Construction and Characterization of Isogenic Series of Saccharomyces cerevisiae Polyploid Strains

Tetraploid cells of Saccharomyces cerevisiae are generated spontaneously in a homothallic MATa/MATα diploid population at low frequency (approximately 10⁻⁶ per cell) through the homozygosity of mating-type alleles by mitotic recombination followed by homothallic switching of the mating-type alleles. To isolate tetraploid clones more effectively, a selection method was developed that used a dye plate containing 40 mg each of eosin Y and amaranth in synthetic nutrient agar per liter. It was possible to isolate tetraploid clones on the dye plate at a frequency of 1 to 3% among the colonies colored dark red in contrast to the light red of the original diploid colonies. Isogenic series of haploid to tetraploid clones with homozygous or heterozygous genomic configurations were easily constructed with the tetraploid strains. No significant differences in specific growth rate or fermentative rate were observed corresponding to differences in ploidy, although the haploid clones showed a higher frequency of spontaneous respiratory-deficient cells than did the others. However, a significant increment in the fermentative rate in glucose nutrient medium was observed in the hybrid strains constructed with two independent homozygous cell lines. These observations strongly suggest that the polyploid strains favored by the brewing and baking industries perform well not because of the physical increment of the cellular volume by polyploidy but because of the genetic complexity or heterosis by heterozygosity of the genome in the hybrid polyploid cells.     

Tumor-specific immunity induced by somatic hybrids. I. Lack of relationship between immunogenicity and tumorigenicity of selected hybrids.

Hybrid clones were derived from fusion of TEPC-15 plasmacytoma cells of BALB/c mice with mouse L cells of C3H origin. The morphology, tumorigenicity, and immunogenicity of three representative clones were extensively studied. One clone (LTC-1) showed a morphology intermediate to that of either parental cell and possessed the highest tumorigenic and immunogenic properties. The other two clones displayed a "flat" morphology which differed significantly from that of either parent. One of these two, LTC-4, eventually induced tumors in some (BALB/c X C3H)F1 mice but failed to stimulate protective immunity against TEPC-15 tumor cells in BALB/c mice. The other hybrid clone, LTC-2, has a "very flat" morphology and did not induce tumors, although it was capable of stimulating a significant level of tumor immunity. Histologically, all the tumors induced by hybrid cells were fibrosarcomas rather than plasmacytomas. These results indicate that the morphology of hybrid cells may be correlated with the tumorigenicity as well as the histologic appearance of tumor. In addition, the degree of tumorigenicity of individual hybrid clones does not correspond to their immunogenicity in the host, suggesting that major antigens responsible for immunogenicity may not play an important role in induction of tumors.     

Analysis of myogenesis by somatic cell hybridization : I. Myogenic competence of homotypic hybrids derived from rat L6 myoblasts

Several authors have described the extinction of myogenic competence in hybrids produced by fusion of myogenic and non-myogenic cells. Interpretations of such experiments rest upon the assumption that extinction does not occur with any appreciable frequency as a non-specific consequence of the cell hybridization process itself. In order to test this assumption we have analyzed the myogenic competence of over 140 independent homotypic hybrid clones produced by PEG-mediated fusion of rat L6 myoblasts. Based upon an evaluation of myotube formation in hybrid colonies, we demonstrate that 99% of primary hybrid clones are myogenic. The fact that 97% of secondary hybrid colonies also differentiate indicates that myogenic competence is a stable characteristic of the hybrids. Four hybrid clones were isolated and expanded for analyses of chromosome numbers, myotube formation, creatine kinase activities, and microfluorimetric DNA determinations of myotube nuclei. Our results demonstrate that polyploid homotypic hybrid cells produced by fusion of non-neoplastic, developmentally determined rat myoblasts retain and express their program of differentiation. This work provides a foundation for future studies which will investigate the expression of myogenic properties in hybrids between myogenic and non-myogenic cells.     

