Further data on chromosomal assignments of pig enzyme loci LDHA, LDHB, MPI, PEPB and PGM1, using somatic cell hybrids

Summary. Clear interspecies differentiation between the chromosomes in pig-mouse somatic cell hybrids was achieved by using the THA-technique for the cytogenetic analysis. The assignments of LDHB and MPI to pig chromosomes nos 5 and 7 respectively, reported previously, were confirmed by analysis of 34 hybrid clones. The LDHA, PEPB and PGM1 genes were assigned to pig chromosomes nos 2, 5 and 6 respectively. Both LDHB and PEPB were indicated to be located on the long arm, except the most proximal part, of pig chromosome no. 5. The proposed synteny between LDHB and PEPB in pigs is in accordance with the synteny observed between these two loci in several other mammalian species.     

Expression of the human adenylate kinase isozymes,phosphopyruvate hydratase, 6-phosphogluconate dehydrogenase,and phosphoglucomutase-1 in man-rodent somatic cell hybrids

The expression of the adenylate kinase isozymes and of phosphopyruvate hydratase was studied in man-mouse and man-hamster hybrid clones. Concordant segregation of the loci coding for AK-2 and PPH was observed in 54 of 55 primary hybrid clones, and these loci were demonstrated to be synthetic with the loci specifying PGM-1 and PGD. The pattern of expression of the four enzymes in discordant clones suggests the gene order 1pter-(PGD,PPH)-AK-2-PGM-1-centromere. In addition, AK-1 was found to be expressed independently of AK-2.This work was supported by the NIH Grants 5 PO1 HB 06276-04, HD 04807-06, and HD 06285-04.     

Analysis of chromatin, nuclear DNA and organelle composition in somatic hybrids between Solanum tuberosum and Solanum sanctae-rosae

A protoplast fusion strategy has been applied to advance aspects of a potato breeding programme. A sub-population of somatic hybrids, selected for agronomic potential, between tetraploid Solanum tuberosum cv. Brodick and a diploid EBN2 accession, S. sanctae-rosae was subjected to detailed molecular analysis. This study reports the use of simple sequence repeats (SSRs) to identify nuclear hybrid genomes and PCR and DNA-DNA analysis to determine organelle composition in somatic hybrids derived from these parents. SSR analysis revealed somatic hybrids containing the genetic background of S. tuberosum cv. Brodick with some specific markers from S. sanctae-rosae. One somatic hybrid contained the chloroplasts derived from S. sanctae-rosae, and several hybrids had detectable RFLP mitochrondrial DNA profiles, indicating genetic re-arrangements. We also examined the use of DNase I sensitivity to the genomic and ribosomal RNA sequences in these somatic hybrids as an indicator of changes in chromatin structure. Chromatin and DNAse I analysis showed differential sensitivity to increasing levels of nuclease; DNA from several somatic hybrids was found to be resistant to DNase I compared to the parental plants. The significance of the findings to somatic cell genetics and plant breeding studies is discussed. Received: 6 July 1999 / Accepted: 29 February 2000     

An immunochemical method for the detection of the expression of human gene loci in human-rodent somatic cell hybrids with special reference to the GPI locus

Species-specific antibodies, prepared against unpurified human and Chinese hamster fibroblast extracts, were used to identify the parental origins of enzymes in human-hamster somatic cell hybrids. Results of the detection of the expression of the human glucosephosphate isomerase gene locus (GPI) by electrophoretic and immunochemical techniques were concordant in 17 instances. The human GPI synthesized by fibroblasts derived from skin explants and by somatic cell hybrids retaining the human GPI locus, regardless of whether the human parental cells were lymphocytes or fibroblasts, appeared to be antigenically identical.This work was supported by the Medical Research Council of Canada (Grant MA-4061). Personnel and operating support were provided by The Children's Hospital of Winnipeg Research Foundation, Inc.     

Intermediate filament expression and lifespan potential in human somatic cell hybrids

Summary Limited lifespan human diploid fibroblast cells have been fused with the HeLa derived cell line HEB 7A which possesses transformed growth characteristics and unlimited division potential. HEB 7A expresses keratin intermediate filaments, while the fibroblast cells express only vimentin intermediate filaments. Independently arising clones of hybrids were examined for the presence of keratin by indirect immunofluorescence. Of 11 limited lifespan hybrids, all were keratin negative and possessed the growth characteristics of the fibroblast parent. Of 8 transformed hybrids, 6 arising early after fusion and 2 arising late, all were keratin-positive and simultaneously expressed the transformed growth characteristics of loss of density dependent growth inhibition, low serum dependence, and anchorage independence. It is concluded that the growth properties of these hybrids are associated with the type of intermediate filament expressed. The intermediate filament expression is therefore a marker of proliferative potential in these hybrids. This work was supported by grant no. AG 02664 from NIA (to C.L.B.) and by grant nos. 1R01 HD 18129-01 from NIH and PCM83-09068 from NSF (to R.H.S.). Editor’s Statement The tight correlation between the expression of the intermediate filaments of the immortal parent in hybrids of limited lifespan fibroblasts and HeLa cells with the transformed phenotype is of interest. It may offer important clues to the mechanism involved in cellular senescence. Gordon H. Sato     

