Selection of mouse X hamster hybrids using HAT medium and a polyene antibiotic.

In the present study we tested the feasibility of utilizing a structurally modified polyene antibiotic, amphotericin B methyl ester (AME), as a half-selection agent for isolating somatic cell hybrids. By using HAT medium supplemented with AME we have isolated interspecific mouse-hamster hybrids from mixed cultures of mouse (TK-C1 ID or HPRT-A9) and hamster (BHK/C 13) cells fused with Sendai virus, lysolecithin or polyethylene glycol. Hybrid cells proliferated and clones were isolated after 2 to 3 weeks growth in three changes of HAT-AME medium and subsequent growth in HAT medium alone. In contrast, genetically deficient parental C1 1D or A9 cells and AME-sensitive BHK/C 13 cells were killed using a similar growth protocol. The described technique is simple, efficient and permits one to use a cell line without a genetic defect in combination with a genetically deficient cell type in hybrid formation.     

Galactocerebrosidase activity in somatic cell hybrids derived from twitcher mouse/control human fibroblasts is associated with human chromosome 17.

Somatic cell hybrids derived from twitcher mouse cells and from control human fibroblasts were selected by two different methods. One method utilized 6-thioguanine-resistant twitcher cells as a parental line and the other used neomycin-resistant control human fibroblasts as a parental line so that hybrid lines could be selected in either HAT or in G-418 medium, respectively. The hybrid lines were analyzed for galactocerebrosidase activity. Since the twitcher cell lines are deficient in galactocerebrosidase activity, the presence of this activity in these hybrid lines depends upon the presence of human chromosome contents. Both galactocerebrosidase-positive and -deficient hybrid lines were analyzed for their human chromosome contents by the use of isozyme markers. In hybrids derived from both selection methods the expression of galactocerebrosidase activity was associated with the presence of human chromosome 17 marker isozymes. This was confirmed cytogenetically by means of trypsin-banded Giemsa staining of intact human chromosome 17 in three galactocerebrosidase-positive hybrid lines.     

Selection of mouse x hamster hybrids using hat medium and a polyene antibiotic

Summary In the present study we tested the feasibility of utilizing a structurally modified polyene antibiotic, amphotericin B methyl ester (AME), as a half-selection agent for isolating somatic cell hybrids. By using HAT medium supplemented with AME we have isolated interspecific mouse-hamster hybrids from mixed cultures of mouse (TK⁻C1ID or HPRT⁻ A9) and hamster (BHK/C 13) cells fused with Sendai virus, lysolecithin or polyethylene glycol. Hybrid cells proliferated and clones were isolated after 2 to 3 weeks growth in three changes of HAT-AME medium and subsequent growth in HAT medium alone. In contrast, genetically deficient parental C1 1D or A9 cells and AME-sensitive BHK/C 13 cells were killed using a similar growth protocol. The described technique is simple, efficient and permits one to use a cell line without a genetic defect in combination with a genetically deficient cell type in hybrid formation. This investigation was supported in part by Contract NIH 69-2161, NIH Grant No. AI-2095 and NIH Training Grant No. GM 507 from the National Institute of General Medical Sciences.     

Transmission of organelle genomes in citrus somatic hybrids

Restriction fragment length polymorphisms (RFLPs), were used to analyze the organelle composition of two-year-old trees, recovered from two different experiments: protoplasts from embryogenic cell suspensions of `Succari' sweet orange (C. sinensis L. Osbeck) were fused with leaf protoplasts of Citropsis gilletiana Swingle & M. Kell or to leaf protoplasts of Atalantia ceylanica(Arn.) Oliv. The somatic hybrids of both fusion combinations had the mitochondrial genome from the embryogenic partner. In some somatic hybrids, non-parental fragments were observed among the mitochondrial patterns. Somatic hybrids between `Succari' + Atalantia had plastid DNA from the embryogenic parent, while the somatic hybrids of `Succari' + Citropsis all had both parental chloroplast genomes. The relative abundance of organelle DNAs in the donor embryogenic and leaf cells may explain the consistent transmission of the embryogenic parent mitochondrial DNA and the inheritance of the chloroplast genome from either parent. This revised version was published online in June 2006 with corrections to the Cover Date.     

