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PubMed2004

Proteome comparative analysis of gynogenetic haploid and diploid embryos of goldfish (Carassius auratus).

Recently, it was found that in the gynogenetic haploid and diploid embryos of goldfish, which have exactly the same genome, the haploid condition results in obstruction of gene expression and abnormal development while the diploid embryos have normal gene expression and development. A diploid-dependent regulatory apparatus was proposed to regulate gene expression. To study the difference at the protein expression level of the embryos of haploid and diploid in development, we extracted the total proteins of both the gynogenetic haploid and diploid embryos of goldfish in the same eye formation stage. Two-dimensional polyacrylamide gel electrophoresis was used to separate proteins. The stained gel images were analyzed with the PDQUEST software. A part of protein spots that were differentially expressed in haploid and diploid embryos were identified by matrix assisted laser desorption/ionisation-time of flight-mass spectrometry and database analysis. Sixteen protein spots that were absolutely different (only expressed in diploid embryos but not in haploid embryos or vice versa) and 16 protein spots that were up- and downregulated were identified unambiguously, which include some proteins that are correlative with eyes development, nerve development, developing regulation, cell differentiation, and signal transduction. The different significantly gene expression during embryos developing between diploid and haploid is demonstrated.

Animals↗

PubMed1983

Towards an understanding of the molecular mechanisms regulating gene expression during diploidization in phylogenetically polyploid lower vertebrates.

Polyploidization and regional gene duplication have occurred frequently during vertebrate evolution, providing the genetic material necessary for creating evolutionary novelties. Mammals, including man, can be regarded as diploid species with a polyploid history of evolution. Polyploidization steps during the phylogeny of mammals probably took place in the genomes of amphibian- or fish-like mammalian ancestors. The polyploid status has subsequently been shaped by the process of diploidization, leading to genomes that are polyploid with respect to the amount of genetic material and the number of gene copies, and diploid with respect to the level of gene expression and chromosomal characteristics. Phylogenetically tetraploid amphibian and teleost species together with their diploid close relatives can be used as a model system to study the effect of polyploidization and the mechanisms of diploidization of a parallel event during early mammalian evolution. Experimental evidence permits the assumption that the diploidization of gene expression in tetraploid cyprinid fish may be functionally correlated with structural modifications of the ribosomal components, RNA and protein. These findings are discussed in the light of reduced protein synthesis in diploidized tetraploid species and a mechanism to explain diploidization during mammalian evolution.

Amphibians↗

PubMed2002

Studies of the transferability of microsatellites derived from Triticum tauschii to hexaploid wheat and to diploid related species using amplification, hybridization and sequence comparisons.

Hexaploid wheat ( Triticum aestivum L em Thell) is derived from a complex hybridization procedure involving three diploid species carrying the A, B and D genomes, respectively. We recently isolated microsatellites from a T. tauschii library enriched for various motifs and evaluated the transferability of these markers to several diploid species carrying the A, B or D genomes. All of the primer pairs amplifying more than one locus on bread wheat and half of those giving D-genome-specific loci gave an amplification product on A-and/or B-diploid species. All of the markers giving a single amplification product for T. tauschii and no amplification on the other diploid species were D-genome-specific at the hexaploid level. The non-specific microsatellite markers (which gave an amplification product on diploid species carrying the A, B or D genome) gave either a complex amplification pattern on bread wheat (with several bands) or generated a single band which mapped to the D genome. Southern blot hybridizations with probes corresponding to the microsatellite flanking regions gave a signal on all diploid and hexaploid species, whatever the specificity of the microsatellite. The patterns observed on bread wheat were generally in accordance with those observed for diploid species, with slight rearrangements. This suggests that the specificity of microsatellite markers is probably due to mutations in microsatellite flanking regions rather than sequence elimination during polyploidization events and that genome stringency is higher at the polyploid than at the diploid level.

Journal Article↗

PubMed1980

Genetic control of diploid recovery after gamma-irradiation in the yeast Saccharomyces cerevisiae.