Inheritance of Arabidopsis DNA in offspring from Brassica napus and A. thaliana somatic hybrids

 Chromosome counts and RFLP markers mapped to Arabidopsis thaliana were used to determine the proportion of eliminated chromosomes and retained A. thaliana DNA in the back-crossed (BC) progeny derived from symmetric and asymmetric somatic hybrids between Brassica napus and A. thaliana. All plants were analysed for the presence of two RFLP markers per chromosome, preferably with one located on each chromosome arm. A reduction in both A. thaliana RFLP markers and chromosome numbers was found in the BC₁ and BC₂ generations of the symmetric hybrids as well as in the BC₁ generation of the asymmetric hybrids. In the symmetric hybrids, two back-crosses to B. napus were required to reduce the frequency of retained A. thaliana loci to 42.4% and mean chromosome number to 39.4. In comparison, the BC₁ progeny of the asymmetric hybrids had 16% of the analysed A. thaliana loci present and an average of 38.4 chromosomes maintained. When the frequency of A. thaliana chromosomes with both analysed loci maintained was compared with the frequency of chromosomes with one locus lost and one kept, a reduction in the number of complete chromosomes between BC₁ and BC₂ derived from the symmetric hybrids was observed. Among the BC₁ plants in the asymmetric group the situation was different, with higher amounts of incomplete donor chromosomes compared to whole chromosomes. The results indicate that A. thaliana chromosome fragments are more often found in the progeny of irradiated hybrids, while back-crossed symmetric hybrids have more complete chromosomes. Received: 2 April 1998 / Accepted: 14 July 1998     

Regeneration of intergeneric somatic hybrids by protoplast fusion between Onobrychis viciaefolia and Medicago sativa

Protoplast fusion was induced between sainfoin and alfalfa by an improved polyethyleneglycol (PEG) method. The intergeneric somatic calluses were selected based on complementation of hydroxyproline-resistance of sainfoin and hormone autonomy growth of alfalfa transformation cell line. 17 somatic hybrid plantlets were regenerat-ed. PEG could induce the tight agglutination of protoplasts. During diluting and washing process, cyclization of the linked membrane and formation of vesicle-like structures were observed, resulting in protoplast fusion. 5%-10% glycerol supplemented in the fusion inducing solution markedly increased the frequency of heterogeneous fusion. Better fusion results were obtained when mixed protoplast suspension was dripped in petri dishes in which PEG solution was previously placed. Chromosome number of regenerated hybrid buds varied from 30 to 60. The genome of hybrids in-cluded the small chromosome from sainfoin and two chromosomes with two clear constrictions from alfalfa. The hybrid     

Chromosomal analysis of Nicotiana asymmetric somatic hybrids by dot blotting and in situ hybridization

Summary A species-specific, dispersed repetitive DNA sequence was cloned from Nicotiana plumbaginifolia and used in dot blots and in situ hybridizations to analyze asymmetric somatic hybrids of N. tabacum(+)kanamycin-resistant N. plumbaginifolia. Dot blot hybridization data, using the cloned, species-specific repetitive DNA as a probe, showed that some of the hybrids contain only 1%–5% N. plumbaginifolia DNA, whereas others contain 15%–25%. In situ hybridization of the probe to chromosome spreads showed that the extremely asymmetric hybrids retain a single N. plumbaginifolia chromosome; the hybrids with higher dot blot values were found to have 8 to 12 N. plumbaginifolia chromosomes and chromosome fragments. In situ hybridization also revealed translocations between N. plumbaginifolia and N. tabacum chromosomes in 3 of 8 hybrids studied. RFLP analysis using a 5S gene probe showed the presence of N. plumbaginifolia-specific 5S banding patterns in most hybrids examined, including those that retain only a single N. plumbaginifolia chromosome.     