Non-random inheritance of organellar genomes in symmetric and asymmetric somatic hybrids between Lycopersicon esculentum and L. pennellii

Summary The organization of the mitochondrial genome and the genotype of the chloroplast genome was characterized using restriction fragment length polymorphisms in a population (82 individuals) of symmetric and asymmetric somatic hybrids of tomato. The protoplast fusion products were regenerated following the fusion of leaf mesophyll protoplasts of Lycopersicon esculentum (tomato cv UC82) with suspension cell protoplasts of L. pennellii that had been irradiated with 5, 10, 15, 25, 50, or 100 kRads from a gamma source. The chloroplast genome in the somatic hybrids showed a random pattern of inheritance, i.e., either parental genome was present in equal numbers of regenerants, while in asymmetric somatic hybrids, the chloroplast genotype reflected the predominant nuclear genotype, i.e., tomato. The mitochondrial genome in the symmetric somatic hybrids showed a non-random pattern of inheritance, i.e., predominantly from the L. pennellii parent; asymmetric somatic hybrids had more tomato-specific mitochondrial sequences than symmetric somatic hybrids. The non-random inheritance of the chloroplast and mitochondrial DNA in these tomato protoplast fusion products appears to be influenced by the nuclear background of the regenerant.     

Profiling mitochondrial proteins in radiation-induced genome-unstable cell lines with persistent oxidative stress by mass spectrometry

Previous work by Morgan and coworkers on radiation-induced genome instability in Chinese hamster ovary (CHO) cell lines showed that unstable LS-12 cells had persistently elevated levels of reactive oxygen species (ROS) that were likely due to dysfunctional mitochondria. To further investigate the correlation between radiation-induced genome instability and dysfunctional mitochondria, we performed quantitative high-throughput mass spectrometry on samples enriched in mitochondrial proteins from three chromosomally unstable CHO cell lines and their stable unirradiated GM10115 parental cell line. Out of several hundred identified proteins, sufficient data were collected on 74 mitochondrial proteins to test for statistically significant differences in their abundance between unstable and stable cell lines. The LS-12 cell line, which exhibited the highest level of ROS among the three unstable cell lines, was characterized by eight significantly down-regulated mitochondrial proteins, all associated with the TCA (tricarboxylic acid). Elevated levels of ROS relative to the unirradiated parental control were also statistically significant for the CS-9 cell line. The protein profile of CS-9 revealed five significantly up-regulated mitochondrial proteins, three of which are involved in oxidative phosphorylation. Elevation of ROS in the unstable 115 cell line was nearly as large as that seen in CS-9 cells but was not statistically significant. The mitochondrial protein profile of 115 cells showed significant down-regulation of acetyl-CoA-acetyltransferase, which was also down-regulated in LS-12, and two other proteins with abundances that were significantly different from control levels but were not directly related to either the TCA or oxidative phosphorylation. These results provide further evidence that elevated ROS and mitochondrial dysfunction are associated with radiation-induced genome instability; however, additional work is required to establish a firm mechanistic relationship between these end points.     

Gametogenesis of intergroup hybrids of hemiclonal frogs

European water frog hybrids Rana esculenta (R. ridibundaxR. lessonae) reproduce hemiclonally, by hybridogenesis: in the germ line they exclude the genome of one parental species and produce haploid gametes with an unrecombined genome of the other parental species. In the widespread L-E population system, both sexes of hybrids (E) coexist with R. lessonae (L). They exclude the lessonae genome and produce ridibunda gametes. In the R-E system, hybrid males coexist with R. ridibunda (R); they exclude either their ridibunda or their lessonae genome and produce sperm with a lessonae or with a ridibunda genome or a mixture of both kinds of sperm. We examined 13 male offspring, 12 of which were from crosses between L-E system and R-E system frogs. All were somatically hybrid. With one exception, they excluded the lessonae genome in the germ line and subsequently endoreduplicated the ridibunda genome. Spermatogonial metaphases contained a haploid or a diploid number of ridibunda chromosomes, identified through in situ hybridization to a satellite DNA marker, and by spermatocyte I metaphases containing a haploid number of ridibunda bivalents. The exception, an F1 hybrid between L-E system R. lessonae and R-E system R. ridibunda, was not hybridogenetic, showed no genome exclusion, and evidenced a disturbed gametogenesis resulting from the combination of two heterospecific genomes. None of the hybridogenetic hybrids showed any cell lines excluding the ridibunda genome, the pattern most frequent in hybrids of the R-E system, unique to that system, and essential for its persistence. A particular combination of R-E system lessonae and R-E system ridibunda genomes seems necessary to induce the R-E system type of hemiclonal gametogenesis.     