Genetic mechanisms(s) of gamma-ray resistance of the diploid and budding haploid cells of S. cerevisiae were investigated, with special reference to mitotic recombination, by examining 11 rad mutant strains. The radiosensitivity of the diploid was markedly enhanced in certain gamma-ray-sensitive rad mutants, whereas the sensitivity of the haploid was not so enhanced in these rad mutants. These enhanced sensitivities of diploids were irrespective of their own haploid sensitivities. From these results, the existence of a mechanism of diploid-specific recovery was postulated. The magnitude of diploid radioresistance in rad mutants was positively correlated with the ability for the induction of mitotic recombinational events which were controlled by RAD genes belonging to the RAD-51 genetic pathway. The genetic mechanism(s) of the diploid recovery after gamma-irradiation are probably related to recombinational processes between the homologous chromosomes leading to reciprocal recombination or non-reciprocal gene conversion. Furthermore, the higher radioresistance of budding cells in comparison with the non-budding cells was also correlated to the diploid radioresistance with a few exceptions. Consequently, the mechanism(s) of budding radioresistance similar to the diploid recovery seems to be related to mitotic recombinational processes.

Diploidy↗

PubMed2000

Allelic losses of 17p, 5q, and 18q loci in diploid and aneuploid populations of multiploid colorectal carcinomas.

17p, 5q, and 18q allelic losses are involved in the pathogenesis and progression of colorectal carcinoma, and DNA aneuploidy in this type of cancer is thought to result from alterations of these chromosomal loci. However, genetic differences between diploid and aneuploid populations of multiploid carcinoma, defined as the coexistence of diploid and aneuploid populations in the same area, remain unclear. The differences in 17p, 5q, and 18q allelic losses between the diploid and aneuploid populations in 24 sporadic DNA multiploid colorectal carcinomas were analyzed by use of crypt isolation coupled with DNA cytometric sorting and polymerase chain reaction assay. 17p Allelic loss was observed in 7 of 22 diploid populations excluding 1 case of microsatellite instability but was found in 21 of 23 aneuploid populations. Although 5q allelic loss was detected in only 3 of 22 diploid populations, 13 of 22 aneuploid populations had 5q allelic loss. Losses of the 18q allele were frequently found in aneuploid populations (15 of 20), although no 18q allelic loss was detected in corresponding diploid populations. 17p Allelic losses may play an important role in the progression from a diploid status to an aneuploid status in a specific subset of colorectal cancer. However, 18q or 5q allelic losses do not appear to precede nor to facilitate the aneuploid clonal divergence of cancer cells. Multiploidy is a useful model to study genetic alterations between diploid and aneuploid populations.

Adult↗

PubMed2005

Diploid males and their triploid offspring in the paper wasp Polistes dominulus.

Although the hymenopteran sex-determining mechanism generally results in haploid males and diploid females, diploid males can be produced via homozygosity at the sex-determining locus. Diploid males have low fitness because they are effectively sterile or produce presumably sterile triploid offspring. Previously, triploid females were observed in three species of North American Polistes paper wasps, and this was interpreted as indirect evidence of diploid males. Here we report what is, to our knowledge, the first direct evidence: four of five early male-producing Polistes dominulus nests from three populations contained diploid males. Because haploid males were also found, however, the adaptive value of early males cannot be ignored. Using genetic and morphological data from triploid females, we also present evidence that both diploid males and triploid females remain undetected throughout the colony cycle. Consequently, diploid male production may result in a delayed fitness cost for two generations. This phenomenon is particularly relevant for introduced populations with few alleles at the sex-determining locus, but cannot be ignored in native populations without supporting genetic data. Future research using paper wasp populations to test theories of social evolution should explicitly consider the potential impacts of diploid males.

Animals↗

PubMed2002

Pollen competition as a unilateral reproductive barrier between sympatric diploid and tetraploid Chamerion angustifolium.