Non-equivalence of embryonic and somatic cell nuclei affecting spindle composition in clones

Cloning by nuclear transfer remains inefficient but is more efficient when nuclei from embryonic cells or embryonic stem cells (ECNT) are employed as compared with somatic cells (SCNT). The factors determining efficiency have not been elucidated. We find that somatic and embryonic nuclei differ in their ability to organize meiotic and mitotic spindles of normal molecular composition. Calmodulin, a component of meiotic and mitotic spindle chromosome complexes (SCCs), displays sharply reduced association with the SCC forming after SCNT but not ECNT. This defect persists in mitotic spindles at least through the second mitosis, despite abundant calmodulin expression in the cell, and correlates with slow chromosome congression. We propose that somatic cell nuclei lack factors needed to direct normal SCC formation in oocytes and early embryos. These results reveal a striking control of SCC formation by the transplanted nucleus and provide the first identified molecular correlate of donor stage-dependent restriction in nuclear potency.     

Cellular expression of the beige mouse mutation and its correction in hybrids with control human fibroblasts

Summary Fibroblasts from a beige mouse (C57BL/6J;bg ^J bg^J) have been established and maintained in culture for more than 3 yr. At early passages, the mutant cells were distinguishable from C57BL/6J control mouse fibroblasts at the ultrastructural level by the presence of enlarged cytoplasmic granules. After continuous passaging, this distinguishing feature was lost from the mutant cells, correlated with their increased growth rate. Clustered, perinuclear distribution of lysosomes was retained, however, and was quantitatively different at any passage number of the beige cell line from the dispersed distribution of these organelles in control mouse fibroblasts, as analyzed by computer-aided, video-enhanced light microscopy. In somatic cell hybrids between the established beige cell line and a control human diploid fibroblast cell strain, seven uncorrected hybrid lines retained a lysosomal dispersion pattern statistically indistinguishable from that of the beige mouse cell lines. Three corrected hybrid lines had lysosomal dispersion patterns that were significantly different from the beige parent line and indistinguishable from that of the control mouse fibroblast line. Thus, lysosomal dispersion can be used objectively and quantitatively to distinguish mutant beige and control mouse fibroblasts and corrected vs. uncorrected cell hybrids made from the beige/control human somatic cell crosses.     

Regeneration of intergeneric somatic hybrids by protoplast fusion between Onobrychis viciaefolia and Medicago sativa

Protoplast fusion was induced between sainfoin and alfalfa by an improved polyethyleneglycol (PEG) method. The intergeneric somatic calluses were selected based on complementation of hydroxyproline-resistance of sainfoin and hormone autonomy growth of alfalfa transformation cell line. 17 somatic hybrid plantlets were regenerated. PEG could induce the tight agglutination of protoplasts. During diluting and washing process, cyclization of the linked membrane and formation of vesicle-like structures were observed, resulting in protoplast fusion. 5%–10% glycerol supplemented in the fusion inducing solution markedly increased the frequency of heterogeneous fusion. Better fusion results were obtained when mixed protoplast suspension was dripped in petri dishes in which PEG solution was previously placed. Chromosome number of regenerated hybrid buds varied from 30 to 60. The genome of hybrids included the small chromosome from sainfoin and two chromosomes with two clear constrictions from alfalfa. The hybridity of obtained hybrid calluses was confirmed by their isayrne banding patterns and their nopaline synthetase activity. Project supported by the National Natural Science Foundation of China.     

Establishment of a galactocerebrosidase-deficient twitcher mouse cell line that expresses galactocerebrosidase activity in hybrids with control human fibroblasts

Summary Primary cell cultures from twitcher (galactocerebrosidase deficient) mice were made by enzymatic dispersion and explantation of skin obtained from 3-d-old littermates of atwi+/twi×twi+/twi mating. Galactocerebrosidase activity remained deficient for two twitcher cell lines, TM-1 and TM-2, and both lines demonstrated an initial period of growth decline, followed by accelerated growth. The TM-2 line has been subcultured for more than 3.5 yr, has a modal chromosome number of 63, a doubling time of approximately 16 h, and has remained galactocerebrosidase deficient throughout its life span. These data indicate this to be an established twitcher cell line that can be continuously maintained in culture as a transformed galactocerebrosidase-deficient mouse cell line. This established line was rendered 6-thioguanine resistant so that the cells could be fused with control human fibroblasts and selected for hybrid lines in hypoxanthine-aminopterin-thymidine medium. Also, the established twitcher cells were crossed with neomycin-resistant control human fibroblasts and selected in G418 medium. Several of the hybrid lines from both crosses had higher than deficient levels of galactocerebrosidase activity initially, followed by a decrease to twitcher levels during subculture, whereas other lines retained high levels of activity. These results indicate that twitcher-human somatic cell hybrids will express galactocerebrosidase activity and thus may be useful for determining the human chromosome or chromosomes associated with this expression. Partial support for these studies was provided by a National Institutes of Health AREA grant (HD21222-01) and a NIH subcontract to Clark University from the Shriver Center for Mental Retardation This research forms a portion of studies performed to fulfill the requirements for the Ph.D. degree in biology at Clark University for J. T. K.     