Characteristics of somatic cell hybrids (mouse x Chinese hamster) with a different ratio of chromosome sets from the parent species. II. Thymidine kinase activity]

The activity of thymidine kinase (TK) was studied in series of somatic cell hybrids between the mouse cell line 3T3-4E (TK-) and Chinese hamster cells M-15-1 (HGPRT-). Four groups of hybrid lines with different ratio of parental chromosome sets have been investigated: 1) three lines containing one hamster and one mouse chromosome set (1 hs+1 ms); 2) one line with 2 hs+1 ms; 3) one line containing 3 hs+1 ms and 4) one line containing 1 hs+2 ms. Mixtures of extracts from the parental cells were shown to possess the expected TK activity. The calculation of the activity per cell revealed that the 1 hs+1 ms and 2 hs+1 ms hybrid lines possessed about 50% of the initial hamster cell TK activity. The decreased TK activity in these hybrids might be due either to a loss of hamster chromosomes or to some inhibitory effect of mouse genome in cells with the studied ratio of parental sets. The enzyme activity in the 3 hs+1 ms hybrid was as expected, about three times greater than that of hamster cells.     

Molecular study and C-banding of chromosomes in common wheat alloplasmic lines obtained from the backcross progeny of barley-wheat hybrids Hordeum vulgare L. (2n = 14) x Triticum aestivum L. (2n = 42) and differing in fertility

We studied common wheat alloplasmic lines differing in fertility traits, which had been obtained from the backcross progeny of barley-wheat hybrids Hordeum vulgare L. (2n = 14) x Triticum aestivum L. (2n = 42), using molecular analysis and chromosome C-banding. It was found that the nuclei of all alloplasmic lines studied, regardless of their fertility traits, contained only the common wheat chromosomes (2n = 42). The formation of line L-79(10)(3)F6, stable for self-fertility, from line L-79(10)(3)F6 was accompanied by changes of the proportions of simple sequence repeats of the parental common wheat varieties in the nuclear genome. The presence of barley genome fragments in line accessions with incomplete self-fertility was shown by RAPD. Heteroplasmy for mitochondrial genome loci was detected in these lines with the use of primers specific to the tMet-18S-5S repeat of mitochondrial ribosomal genes.     

Genetic and adaptive differences in the expression of drug resistance in hybrid cells

Hybrids between Chinese hamster cells were isolated and maintained in media that were selective or nonselective for markers present in the parent cells (HGPRT and TK deficiencies, respectively). Segregation frequencies for resistance to azaguanine (AZG), thioguanine (THG), or bromodeoxyuridine (BrdU) could be enhanced for some groups of hybrids if the stock cells were maintained under nonselective conditions rather than in HAT medium. In these populations the expression of resistance was dominant or codominant even though marker patterns were recessive for the same cells in HAT. Clonal analysis showed that enhancement took place by adaptive shifts rather than by variation and selection. Segregation frequencies in hybrids were also found to differ significantly between clones isolated by replicate fusions of any two parental cell types. The basis for this heterogeneity is unknown and deserves further study.     

Establishment and characterization of murine macrophage hybrids

Proteose peptone-induced peritoneal macrophages from CBA/J (H-2k) mice have been fused to a hypoxanthine phosphoribosyltransferase-negative variant of the P388D1 (H-2d) murine macrophage cell line. Six hybrid clones were isolated following HAT selection and further characterized. Five of the six clones express class I antigens of both parental haplotypes by microelisa and by flow cytometric analysis. Class II antigen expression of both haplotypes was apparent following a 72-hr incubation of the hybrids with concanavalin A-stimulated rat spleen cell supernatant. However, I-Ad was expressed in all hybrids to a greater extent than I-Ak. Three clones with the highest level of I-Ak expression, E5, C2, and C4, were capable of antigen presentation to the I-Ak-restricted T-cell line, D10.G4.1. LPS induction of the hybrids resulted in a 2- to 15-fold increase in the amount of IL-1 produced relative to the P388D1 parent. Finally, in distinction to P388D1, all hybrids demonstrated increased Fc-mediated erythrophagocytosis of chromium-labeled antibody-coated erythrocytes. These murine macrophage hybrids appear stable and should serve as useful models in understanding the regulation of macrophage function.     