Expression of malignancy traits in the interspecific somatic hybrids of tumor and normal cells

Tumorigenicity and anchorage independence in two types of the interspecies hybrids of the tumor and normal mammalian cells were studied. One hybrid type was derived from fusion of spontaneously transformed Chinese hamster and normal mouse cells; the second type was obtained by fusion of SV40-transformed Djungarian hamster and the same mouse cells. The tumorigenicity in the athymic nude mice was suppressed in the first type of hybrids. The hybrid clones derived from fusion of SV40-transformed and normal cells could form tumor in nude mice. Testing of hybrid clones for their ability to form colonies in soft agar showed that all hybrids grew well in the medium, similar to tumor parental cells. These data suggest that malignancy and anchorage independence are under separate genetic control. The influence of the origin of the tumor parental cells (spontaneous or SV40-virus transformation) on the expression of the malignancy in hybrids of the tumor and normal cells is discussed.     

Genetic characterization of an arctic zooplankter: insights into geographic polyploidy

Species of Bosmina from the temperate regions of North America and Europe are diploid and reproduce by cyclical parthenogenesis. By contrast, this study provides evidence that the dominant bosminid taxon in High Arctic lakes reproduces by obligate parthenogenesis and is a polyploid derived from interspecific hybridization. Sinobosmina liederi, a species common in temperate North America, is likely to have been one parent of these hybrids, but the other parent is unknown. As neither parent was detected in the Arctic, it seems unlikely that the hybrid clones that now occupy arctic lakes were synthesized locally. Most habitats contained only one or two clones, despite a total of 38 clones in the region, suggesting that priority effects have been important in restricting diversity within single lakes. The high regional diversity of arctic bosminids could reflect either repeated hybridization between the parent taxa or the genetic instability of newly formed polyploid lineages. These processes would produce hybrid polyploids that are considerably more diverse than their sexual parent taxa, and this difference in genetic diversity may confer an advantage to the polyploid biotype. As many zooplankton taxa from the arctic possess genetic characteristics similar to those of bosminids, these processes may provide a general explanation for the widespread occurrence of polyploids in the Arctic.     

Association of human chromosome 14 with a ts defect in G1 of Chinese hamster K12 cells.

Somatic cell hybrids between human lymphoblastoid cells (Raji) and temperature-sensitive Chinese hamster cells (K12) were selected from monolayer cultures in MEM at 40 degrees C. A total of 21 hybrid clones were isolated and karyotyped. All clones contained a near complete set of Chinese hamster chromosomes and 1 to 5 human chromosomes. Human chromosome 14 present in the hybrid cells of all clones; and was the only human chromosome retained in 10 clones. The presence of human chromosome 14 in hybrids was further confirmed by the demonstration of human nucleoside phosphorylase activity in the hybrid cells. Only one hybrid clone was positive for EBNA, the Epstein-Barr virus antigen present in Raji cells. These findings indicate that human chromosome 14 contains the necessary information for the K12 cells to overcome their G1 defect in the cell cycle and grow at non-permissive temperature. The present study lends strong support to the possibility that different steps in the G1 phase of the cell cycle are controlled by genes located on different chromosomes.     

Polyploid rat glioma cells. Production, oscillations of membrane potential and response to neurohormones

Due to their small size, electrophysiological investigation of C6 rat glioma cells by intracellular recording with microelectrodes is very difficult. In order to facilitate such electrophysiological studies, the degree of ploidy of C6 cells was increased in three successive steps. At each step complementary drug-resistant mutants of C6 were fused and hybrid cell clones isolated in selective culture medium. Cell volume was measured by electronic cell sizing and DNA content per cell assessed by impulse cytofluorimetry. In the final polyploid line, C6-4-2, both parameters were found to be increased 6-fold in comparison with the original cell line C6. In response to β-adrenergic agonists and prostaglandin E₁ the intracellular concentration of cAMP was raised in C6-4-2 cells. Stable membrane potentials could be recorded from the polyploid glioma cells much more easily than from the original C6 cells. The plasma membrane of the C6-4-2 cells could hyperpolarize spontaneously with a frequency of about 1 cycle/min. Elevation of the concentration of Ca²⁺ ions (but not other earth alkali ions) in the medium induced the onset of the spontaneous hyperpolarizations. The membranes of the C6-4-2 cells also hyperpolarized in response to acetylcholine, γ-aminobutyric acid (GABA) and glutamate.     