Speciation requires the evolution of barriers to gene exchange between descendant and progenitor populations. Cryptic reproductive barriers in plants arise after pollination but before fertilization as a result of pollen competition and interactions between male gametophytes and female reproductive tissues. We tested for such gametic isolation between the polyploid Chamerion angustifolium and its diploid progenitor by conducting single (diploid or tetraploid) and mixed ploidy (1 : 1 diploid and tetraploid) pollinations on both cytotypes and inferring siring success from paternity analysis and pollen-tube counts. In mixed pollinations, polyploids sired most (79%) of their own seeds as well as those of diploids (61%) (correcting for triploid block, siring success was 70% and 83%, respectively). In single donor pollinations, pollen tubes from tetraploids were more numerous than those from diploids at four different positions in each style and for both diploid and tetraploid pollen recipients. The lack of a pollen donor x recipient interaction indicates that the tetraploid siring advantage is a result of pollen competition rather than pollen-pistil interactions. Such unilateral pollen precedence results in an asymmetrical pattern of isolation, with tetraploids experiencing less gene flow than diploids. It also enhances tetraploid establishment in sympatric populations, by maximizing tetraploid success and simultaneously diminishing that of diploids through the production of inviable triploid offspring.

Crosses, Genetic↗

PubMed2001

Origin of Cryptococcus neoformans var. neoformans diploid strains.

The basidiomycetous yeast Cryptococcus neoformans is an important human fungal pathogen. Two varieties, C. neoformans var. neoformans and C. neoformans var. gattii, have been identified. Both are heterothallic with two mating types, MATa and MATalpha. Some rare isolates are self-fertile and are considered occasional diploid or aneuploid strains. In the present study, 133 isolates, mostly from Italian patients, were investigated to detect the presence of diploid strains in the Igiene Università Milano culture collection. All of the diploid isolates were further investigated by different methods to elucidate their origins. Forty-nine diploid strains were identified by flow cytometry. PCR fingerprinting using the (GACA)(4) primer showed that the diploid state was associated with two specific genotypes identified as VN3 and VN4. Determination of mating type on V8 juice medium confirmed that the majority of the strains were sterile. PCR and dot blotting using the two pheromone genes (MFa and MFalpha) as probes identified 36 of the 49 diploid isolates as MATa/alpha. The results of pheromone gene sequencing showed that two allelic MFalpha genes exist and are distinct for serotypes A and D. In contrast, the MFa gene sequence was conserved in both serotype alleles. Amplification of serotype-specific STE20 alleles demonstrated that the diploid strains contained one mating locus inherited from a serotype A parent and one inherited from a serotype D parent. The present results suggest that diploid isolates may be common among the C. neoformans population and that in Italy and other European countries serotype A and D populations are not genetically isolated but are able to recombine by sexual reproduction.

Cryptococcosis↗

PubMed1987

Live haploid-diploid and other unusual mosaic chickens (Gallus domesticus).

Sixteen haploid-diploid chickens, four diploid-triploids, and one haploid-diploid-triploid chicken have been found in a layer line of crossbred origin. Live haploid-euploid vertebrates have not been reported previously. Flow cytometric analysis revealed 1-80% haploid red blood cells (RBCs) in the haploid-euploid chickens and 5-30% triploid RBCs in the diploid-triploids. Mosaic cell lines were also found in leukocytes and many tissues. The haploid-diploid chickens were all phenotypically normal and fertile, but the phenotype and fertility of the diploid-triploid chickens depended on their sex-chromosome complements and the ratio of diploid to triploid cells. The haploid cell lines are thought to originate from supernumerary spermatozoa, and the diploid-triploids from binucleated oocytes with meiotic errors. It is likely that genetic factors are partly responsible for the occurrence of these mosaic chickens.

Animals↗

PubMed1995

Assessment and significance of diploid-range epithelial populations in DNA aneuploid breast carcinomas using multi-parametric flow cytometry.

A 2-color (PI, cytokeratin--FITC) multi-parametric analysis of intact cells was used to reveal diploid-range epithelial populations by flow cytometry in 108 consecutive DNA aneuploid breast carcinomas. Thirty-eight tumors (35%) contained a significant diploid range epithelial population, defined as cytokeratin-positive cells having a DNA content indistinguishable from that of endogenous lymphocytes and comprising at least 20% of all cytokeratin-positive cells. An additional 23 cases (21%) contained a minor diploid range epithelial population having a normal DNA content and comprising only 5-20% of all cytokeratin-positive cells. Multiple DNA aneuploid stemlines were present in 24 cases (22%). Diploid-range populations were more frequent (91%) in tetraploid cases than in hyperdiploid (32%), hypodiploid (17%) or hypertetraploid cases (20%). The presence of diploid epithelial populations and/or multiple aneuploid stemlines correlated with histologic parameters, including an extensive intraductal component (unimodal--4% vs. multi-modal--57%, P = 0.001), heterogeneous differentiation (unimodal--0% vs. multi-modal--52%, P = 0.001), and multi-focal growth with residual interspersed benign tissue (unimodal--8% vs. multimodal--57%, P = 0.01). These data show that diploid-range epithelial cells are frequent in aneuploid breast carcinomas analyzed by flow cytometry. In some tumors, these populations undoubtedly reflect the presence of residual benign epithelium. The numerical dominance of other histograms by near-diploid measurements suggests the presence of diploid-range neoplastic stemlines which would be 'hidden' by contaminating host-derived cells in single parameter DNA histograms. Finally, the correlation of DNA content heterogeneity with distinctive histologic patterns of breast neoplasia implies that co-existing stemlines may have biological significance in the progression of some tumors.

