. In the first experiment, nuclei from blastula cells of a medaka stock with the wild-type body color were transplanted into 1722 eggs from the orange-red variety. Of 26 adult nuclear transplants with the wild-type body color, 22 were, as expected, triploid and sterile, but the other four were fertile. Three of the four were diploid, and the last one was tetraploid. They transmitted the wild-type body color to the F1 and F2 progenies in a Mendelian fashion. In the second experiment, cell nuclei from four-somite-stage embryos of the orangered variety carrying the green fluorescent protein (GFP) transgene were transplanted into 1688 recipients of the same strain. Three adult nuclear transplants expressing GFP were obtained. Two of them were triploid and sterile, but the remaining one was fertile and diploid. The transgene of the donor nuclei was transmitted to the F(1) and F(2) offspring in a Mendelian fashion. These observations that diploid and fertile nuclear transplants could be obtained without enucleation of the recipient eggs may have important implications for future nuclear transplantation in medaka.

Animals↗

PubMed1984

Emergence of a population of small, diploid hepatocytes during hepatocarcinogenesis.

The sequential treatment of young Wistar rats with two different carcinogens (diethylnitrosamine - plus partial hepatectomy - as an initiator, and 2-acetylaminofluorene as a cytotoxic selection pressure) induces the appearance of foci and nodules of liver cells which are phenotypically altered. By means of an algorithm which takes into account binuclearity as well as cell-to-cell aggregation it is possible to compute cellular ploidy distributions from flow-cytometric analysis of either hepatocyte suspensions or suspensions of hepatocytic nuclei. Cell suspensions isolated from carcinogen-treated rats can be shown to contain, already after 8 weeks, approximately 70% small, diploid hepatocytes, whereas suspensions from normal or partially hepatectomized control livers contain only approximately 10% diploid cells (the remainder being mostly tetraploid). Isolated nodules, i.e., expanding clones of proliferating cells, believed to be neoplastic precursor lesions, contained almost only diploid cells. These observations suggest that the selective outgrowth of a population of small, diploid hepatocytes may be a significant early step in the development of liver cancer.

2-Acetylaminofluorene↗

PubMed1999

Unpredictable fitness transitions between haploid and diploid strains of the genetically loaded yeast Saccharomyces cerevisiae.

Mutator strains of yeast were used to accumulate random point mutations. Most of the observed changes in fitness were negative and relatively small, although major decreases and increases were also present. The average fitness of haploid strains was lowered by approximately 25% due to the accumulated genetic load. The impact of the load remained basically unchanged when a homozygous diploid was compared with the haploid from which it was derived. In other experiments a heterozygous diploid was compared with the two different loaded haploids from which it was obtained. The fitness of such a loaded diploid was much less reduced and did not correlate with the average fitness of the two haploids. There was a fitness correlation, however, when genetically related heterozygous diploids were compared, indicating that the fitness effects of the new alleles were not entirely lost in the heterozygotes. It is argued here that to explain the observed pattern of fitness transitions it is necessary to invoke nonadditive genetic interactions that go beyond the uniform masking effect of wild-type alleles. Thus, the results gathered with haploids and homozygotes should be extrapolated to heterozygotes with caution when multiple loci contribute to the genetic load.

Adaptation, Biological↗

PubMed1973

Selection for mating reluctance in females of a diploid parthenogenetic strain of Drosophila mercatorum.

A diploid parthenogenetic strain of Drosophila mercatorum was outcrossed to produce genetic variance among the impaternate female offspring. Selection experiments were carried out for reluctance of the parthenogenetic females to mate. After only two cycles of selection, a parthenogenetic strain which is significantly less receptive to males from three different bisexual strains was obtained. It was also found that there is some degree of sexual isolation among the three bisexual strains used. The results support the idea that selection can render a newly produced diploid parthenogenetic strain behaviorally different from its bisexual ancestor. This appears to provide a framework which can explain the natural coexistence of diploid bisexual and diploid parthenogenetic biotypes in some species of insects.

Animals↗

PubMed1974

Bisexual mating behavior in a diploid of Saccharomyces cerevisiae: evidence for genetically controlled non-random chromosome loss during vegetative growth.

A diploid strain of Saccharomyces cerevisiae has been isolated which exhabits bisexual mating behavior. The strain mates with either a or alpha strains with a relative mating efficiency of 1 to 2%. The efficiency of mating is correlated with the frequency with which subclones of this strain revert to a single mating type. Crosses of the bisexual diploid with a/a or alpha/alpha diploids yield bisexual segregants with a frequency of approximately 3%. Analysis of the segregation of the mating type alleles and other markers on chromosome III indicates that the primary event which leads to the bisexual phenotype is the loss of one homolog of chromosome III during vegetative growth to produce a monosomic (2n-1) diploid. Evidence is presented that the loss of chromosome III and possibly of other chromosomes during vegetative growth is affected by a recessive nuclear gene-her (hermaphrodite)-which is not closely linked to the mating type locus.

Aneuploidy↗

PubMed1997

Reliability of estimates of diploid human spermatozoa using multicolour fluorescence in-situ hybridization.