Embryonic stem cell/fibroblast hybrid cells with near-tetraploid karyotype provide high yield of chimeras

Ten primary clones of hybrid cells were produced by the fusion of diploid embryonic stem (ES) cells, viz., line E14Tg2aSc4TP6.3 marked by green fluorescent protein (GFP), with diploid embryonic or adult fibroblasts derived from DD/c mice. All the hybrid clones had many characteristics similar to those of ES cells and were positive for GFP. Five hybrid clones having ploidy close to tetraploidy (over 80% of cells had 76–80 chromosomes) were chosen for the generation of chimeras via injection into C57BL blastocysts. These hybrid clones also contained microsatellites marking all ES cell and fibroblast chromosomes judging from microsatellite analysis. Twenty chimeric embryos at 11–13 days post-conception were obtained after injection of hybrid cells derived from two of three clones. Many embryos showed a high content of GFP-positive descendents of the tested hybrid cells. Twenty one adult chimeras were generated by the injection of hybrid cells derived from three clones. The contribution of GFP-labeled hybrid cells was significant and comparable with that of diploid E14Tg2aSc4TP6.3 cells. Cytogenetic and microsatellite analyses of cell cultures derived from chimeric embryos or adults indicated that the initial karyotype of the tested hybrid cells remained stable during the development of the chimeras, i.e., the hybrid cells were mainly responsible for the generation of the chimeras. Thus, ES cell/fibroblast hybrid cells with near-tetraploid karyotype are able to generate chimeras at a high rate, and many adult chimeras contain a high percentage of descendants of the hybrid cells. A. A. Kruglova and E. A. Kizilova contributed equally to this work. This study was financially supported by grants from the Russian Academy of Sciences, Siberian Branch 5.2 and 14.0.     

Cellular alterations dependent upon the polyoma virus Hr-t function: separation of mitogenic from transforming capacities.

Hr-t mutants of polyoma virus are restricted in their growth properties (host range) and defective in cell transformation and tumor induction. The present study indicates that these mutants have lost the ability to induce morphological transformation, but have retained a mitogenic function. Thus an early and dramatic difference between wild-type virus and hr-t mutant-infected cultures of rat fibroblasts is the morphological change in individual cells observed by light, fluorescence and scanning electron microscopy. Viruses containing an intact hr-t function (wild-type virus and ts-a mutants) induce a transformed phenotype consisting of stellate cell shape, loss of defined cytoplasmic actin architecture, cellular "underlapping," and increased nuclear and nucleolar sizes. These prominent alterations constitute an abortive transformation, peaking 24-48 hr post-infection, and subsequently resolving in most or all of the cells. In contrast, cells infected with hr-t mutants do not develop the above structural changes, but rather retain their preinfection appearance. Both wild-type virus and hr-t mutants induce cellular DNA synthesis in confluent monolayers of rat cells beginning 12-14 hr post-infection. Flow microfluorometric (FMF) analysis confirms the viral mediated transit of cells from the G1 to the S and G2 phases of the cell cycle, as well as an increase in the proportion of cells with an 8N (octaploid) DNA content. Approximately 50% of the clones isolated from wild-type-infected cultures are polyploid. Stable transformants are found among these polyploid clones, but the majority of the latter resemble the parental cells in their morphology and growth properties. Polyploid clones are derived from hr-t mutant-infected cultures at a much lower frequency, similar to that of mock-infected cultures. Data obtained by sequential labeling of infected cultures with 3 H-thymidine and 5-bromo-deoxyuridine, together with cell number quantitation, indicate that hr-t mutants promote only a single round of cell division, while the wild-type virus and ts-a mutants promote multiple rounds. Loss of the hr-t function in polyoma virus therefore reveals a residual viral mitogenic activity, but prevents the virus from effecting morphological transformation of cells with concomitant loss of defined actin cables, polyploidization and multiple cycles of cell division in confluent cultures.     