Aneuploidy↗

PubMed1998

Image cytometry of breast carcinomas that are DNA diploid by flow cytometry: time to revise the concept of DNA diploidy?

OBJECTIVE: To investigate whether breast carcinomas found to be DNA diploid by flow cytometry (FCM) are still diploid if reassessed by image cytometry (ICM). STUDY DESIGN: In a series of 286 breast cancers analyzed by FCM there were 100 (35%) cancers that were classified as DNA diploid. Fourteen of the 100 diploid cases were selected for further analysis with ICM because the patient had died of breast cancer within 11-84 months after the diagnosis (a group with unfavorable outcomes), and 19 cases were selected at random from the cases who had no recurrence of cancer during follow-up of six or more years (a favorable group). RESULTS: Eleven (33%) of the 33 cases turned out to be DNA nondiploid, with a DNA index > or = 1.2 when analyzed by ICM. Nine of the 11 DNA aneuploid samples by ICM were found among the 14 patients with unfavorable prognoses and only 2 among the 19 patients with favorable outcomes (P = .002). The five-year survival rate of the women with DNA diploid cancer by both methods was 86%, whereas that of patients with DNA aneuploid cancer by ICM was 36% (P = .002). CONCLUSION: The results show that some breast carcinomas classified as DNA diploid based on FCM are not DNA diploid by ICM and that such carcinomas are associated with poorer outcomes than the ones that are DNA diploid also by ICM. The prognostic significance of DNA ploidy in breast cancer may need to be reexamined in studies where both FCM and ICM are used.

Breast Neoplasms↗

PubMed2001

Clone heterogeneity in diploid and aneuploid breast carcinomas as detected by FISH.

We investigated the relationship between DNA ploidy and alterations in chromosomes 1, 8, 12, 16, 17, and 18 in 63 breast carcinoma samples by static cytofluorometry and fluorescence in situ hybridization. Thirty specimens were diploid and 33 were aneuploid. In aneuploid samples, the DNA index value ranged from 1.3 to 3.1, with a main peak near tetraploid values. Diploid clones were present in 21 of 33 aneuploid specimens. Fluorescence in situ hybridization analysis showed a heterogeneous degree of alterations in diploid specimens: one sample was normal, 16 samples had one to three chromosome alterations involving mostly chromosomes 1, 16, and 17, and 13 samples an even higher degree of alterations. The 33 aneuploid specimens showed a very high number of signals (four, five, or more). All the investigated chromosomes were affected in 23 of 33 specimens. Alterations in chromosomes 1 and 17 were detected to a similar percentage in diploid and aneuploid samples, whereas chromosome 16 monosomy was more frequent in diploid samples. Overrepresentation of chromosomes 8, 12, 16, and 18 was significantly higher in aneuploid than in diploid samples. Based on these results, we suggest that diploid and aneuploid breast carcinomas are genetically related. Chromosome 1 and 17 alterations and chromosome 16 monosomy are early changes. Allelic and chromosomal accumulations occur during progression of breast carcinoma by different mechanisms. The high clone heterogeneity found in 17 of 33 aneuploid samples could not be completely explained by endoreduplication and led to the suggestion that chromosomal instability concurs with aneuploidy development. This different evolutionary pathway might be clinically relevant because clone heterogeneity might cause metastasis development and resistance to therapy.