Multicolour fluorescence in-situ hybridization (FISH) analysis permits distinction between disomic and diploid spermatozoa. Thus estimates of the frequency of diploid spermatozoa can be obtained for human semen samples. The issue of the accuracy and reliability of these diploidy estimates has been addressed by analysing diploidy frequencies in 10 men using the same sperm sample to estimate diploidy twice-once during two-colour FISH analysis of disomy for chromosomes 1 and 12 and a second independent analysis of three-colour FISH for disomy estimates for chromosomes X and Y (with chromosome 1 used as the autosomal control). A minimum of 10,000 spermatozoa per hybridization per male was counted for a total of over 200,000 spermatozoa analysed. The mean frequency of diploid spermatozoa was 0.13% for the autosomal study and 0.14% for the sex chromosomal study, which were not significantly different. One donor had extremely divergent values of diploidy in the two studies. Analysis of values in the other nine donors demonstrated no significant difference in the two diploidy estimates. These results indicate that the FISH technique is an accurate and reliable method for determining diploid frequencies in human spermatozoa.

Cell Nucleus↗

PubMed2006

A human tetraploid pachytene spermatocyte as the possible origin of diploid sperm: a case report.

Diploid spermatozoa represent 0.2-0.3% of all spermatozoa in the normal population and cause 8.3% of diandric triploids. Errors in meiosis I and II are the most common mechanisms by which diploid spermatozoa are produced. Endoreduplication before meiosis has been suggested as a possible origin for tetraploid meiocytes, which might, in turn, produce diploid sperm. Synaptonemal complex (SC) spreads of a fertile man were immunolabelled (SCP3, MLH1 and CENP) and hybridized with subtelomere-specific multiplex fluorescent in situ hybridization (stM-FISH) assay for SCs identification. The unexpected finding of a tetraploid pachytene cell and the identification of all of its SCs demonstrate that synapsis and crossover events can occur in human tetraploid cells. Moreover, it indicates that diploid sperm may also originate from mitotic errors (endoreduplication) occurring before meiosis.

Aneuploidy↗

PubMed1986

Disomic and diploid meiotic products induced in Saccharomyces cerevisiae by the salts of 27 elements.

The effects of salts of 27 elements on recombination and on the production of disomic and/or diploid spores during meiosis of Saccharomyces cerevisiae has been investigated. Be(NO3)2, MgSO4, FeSO4, CuSO4, AgNO3, Na2HAsO4 were inactive on the events studied during meiotic cell division. AuCl4, CdCl2, C4H6O4Pb, SnCl2, K2Cr2O7, RbCl induced both disomic and diploid spores. LiCl acted similarly and also affected recombination. Activity in the induction of disomic spores was shown by MnSO4, HgCl2 and SrCl2. CsCl, CaCl2, Na2MoO4, NiCl2, K2PtCl4 increased the frequency of diploid spores, while NaWO4, VOSO4, KCl, BaCl2 already increased recombination frequency. NaBiO3 showed an effect on meiotic recombination only. A decrease in the occurrence of both diploid and disomic spores was suggested by the data obtained with CoCl2.

Meiosis↗

PubMedDate not supplied

Gene dosage effects on MDH isozyme expression in diploid, triploid, and tetraploid treefrogs of the genus Hyla.

Electrophoretic staining intensities at a polymorphic malate dehydrogenase locus (MDH-1) in the tetraploid treefrog Hyla versicolor and diploid Hyla chrysoscelis were compared using intraspecific and interspecific crosses. Gene dosage levels within the three ploidies were additive for the expression of isozymes encoded by this locus. Using densitometry, phenotypes from intraspecific tetraploid progeny conformed to a tetraploid gene dosage model, while phenotypes from interspecific hybrids conformed to a triploid gene dosage model. Intra- and interspecific progeny phenotypes and ratios agree with theoretical models predicting differences in such phenotypes and ratios, depending on the ploidy of the parents involved. Heterozygous phenotypes revealed that diploid crosses produced only symmetrically heterozygous progeny while tetraploid crosses produced two classes of asymmetrically heterozygous progeny in addition to symmetrically heterozygous progeny. Interspecific crosses produced only asymmetrically heterozygous progeny. Interspecific crosses involving a homozygous diploid genotype and symmetrically heterozygous tetraploid genotype produced three progeny phenotypes, while the reciprocal cross produced only two progeny phenotypes. the value of interspecific crosses between diploids and tetraploids in determining gene silencing at duplicated loci is considered.

Animals↗

PubMed2004

Variation in the membrane transport properties and predicted optimal rates of freezing for spermatozoa of diploid and tetraploid Pacific oyster, Crassostrea gigas.