Segregation of the NK-sensitive phenotype in human x mouse somatic cell hybrids reveals separate genetic control of recognition and postrecognition determinants

In an attempt to investigate the nature of tumor cell-derived membrane surface determinants involved in natural killer cell (NK) recognition or postrecognition events, we have constructed human X mouse interspecies somatic cell hybrids. Highly NK-sensitive (NKs) human tumor cells were fused with NK resistant (NKr) mouse fibroblasts (LMTK-) in polyethylene glycol and selected in hypoxanthine/aminopterin/thymidine medium and ouabain. Hybrids generated from NKs erythroleukemia cells (K-562) or NKs retinoblastoma cells (Y-79) with LMTK- displayed an intermediate NK-sensitive phenotype. One Y-79 X LMTK- hybrid (YL-22) retained a high level of susceptibility to NK binding and cytolysis, as determined by 51Cr release and in cold-target inhibition assays. On the other hand, human NKr RAJI cells generated NK-resistant hybrids when fused with LMTK- fibroblasts. Four hybrids (KL-12, YL-2, YL-22, and YL-43) displaying consistent NK sensitivity were subsequently cloned by limiting dilution. Various hybrid clones derived from the KL-12 hybrid (K-562 X LMTK-) demonstrated a range of NK-sensitive phenotypes. However, the uncloned KL-12 and most cloned lines derived from this hybrid competed against 51Cr-labeled K-562 targets as well as unlabeled K-562 parental cells, regardless of their NK-sensitive phenotype. These findings raise the possibility that chromosomal segregation may be affecting a postbinding step in this hybrid system. The NK-sensitive hybrids exhibited a limited number of human chromosomes as assessed by quinacrine banding. Furthermore, human transferrin receptor (TfR) expression, as monitored by flow cytometry using the B3/25 monoclonal antibody, demonstrated no clear correlation with NK sensitivity or competitive ability in either KL or YL hybrid clones, thus arguing against the involvement of the TfR in human NK recognition. These results suggest that the NK-sensitive phenotype in human tumor cells may be regulated by genes encoded by a limited number of human chromosomes.     

Tumor cells can escape DNA-damaging cisplatin through DNA endoreduplication and reversible polyploidy

Cancer chemotherapy can induce tumor regression followed, in many cases, by relapse in the long-term. Thus this study was performed to assess the determinants of such phenomenon using an in vivo cancer model and in vitro approaches. When animals bearing an established tumor are treated by cisplatin, the tumor initially undergoes a dramatic shrinkage and is characterized by giant tumor cells that do not proliferate but maintain DNA synthesis. After several weeks of latency, the tumor resumes its progression and consists of small proliferating cells. Similarly, when tumor cells are exposed in vitro to pharmacological concentrations of cisplatin, mitotic activity stops initially but cells maintain DNA duplication. This DNA endoreduplication generates giant polyploid cells that then initiate abortive mitoses and can die through mitotic catastrophe. However, many polyploid cells survive for weeks as non-proliferating mono- or multi-nucleated giant cells which acquire a senescence phenotype. Prolonged observation of these cells sheds light on the delayed emergence of a limited number of extensive colonies which originate from polyploid cells, as demonstrated by cell sorting analysis. Theses colonies are made of small diploid cells which differ from parental cells by stereotyped chromosomal aberrations and an increased resistance to cytotoxic drugs. These data suggest that a multistep pathway, including DNA endoreduplication, polyploidy, then depolyploidization and generation of clonogenic escape cells can account for tumor relapse after initial efficient chemotherapy.     

Selective growth, in vitro and in vivo, of individual T cell clones from tumor-infiltrating lymphocytes obtained from patients with melanoma

In recent clinical trials in patients with metastatic melanoma, adoptive transfer of tumor-reactive lymphocytes mediated the regression of metastatic tumor deposits. To better understand the role of individual T cell clones in mediating tumor regression, a 5' RACE technique was used to determine the distribution of TCR beta-chain V region sequences expressed in the transferred cells as well as in tumor samples and circulating lymphocytes from melanoma patients following adoptive cell transfer. We found that dominant T cell clones were present in the in vitro-expanded and transferred tumor-infiltrating lymphocyte samples and certain T cell clones including the dominant T cell clones persisted at relatively high levels in the peripheral blood of the patients that demonstrated clinical responses to adoptive immunotherapy. However, these dominant clones were either undetected or present at a very low level in the resected tumor samples used for tumor-infiltrating lymphocyte generation. These data demonstrated that there was selective growth and survival, both in vitro and in vivo, of individual T cell clones from a relatively small number of T cells in the original tumor samples. These results suggest that the persistent T cell clones played an active role in mediating tumor regression and that 5' RACE analysis may provide an important tool for the analysis of the role of individual T cell clones in mediating tumor regression. A similar analysis may also be useful for monitoring autoimmune responses.     