Aneuploidy↗

PubMed2003

Near-diploid and near-triploid human sporadic colorectal adenocarcinomas differ for KRAS2 and TP53 mutational status.

Mutations of the KRAS2 protoncogene and inactivation of the TP53 oncosuppressor gene have been suggested to contribute to chromosomal instability (CIN) and aneuploidy in colorectal cancer (CRC). Previous work has also shown that the degree of DNA ploidy [DNA index (DI)], as obtained by flow cytometry in CRC, is non-randomly distributed and, in particular, that DI near-diploid and near-triploid values are well separated by a low-probability valley region. At present, it is not known whether a relationship exists between DI and the mutational status of KRAS2 and TP53. Multiple samples obtained from 35 human sporadic CRCs have been used to provide nuclei suspensions for flow cytometric analysis and sorting of specific DI subpopulations. Sorted nuclei were then used to analyze the high-microsatellite-instability (MSI-H) phenotype and the mutation spectrum of the KRAS2 and TP53 genes. A single MSI-H case was detected. There were 6 DNA diploid (DI = 1) and 29 aneuploid (DI not equal 1) CRCs, with the DI aneuploid cases non-randomly subdivided in 9 near-diploid (DI not equal 1 and DI </= 1.4), 8 near-triploid (1.4 < DI < 1.6), and 12 high-aneuploid (DI >/= 1.6) cases. Proximal CRCs were more often DNA diploid and near-diploid than distal ones, and Dukes' C cases were more commonly high-aneuploid than Dukes' B. Moreover, the incidence of mutations of the KRAS2 and TP53 genes was lowest among the DNA near-triploid subpopulations and highest among the near-diploid ones. We suggest that DNA near-diploid and near-triploid subpopulations in human sporadic CRC reflect different genetic mechanisms of CIN and have a potentially different clinical behavior.

Adenocarcinoma↗

PubMed2000

Diploid strains of the pathogenic basidiomycete Cryptococcus neoformans are thermally dimorphic.

Cryptococcus neoformans is an opportunistic human pathogenic fungus with a defined sexual cycle. Clinical and environmental isolates of C. neoformans are haploid, and the diploid stage of the lifecycle is thought to be transient and unstable. In contrast, we find that diploid strains are readily obtained following genetic crosses of congenic MATalpha and MATa strains. At 37 degrees C, the diploid strains grow as yeast cells with a single nucleus that is larger than a haploid nucleus, contains a 2n content of DNA by FACS analysis, and is heterozygous for the MATalpha and MATa loci. At 24 degrees C, these diploid self-fertile strains filament and sporulate, producing recombinant haploid progeny in which meiotic segregation has occurred. In contrast to dikaryotic filament cells that are typically linked by fused clamp connections during mating, self-fertile diploid strains produce monokaryotic filament cells with unfused clamp connections. We also show that these diploid strains can be transformed and sporulated and that an integrated selectable marker segregates in a mendelian fashion. The diploid state could play novel roles in the lifecycle and virulence of the organism and can be exploited for the analysis of essential genes. Finally, the observation that dimorphism is thermally regulated suggests similarities between the lifecycle of C. neoformans and other thermally dimorphic human pathogenic fungi, including Histoplasma capsulatum, Blastomyces dermatitidis, Coccidioides immitis, Paracoccidioides brasiliensis, and Sporothrix schenkii.

Crosses, Genetic↗

PubMed2004

Chromosomes associate premeiotically and in xylem vessel cells via their telomeres and centromeres in diploid rice ( Oryza sativa).

Studies of the meiosis of diploid plants such as Arabidopsis, maize and diploid progenitors of wheat have revealed no premeiotic association of chromosomes. Premeiotic and somatic association of chromosomes has only been previously observed in the anther tissues and xylem vessel cells of developing roots in polyploid plants such as hexaploid and tetraploid wheat, polyploid relatives of wheat and artificial polyploids made from the progenitor diploids of wheat. This suggested that this association was confined specifically to polyploids or was induced by polyploidy. However, we developed procedures for in situ hybridization on structurally well-preserved tissue sections of rice, and analysed two diploid rice species ( Oryza sativa and O. punctata). Contrary to expectation, this has revealed that centromeres and telomeres also associate both in the xylem vessel cells of developing root and in undifferentiated anther cells in these diploids. However, in contrast to wheat and related polyploids, where the initial association in undifferentiated anthers is between either non-homologous or related chromosomes, and not homologous chromosomes, the initial association of rice chromosomes seems to be between homologues. Thus, in contrast to the diploid dicot model Arabidopsis, meiotic studies on the diploid model cereal, rice, will now need to take into account the effects of premeiotic chromosome association.