In the present study, a shape-independent differential scanning calorimeter (DSC) technique was used to measure the dehydration response during freezing of sperm cells from diploid and tetraploid Pacific oysters, Crassostrea gigas. This represents the first application of the DSC technique to sperm cells from nonmammalian species. Volumetric shrinkage during freezing of oyster sperm cell suspensions was obtained at cooling rates of 5 and 20 degrees C/min in the presence of extracellular ice and 8% (v/v) concentration of dimethyl sulfoxide (DMSO), a commonly used cryoprotective agent (CPA). Using previously published data, sperm cells from diploid oysters were modeled as a two-compartment "ball-on-stick" model with a "ball" 1.66 microm in diameter and a "stick" 41 microm in length and 0.14 microm wide. Similarly, sperm cells of tetraploid oysters were modeled with a "ball" 2.14 microm in diameter and a "stick" 53 microm in length and 0.17 microm wide. Sperm cells of both ploidy levels were assumed to have an osmotically inactive cell volume, Vb, of 0.6 Vo, where Vo is the isotonic (or initial) cell volume. By fitting a model of water transport to the experimentally obtained volumetric shrinkage data, the best-fit membrane permeability parameters (Lpg and ELp) were determined. The combined-best-fit membrane permeability parameters at 5 and 20 degrees C/min for haploid sperm cells (or cells from diploid Pacific oysters) in the absence of CPAs were: Lpg = 0.30 x 10(-15) m(3)/Ns (0.0017 microm/min-atm) and ELp = 41.0 kJ/mole (9.8 kcal/mole). The corresponding parameters in the presence of 8% DMSO were: Lpg[cpa] = 0.27 x 10(-15) m(3)/Ns (0.0015 microm/min-atm) and ELp[cpa] = 38.0 kJ/mole (9.1 kcal/mole). Similarly, the combined-best-fit membrane permeability parameters at 5 and 20 degrees C/min for diploid sperm cells (or cells from tetraploid Pacific oysters) in the absence of CPAs were: Lpg = 0.34 x 10(-15) m(3)/Ns (0.0019 microm/min-atm) and ELp = 29.7 kJ/mole (7.1 kcal/mole). The corresponding parameters in the presence of 8% DMSO were: Lpg[cpa] = 0.34 x 10(-15) m(3)/Ns (0.0019 microm/min-atm) and ELp[cpa] = 37.6 kJ/mole (9.0 kcal/mole). The parameters obtained in this study suggest that optimal rates of cooling for Pacific oyster sperm cells range from 40 to 70 degrees C/min. These theoretical cooling rates are in close conformity with empirically determined optimal rates of cooling sperm cells from Pacific oysters, C. gigas.

Animals↗

PubMed1980

Photosynthetic and Photorespiratory Carbon Metabolism in Mesophyll Protoplasts and Chloroplasts Isolated from Isogenic Diploid and Tetraploid Cultivars of Ryegrass (Lolium perenne L.).

Photosynthetic (14)CO(2) fixation, [(14)C]glycolate formation, and the decarboxylation of [1-(14)C]glycolate and [1-(14)C]glycine by leaf mesophyll protoplasts isolated from isogenic diploid and tetraploid cultivars of ryegrass (Lolium perenne L.) were examined. The per cent O(2) inhibition of photosynthesis in protoplasts from the tetraploid cultivar was less than that of the diploid line at both 21 and 49% O(2). Kinetic studies revealed that the K(m) (CO(2)) for photosynthesis by the diploid protoplasts was about twice that of the tetraploid line. In contrast, the K(i) (O(2)) for protoplast photosynthesis was similar in both cultivars, as was the potential for oxidizing glycolate and glycine to CO(2) via the photorespiratory carbon oxidation cycle. Although the maximal rates of glycolate accumulation by the isolated protoplasts in the presence of 21% O(2) and a glycolate oxidase inhibitor were similar in the two cultivars, the percentage of total fixed (14)C entering the [(14)C]glycolate pool and the ratio of the rate of [(14)C]glycolate formation to (14)CO(2) fixation at 21% O(2) and low pCO(2) were about two times greater in protoplasts and intact chloroplasts isolated from the diploid line compared to the tetraploid. These results fully support the recent observation that a doubling of ploidy in various ryegrass cultivars reduced the K(m) (CO(2)) of purified ribulose bisphosphate carboxylase-oxygenase by about one-half without affecting the K(i) (O(2)) (Garrett 1978 Nature 274: 913-915).

Journal Article↗

PubMed1992

The spo0A gene is implicated in the maintenance of non-complementing diploids in Bacillus subtilis.

Bacillus subtilis can exist in a diploid state in which two genetically distinct chromosomes co-exist in the same cell and yet only one of them is expressed, thereby determining the phenotype. Such cells are called non-complementing diploids (Ncds). In this study, two types of experiments are reported which indicate that a previously known pleiotropic gene, spo0A, plays a role in the maintaining the diploid state, as follows. (i) When protoplasts of two Spo0A mutant strains were fused, the resulting products continued to segregate cells of both parental phenotypes for many more divisions than had been reported previously. (ii) When a stable Ncd (an Ncd in which the unexpressed markers are not spontaneously activated at a detectable level) harbouring a chloramphenicol acetyltransferase gene on the silent chromosome was transformed with spo0A null alleles the transformants often expressed chloramphenicol acetyltransferase activity. Together these results indicate that the spo0A gene is involved in maintenance of the diploid state in both unstable and stable Ncds.

Bacillus subtilis↗