Cytogenetic characteristics of the cell line BHK-21 (C-13) and three of its clones

Cell clones (C-9, C-24, C-36) of the cell line BHK-21 (C-13) were obtained and characterized. Compared to the original line, cells of these clones are larger in size, display an increased content of nuclear DNA and have more chromosomes. The G-bands were obtained by Giemsa staining following mild trypsin treatment. Marker chromosomes were determined in the original cell line BHK-21 (C-13) and in its large-cell clones. All the clones obtained are polyploid.     

Hybridization and Polyploidization of Saccharomyces cerevisiae Strains by Transformation-Associated Cell Fusion

Hybrid or polyploid clones of Saccharomyces cerevisiae produced by protoplast fusion were easily isolated by selecting transformants with the plasmid phenotype because the transformation was directly associated with cell fusion. When haploid cells were used as the original strain, the transformants were mostly diploids with a significant fraction of polyploids (triploids or tetraploids). Repeated transformation after curing the plasmid gave rise to clones with higher ploidy, but the frequency of cell fusion was severely reduced as ploidy increased.     

Isolation of a human von Willebrand factor cDNA from the hybrid endothelial cell line EA.hy926

A permanent human hybrid endothelial cell line (EA.hy926) was shown to produce the von Willebrand factor, a protein of 250,000 relative mass (Mr) which was secreted into the medium as a 220,000 Mr protein. A cDNA library was constructed in lambda gt11 using mRNA from these hybrid cells. Several von Willebrand factor cDNA clones were isolated from this library using a synthetic oligodeoxyribonucleotide as a hybridization probe. These cDNA clones were used to analyze the von Willebrand factor gene in normal individuals and in cultured cells.     

Enhanced expression of glucose transporter GLUT3 in tumorigenic HeLa cell hybrids associated with tumor suppressor dysfunction.

Previous studies on human cell hybrids between HeLa and normal human fibroblasts have indicated that the tumorigenicy may be controlled by a putative tumor suppressor gene on chromosome 11. We previously demonstrated a twofold increase in glucose uptake with a reduced Km by tumorigenic HeLa cell hybrids which expressed a highly glycosylated GLUT1. In this study, we reported that a tumorigenic cell hybrid, CGL4, also expressed a glucose transporter isoform, GLUT3, that was undetectable in nontumorigenic CGL1 cells. The expression of GLUT3 together with GLUT1 of 70 kDa was also evident in three gamma-ray-induced tumorigenic clones isolated from CGL1 cells, while control nontumorigenic irradiated cells expressed 50 kDa GLUT1 alone. In accordance with this, GLUT3 mRNA was specifically expressed in tumorigenic cell hybrids. To examine the role of GLUT3, clones which stably overexpress GLUT3 were developed from both CGL1 and CGL4 cells. In these transfectants, the affinity for 2-deoxyglucose markedly increased, in parallel with the amount of expressed GLUT3 irrespective of its N-glycosylation state. These results suggest that the enhanced GLUT3 expression in HeLa cell hybrids associated with the tumorigenic phenotypes may account for the increased affinity for 2-deoxyglucose. Possible roles of the putative tumor suppressor in control of gene expression and glucose uptake is discussed.     

Tumor-specific immunity induced by somatic hybrids: IV. Relationship between immunogenicity and expression of surface tumor-associated antigens

Semi-allogeneic hybrid clones were derived by fusion of the TEPC-15 plasmacytoma (H-2^d) and mouse L cells of C3H (H-2^k) origin. Three representative clones were chosen to study the relationship between the expression of different membrane antigens and their immunogenicities leading to protection of recipient mice from the parent TEPC-15 plasmacytoma. The level of surface tumor-specific transplantation antigens (TSTA) was measured by radioimmunoprecipitation with syngeneic anti-TSTA and by inhibition of anti-TSTA binding to the TEPC-15 tumor cells. The most immunogenic hybrid clone (LTC-1) expressed the highest level of TSTA and the weakly immunogenic (LTC-2) and nonimmunogenic (LTC-4) hybrid clones exhibited relatively low levels of TSTA on the surface. Moreover, the strongly immunogenic LTC-1 hybrid cells, but not the parent tumor cells, were effective in priming recipient spleen cells to generate TEPC-15 tumor-specific cytotoxic cells upon subsequent in vitro exposure to the TSTA-bearing cells. Therefore, the level of TSTA on the semi-allogeneic hybrid clones may play an important role in enhancing the immunogenicity of TSTA.     