Analysis of chloroplast and mitochondrial DNA in asymmetric somatic hybrids betweenSolanum tuberosum and irradiatedS. brevidens

Organellar DNA of asymmetric somatic hybrids betweenSolanum tuberosum and irradiatedS. brevidens were analysed by DNA hybridization methods using the spinach chloroplast probepSBD, wheat mitochondrial genenad5 and petunia mitochondrial geneorf25. Eight of the 12 asymmetric hybrid plants hadS. tuberosum chloroplast DNA and the remaining fourS. brevidens chloroplast DNA. A novel mitochondrial hybridization pattern was present in eight out of the 17 hybrids tested. In six hybrids, novel combinations of chloroplasts and mitochondria were found, indicating that both organelle types sorted out independently.     

Alkaloid biosynthesis in somatic hybrids of Duboisia leichhardtii F. Muell. and Nicotiana tabacum L.

Somatic hybrids of Duboisia leichhardtii and Nicotiana tabacum were obtained by electrofusion followed by individual cloning. The hybrid nature of the cloned cells and regenerated shoots was confirmed by cytological investigation and ribosomal-DNA analysis, respectively. The hybrid plantlets predominantly produced nicotine, while Duboisia plantlets produced both tropane and nicotine alkaloids. Activities involved in tropane-alkaloid biosynthesis were examined in a series of precursor-feeding experiments. The presence in the hybrid plants of activities responsible for the reduction of tropinone, the hydroxylation and epoxidation of hyoscyamine, and the conversion of nicotine to nornicotine demonstrated the presence of the Duboisia genes for these enzyme activities.We thank Mr. T. Shikanai, Kyoto University, for the preparation of rice rRNA. We also appreciate Dr. J.H. Fitchen, Kyoto University, for critical discussion and English correction.     

Longitudinal analysis of somatic and germ‐cell telomere dynamics in outbred mice

Although telomere length (TL) shortens with age in most tissues, an age‐related increase in length has been described in sperm through a mechanism that is not yet fully understood. Changes in TL with age in the same individual have not been explored. This longitudinal study examines TL dynamics in somatic tissue and gametes during an entire lifespan in an outbred mouse population (from 8 to up to 114 weeks of age). Our findings indicate a reduced life expectancy in males compared to females (84.75 ± 9.23 vs. 113.16 ± 0.20 weeks) and significant variability in TL dynamics between individuals. While with aging, a clear reduction in TL was produced in somatic cells and oocytes, telomeres in sperm cells significantly lengthened. Finally, we found evidence indicating that telomere elongation in sperm during aging may be dependent on different mechanisms, such as the survival of spermatogonia with longer telomeres and the alternative lengthening of telomeres mechanism in meiotic and postmeiotic spermatogenic cells.     

Estimation of economic values and financial losses associated with clinical mastitis and somatic cell score in Holstein dairy cattle

The objective of this study was to develop a method for calculating economic values of clinical mastitis (CM) and somatic cell score (SCS) for inclusion in a dairy cattle breeding goal in the context of a country where farm production and economic data are scarce. In order to calculate the costs and derive economic values for SCS, a new model, 'milk collection method', has been developed and was compared with the Meijering model with individual and average SCS distributions. For the population, estimated economic values using the milk collection method were 1.3 and 2.4 times higher than those of Meijering method with average and individual SCS, respectively. The milk collection method needs no assumptions about normality of the distribution of SCS and because of a lack of normality in Iranian data for SCS, the Meijering method resulted in economic values that were biased downwards. Failing to account for the fact that milk price penalties for SCS are applied at milk collection rather than individual cow level resulted in a further large downward bias in the economic value of SCS. When the distribution of data is unknown or difficult to approximate or when a transformation to normality is not straightforward, the milk collection method would be preferable. Inclusion of SCS and CM in the breeding goal for Iranian dairy cattle is justified based on these results. The model to calculate mastitis costs proposed here could be used to estimate economic values for CM in other developing countries where farm production and economic data are generally poor.     

Separation and analysis of cell types involved in early stages of carrot somatic embryogenesis

The occurrence of the polarized synthesis of DNA in embryogenic cell clusters of carrot on the third and fourth days after transfer to an embryogenesis-inducing medium was observed by labeling with [³H]thymidine and autoradiography. The cells that were actively synthesizing DNA were separated from cells that were not synthesizing DNA by maceration of cell clusters into individual protoplasts and centrifugation in a Percoll density gradient. [³⁵S]Methionine-labeled proteins extracted from the two types of cell were analyzed by SDS-PAGE and fluorography. Three polypeptides (of 69, 98 and 108 kD, respectively) were found only in cells that were actively synthesizing DNA and could be candidates for markers of the polarity of DNA synthesis that is specific to embryogenesis.