Nuclear and cytoplasmic genome composition of Solanum bulbocastanum (+) S. tuberosum somatic hybrids.

Somatic hybrids between the wild incongruent species Solanum bulbocastanum (2n = 2x = 24) and S. tuberosum haploids (2n = 2x = 24) have been characterized for their nuclear and cytoplasmic genome composition. Cytologic observations revealed the recovery of 8 (near-)tetraploid and 3 hexaploid somatic hybrids. Multicolor genomic in situ hybridization (GISH) analysis was carried out to study the genomic dosage of the parental species in 5 somatic hybrids with different ploidy. The GISH procedure used was effective in discriminating parental genomes in the hybrids; most chromosomes were unambiguously colored. Two (near-)tetraploid somatic hybrids showed the expected 2:2 cultivated-to-wild genomic dosage; 2 hexaploids revealed a 4:2 cultivated-to-wild genomic dosage, and 1 hexaploid had a 2:4 cultivated-to-wild genomic dosage. Characterization of hybrid cytoplasmic genomes was performed using gene-specific primers that detected polymorphisms between the fusion parents in the intergenic regions. The analysis showed that most of the somatic hybrids inherited the plastidial and mitochondrial DNA of the cultivated parent. A few hybrids, with a rearranged mitochondrial genome (showing fragments derived from both parents), were also identified. These results confirmed the potential of somatic hybridization in producing new variability for genetic studies and breeding.     

Intergeneric hybridization between Pleurotus ostreatus and Schizophyllum commune by PEG-induced protoplast fusion

Intergeneric hybridization between Pleurotus ostreatus and Schizophyllum commune was studied using PEG-induced fusion. The fusion of protoplasts from auxotrophic mutant strains resulted in the formation of fusion hybrids in the frequencies of 3.6 to 7.3×10^–5. Most of these fusion hybrids were monokaryotic and sterile and no heterokaryosis occurred. Most fusants showed a significantly higher nuclear DNA content when compared to parental strains and no diploids (parent 1 genome plus parent 2 genome) were found. Some fusion hybrids revealed both parental fragments in nuclear and mitochondrial rDNA PCR profiles. AP-PCR (Arbitrarily-primed Polymerase Chain Reaction) fingerprints also indicated that most of the fusion products were recombinant hybrids.     

Coordinate expression of parietal endodermal functions in hybrids of embryonal carcinoma and endodermal cells.

A derivative, FOT5, of the F9 murine embryonal carcinoma cell line which is resistant to ouabain and thioguanine was fused with a near diploid parietal endodermal cell line, PFHR9, Hybrid clones (ENEC1 to ENEC5) were isolated in HAT Medium containing ouabain at a frequency of approximately 2 x 10(-4). The DNA contents and chromosome number of the ENEC hybrids were approximately the sum of those of the parents. Five hybrid cell lines examined in detail expressed the following parietal endodermal functions: plasminogen activator activity, basement membrane proteins, and endodermal cytoskeletal proteins. Embryonal carcinoma characteristic functions (tumorigenicity, a stage specific embryonic antigen, and high alkaline phosphatase activity) were extinguished in the hybrids. No hybrid clones with embryonal carcinoma morphology were observed among 1,358 hybrid clones examined. Hybrids, propagated for over 100 generations, continued to express endodermal functions and not embryonal carcinoma functions. The coordinate expression of endodermal functions and the extinction of embryonal carcinoma functions in the ENEC hybrids suggest that the parietal endodermal cells contain diffusible activities which extinguish embryonal carcinoma functions and possibly cause the embryonal carcinoma genome to express parietal endodermal characteristics.     