Genome size evolution and polyploidy in the Daphnia pulex complex (Cladocera: Daphniidae)

Genome size was estimated in 49 clones of the Daphnia pulex complex from temperate and subarctic locations using flow cytometry and microsatellite DNA analyses. Significant genome size differences were found in diploid species belonging to the two genetically distinct groups (the pulicaria and the tenebrosa groups), with clones from the tenebrosa group having genome sizes 22% larger than those in the pulicaria group. Combined flow cytometry and microsatellite DNA analyses revealed that nearly all polyploid clones in the D. pulex complex are triploid and not tetraploid, as was previously suggested. Sequencing analyses of the ND5 gene to position clones in their respective clades within the D. pulex complex have uncovered three triploid clones of Daphnia middendorffiana with a D. pulex maternal parent. This result was unexpected because Daphnia pulicaria has always been identified as the maternal parent of these hybrid polyploid clones. Triploid clones likely owe their origins to interactions between sexual and asexual populations. Further interactions in the tenebrosa group have generated tetraploid clones but these events have been rare.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 97 , 68–79.     

Genetic processes in intergeneric cell hybrids Atropa + Nicotiana

Summary The genetic constitution of the cell hybrids Atropa belladonna + Nicotiana chinensis, obtained by cloning of individual heteroplasmic protoplast fusion products (Gleba et al. 1982) and cultured in vitro for 12 months, has been studied. The study comprised 11 hybrid cell clones of independent origin and included analysis of a) chromosome number, size, morphology, and relative position in metaphase plates, b) multiple molecular forms of the enzymes esterase and amylase, and c) relative nuclear DNA content. The data obtained permit us to conclude that, after one year of unorganized growth in vitro, the cells of most (8) clones had retained chromosomes of both parents, while species-specific elimination of nearly all Atropa chromosomes had occurred in three clones. About half of the non-segregating clones possess 120–150 chromosomes including 50–70 of Atropa and 50–90 of Nicotiana. Other clones are polyploid and possess 200–250 chromosomes with a predominance of either Atropa or Nicotiana chromosome types. Only a few chromosomal changes (reconstituted chromosomes, ring chromosomes) have been detected. In some metaphase plates, chromosomes of the two parents tend to group separately, indicating non-random arrangement of chromosomes of the two parents within the hybrid nucleus. Cytophotometric studies of the relative nuclear DNA content showed that distribution histograms for cell clones were similar to those of non-hybrid cultured cells. Cell populations were relatively homogenous and do not indicate any genetic instability as a result of hybridization between remote plant species. Biochemical analysis of isoenzyme patterns confirmed that in most cell clones, species-specific multiple molecular forms of esterase and amylase from both parents were present, i.e. genetic material of both parental species was expressed in the cell hybrids.Dedicated to Professor G. Melchers with gratitude     

Selection of Morphologically Normal Cell Lines from Polyoma-transformed BHK21/13 Hamster Fibroblasts

A selective method was devised for the isolation of "revertants" from polyoma-transformed sublines derived from BHK21/13 Syrian hamster fibroblasts. A hybrid, polyploid subline was obtained by growing together, in mixed culture in the presence of aminopterin, two variant BHK21/13 sublines lacking either inosinic acid pyrophosphorylase or thymidine kinase. Whereas these variant sublines were resistant to 6-thioguanine or to 5-bromodeoxyuridine, the hybrid had regained sensitivity to both analogues. By plating a polyoma-transformed subline derived from this hybrid in the presence of 6-thioguanine, resistant clones were obtained with a frequency of about 10(-4). All of these surviving clones had a reduced chromosome complement and some of them had regained a normal phenotype.