Centromere↗

PubMed1992

Uptake of taurocholate, a vecuronium-like organic cation, ORG 9426, and ouabain into carcinogen-induced diploid and polyploid hepatocytes obtained by centrifugal elutriation.

Bile acid uptake, an important function of differentiated hepatocytes, is decreased in hepatocellular carcinomas and gamma-glutamyltranspeptidase-positive, putatively preneoplastic hepatocytes. Whether hepatic uptake is also changed in carcinogen-induced diploid hepatocytes versus polyploid hepatocytes is unknown. The present study has determined whether the hepatic uptake of three model compounds, an anionic bile acid, an organic cation and a neutral organic compound, into diploid cells is different from that in polyploid hepatocytes. These two hepatocyte populations were separated from the parent freshly isolated hepatocyte suspension by centrifugal elutriation. Flow cytometric analysis indicated that the diploid fraction contained approximately 83% diploid cells and that the polyploid fraction had about 84% polyploid hepatocytes. Initial uptake velocity was determined for taurocholate (1-50 microM), ORG 9426 (20-400 microM), a vecuronium-like cation, and ouabain (20-500 microM). Apparent Km was not different between diploid and polyploid cells for the three tested substrates, whereas apparent Vmax was decreased in diploid hepatocytes for taurocholate and ouabain by 42 and 55%, respectively. There were no changes in the hepatic uptake of ORG 9426. These data indicate that uptake by the bile acid/multispecific carrier is compromised in carcinogen-induced diploid cells.

Androstanols↗

PubMed1983

Disomic and diploid meiotic products in Saccharomyces cerevisiae. Effect of vincristine, vinblastine, adriamycin, bleomycin, mitomycin C and cyclophosphamide.

The effect of some antineoplastic drugs on the induction of disomic and diploid meiotic products in Saccharomyces cerevisiae was evaluated. Vincristine and vinblastine turned out not to be effective on any of the four genetic phenomena studied (sporulation, recombination, disomic induction and diploid induction). Adriamycin showed only slight activity in inducing diploids, particularly during the first meiotic division. Cyclophosphamide was active on both phenomena leading to the formation of disomic and diploid spores. Mitomycin C and bleomycin were effective on the induction of diploids. In all of these inductions, the origin of diploids was due to failure of the second meiotic division. No significant effects were found on recombination frequency. As a general conclusion, one may assume that formation of aneuploid and diploid gametes are two distinct phenomena, not necessarily correlated from the point of view of the mechanism and of the specificity of induction.

Antineoplastic Agents↗

PubMed1996

The incidence of bovine diploid oocytes matured in vitro.

The present work describes a cytogenetic study of in vitro matured bovine oocytes to determine the proportion of unreduced oocytes carrying the diploid number of chromosomes. Studied oocytes were derived either from a pool of oocytes collected from several different donors, or from oocytes collected separately from individual donors. In vitro maturation was performed by culturing immature oocytes for 24 h in TCM199-medium supplemented with estrous cow serum and hormones at 39 degrees C in 5% CO2. Chromosomal complement of in vitro matured oocytes was studied by Giemsa-staining and produced analyzable results in approximately 60% of the cases. The results revealed that approximately 75% of oocytes had matured to the MII stage in both groups of oocytes studied. Of these MII oocytes, 11 and 12.4% (from the oocyte pool or from individual cows, respectively) contained the diploid set of chromosomes. The occurrence of diploid MII oocytes was not quite uniform among donors: 40.5% of all cows produced one, 18.9% produced two and 2.7% (one cow) produced three diploid MII oocytes. However, a positive relationship between the number of MII oocytes in general and diploid MII oocytes among individual donors was not found. The possible factors that may lead to the formation of diploid MII oocytes observed under IVM procedures are discussed. The results of this study showed a higher incidence of diploid oocytes in cattle than previously reported.

Journal Article↗