Interspecific hybridization between Volvariella volvacea and V. bombycina by PEG-induced protoplast fusion

Interspecific hybridization between Volvariella volvacea and V. bombycina was studied using the protoplast fusion technique. The fusion frequency was found to be in the range of 0.032 to 0.333%. Protoplasts from various hybrids were released and regenerated to determine whether they were heterokaryons. In all regenerated colonies, both parental types could not be recovered at the same time. The nuclear DNA contents of hybrids were compared with their parents, and no diploid (parent 1 genome plus parent 2 genome) was found. Some hybrids revealed novel fragments in mitochondrial rDNA PCR profiles, which indicated that rearrangement of mtDNA could have occurred after fusion. Results from arbitrarily-primed polymerase chain reaction (AP-PCR) fingerprints also revealed that the majority of hybrids were similar to one parental type, but heterologous fragments were found in some hybrids.     

Insertion of muntjac gene segment into hamster cells by cell fusion

Indian muntjac diploid cells that have only three pairs of easily discernible large chromosomes were fused with hamster cells deficient in hypoxanthine guanine phosphoribosyltransferase (HGPRT) using polyethylene glycol. Cells that survived in hypoxanthine-aminopterine-thymidine (HAT)-oubaine medium were analyzed. Hybrid cells containing both muntjac and hamster chromosomes in a given cell were not found. Instead, the cells had the same chromosomal sets as those of either parental muntjac or hamster cells. A clonal isolate that had the same chromosomal sets as those of parental hamster cells was analyzed in detail and showed the following characteristics: (1) portions of the survival curve in various concentrations of HAT medium were intermediate between those of parental cells; (2) expressions of both muntjac and hamster antigen(s) were detected by immunofluorescence staining; (3) the mobility of the enzyme HGPRT in gel electrophoresis differed from that of parental hamster or muntjac cells. These results indicate that the clonal isolate (AD202h) is a somatic cell hybrid of hamster and muntjac that contains chromosomal sets of hamster with an inserted segment of the muntjac genome, including HGPRT. The formation of such an unusal hybrid and a possible explanation of transfer of some gene segments in the hybrid cell in this system are discussed.     

Mitochondrial genomes in intraspecies mammalian cell hybrids display codominant or dominant/recessive behavior

A unique type of nonstochastic mitochondrial DNA (mtDNA) segregation was found in mammalian cells. In human cell hybrids isolated from the fusion of HeLa cells with 23, GM639, A549, or 293 cells, HeLa mtDNA was always lost from the hybrids, whereas both parental mtDNAs were maintained in hybrids of HeLa X 143BTK-. Similar phenomena were observed in mouse cell hybrids isolated by the fusion of cells with different mtDNA types. Types 1, 2, and 3, can be distinguished from each other by restriction fragment-length polymorphisms. The mouse cell hybrids between cells with type 1 and type 2 mtDNA always lost type 2 mtDNA, whereas the hybrids between cells with type 2 and type 3 mtDNA retained both types stably. These observations suggest that either a codominant or a dominant/recessive relationship may be present in intraspecies mitochondrial genomes of human and mouse cells. When the mitochondrial genomes in cell hybrids are codominant, stochastic segregation occurs while nonstochastic segregation occurs when they are in the dominant/recessive relationship. These concepts may help elucidate organelle heredity in animals.     

Independent segregation of the plastid genome and cytoplasmic male sterility in Petunia somatic hybrids

Summary The chloroplast genomes of three sets of Petunia somatic hybrids were analyzed to examine the relationship between chloroplast DNA (cpDNA) composition and cytoplasmic male sterility (CMS). Chloroplast genomes of somatic hybrid plants were identified either by restriction and electrophoresis of purified cpDNAs or by hybridization of total DNA digests with cloned cpDNA probes that distinguish the parental genomes.The chloroplast genomes of a set of seven somatic hybrids derived from the fusion of Petunia CMS line 2423 and fertile line 3699 were analyzed. All seven plants were fertile, and all exhibited the cpDNA restriction pattern of the sterile cytoplasm. Similarly, four fertile somatic hybrids derived from the fusion of CMS line 3688 and fertile line 3677 were found to contain the CMS chloroplast genome. The cpDNA compositions of four fertile and two sterile somatic hybrids derived from the fusion of CMS line 3688 and fertile line 3704 were determined by restriction analysis of purified cpDNAs; all six plants exhibited the cpDNA restriction pattern of line 3704. Thus the CMS phenotype segregates independently of the chloroplast genome in Petunia somatic hybrids, indicating that CMS in Petunia is not specified by the chloroplast genome.