Normal X chromosome induced reversion in the direction of chromosome segregation in mouse-Chinese hamster somatic cell hybrids

The effect of a normal mouse X chromosome on the chromosome segregation of mouse-Chinese hamster somatic cell hybrids was determined by (i) producing hybrids between the mouse sarcoma line CMS4 and a microcell hybrid (mfe4) of the hamster line E36, containing a mouse X chromosome from a normal cell; (ii) isolating hybrids between CMS4 and a 6-thioguanine selected (X minus) mfe4 subpopulation; (iii) comparing the direction of segregation in the two sets of hybrids. It was found that the normal X chromosome, like the X chromosomes from two MCA-transformed sarcoma lines reported previously [9], has the ability to switch the chromosome segregation of mouse-Chinese hamster somatic cell hybrids. We conclude that the reversal in chromosome segregation is mediated by factors located on the X chromosome. We designate these genetic elements as segregation reversal genes or sr genes.     

Viable hybrids between lethally X-irradiated hamster cells and unirradiated mouse cells.

Senda?-virus-induced fusion between heavily X-irradiated hamster cells, BHK21 or RS2-3 (BHK21 cells transformed by Rous Sarcoma Virus), and unirradiated mouse cells, A9 or c11D, give rise to hybrids. These hybrids possess mouse and hamster surface antigens. However, RS2-3 x mouse hybrids do not form heterokaryons with chick-embryo fibroblasts producing infectious Rous sarcoma virus.     

X chromosome-induced reversion of chromosome segregation in mouse/Chinese hamster somatic cell hybrids : Cellular recognition of native and foreign X chromosomes

The direction of chromosome loss in two sets of mouse-Chinese hamster hybrids was compared with the direction of segregation of the same hybrids, to which an additional X chromosome derived from either of the mouse sarcoma lines MethAa, MethAs, or CMS4, was introduced at the time of the fusion. The addition of the X chromosome was carried out by substituting in place of the Chinese hamster parent a mouse X containing microcell hybrid of the latter. It was found that the addition of an X chromosome reverses the direction of chromosome segregation, but it can do so only if the mouse parent in the hybridization is different from the line from which the X originated. The possible reasons for recognition by the cells of a native and a foreign X are discussed. The existence of a multigene family on the X chromosome, involved in this recognition, is proposed.     

Clonal growth of chinese hamster cell lines in protein-free media

A protein-free synthetic medium, MCDB 301, has been developed for clonal growth of Chinese hamster ovary cell lines. Medium F12 was developed originally for that purpose, but later failed to support good growth without small amounts of serum protein. Growth was restored by the addition of nonphysiological amounts of commercially prepared thyroxine or smaller amounts of the trace element selenium. The thyroxine preparation was shown to contain sufficient selenium to account for all of its growth-promoting activity. MCDB 301 contains increased concentrations of calcium chloride and glutamine, and a smaller amount of cysteine than medium F12. It also has been supplemented with 19 inorganic ions, in addition to selenium and those in medium F12, in order to insure against possible future deficiencies as chemicals are purified further. A Chinese hamster lung line which will not grow in MCDB 301 alone will grow when the medium is supplemented either with methylcellulose or with insulin. The growth-promoting activity is thought to be an impurity shared in common by both substances. The probable "essential" role of impurities in cellular growth in most synthetic media and the problems involved in attempting to develop a truly "defined" medium are discussed.     

Quantitative analysis of human chromosome segregation in man-mouse somatic cell hybrids.

The presumed random and independent process of human chromosome segregation in man-mouse somatic cell hybrids was studied. The results of chromosome analysis on 196 cells from 15 related hybrid strains have provided the first convincing evidence that segregation of human chromosomes can be nonindependent and often concordant. Different human chromosomes were not retained with equal frequency in these hybrid clones. Some were present in 80% of all the cells, whereas others appeared in less than 10% of the same cells. Linear regression analysis was used to test for correlation of the frequencies of all pair-wise combinations of human chromosomes present in these hybrid clones. Twenty-two of 136 possible correlations were statistically significant, indicating that concordant segregation of particular pairs of human chromosomes is a rather frequent occurrence.     

Assignment of the genes for mouse type I procollagen to chromosome 16 using mouse fibroblast-Chinese hamster somatic cell hybrids

Somatic cell hybrids between mouse and Chinese hamster fibroblasts have been used to identify the chromosome responsible for the synthesis of both mouse type I procollagen subunit chains (MCOLA1 and MCOLA2). Thirty-one separate hybrid clones and subclones from ten separate hybridization events were isolated in hypoxanthine-aminopterin-thymidine (HAT) selection medium and were used for detailed gene-mapping studies. ELISA and "Western blotting" immunochemical analysis were used to detect the production of mouse type I procollagen in each hybrid clone. Mouse and Chinese hamster chromosomes were identified in each hybrid clone by trypsin-Giemsa banding of metaphase chromosome spreads and by isozyme analysis. We have found that mouse type I procollagen production segregates concordantly with mouse superoxide dismutase-1, previously mapped to mouse chromosome 16, and with the presence of mouse chromosome 16 karyotypically. Western blotting immunochemical analysis of the separated mouse procollagen chains produced by each hybrid line demonstrated that apparently the genes for both subunit chains are located on the same chromosome. These studies, therefore, assign the structural genes for mouse type I procollagen pro alpha 1 (MCOLA1) and pro alpha 2 (MCOLA2) chains to mouse chromosome 16.     

Somatic cell hybrids between Friend erythroleukemia cells and mouse hepatoma cells.

Somatic cell hybrids between hepatoma and Friend erythroleukemia parental cells were studied for the expression of liver-specific and erythroid properties. Several independent clones were isolated using HAT selection and were shown to be true hybrids by isozyme and chromosome analysis. All displayed a complete extinction of hemoglobin and globin mRNA production, but a retention of albumin and transferrin secretion. The data suggest that erythroid differntiation is being actively inhibited by the hepatoma genome. Possible mechanisms that might explain these results are discussed in the light of current hypotheses regarding the mechanism of cell differentiation.     

Patterns of late chromosomal DNA replication in unbalanced chinese hamster-mouse somatic cell hybrids

The chromosome late-replication patterns of five mouse × Chinese hamster somatic cell hybrids with reduced hamster complements were compared with those of the Chinese hamster parent cell, in order to determine whether the sequential order of chromosome replication is dependent on the presence of the whole chromosome set. In all hybrid clones the parental pattern could be recognized in a variable proportion of cells, although different chromosomes were missing in each clone. The results suggest that sequential order of replication is not the consequence of any sort of interaction between replication units (such as competition for limiting factors), and point to a considerable degree of autonomy in replication of individual chromosomes or chromosome parts.     

Lipopolysaccharide effects on sensitive and resistant variant chinese hamster ovary cell lines

Chinese hamster ovary (CHO · K1 · PRO) cell growth was inhibited by addition of a gram-negative bacterial lipopolysaccharide (LPS) to the cell culture medium. Growth inhibition began after three or four days of incubation, was dose-dependent up to a maximum at an LPS concentration of 500 μg/ml and was accompanied by cell shape changes and enhanced cytoplasmic vacuolization. Formation of bizarre CHO · K1 · PRO cell shapes and vacuole formation were most pronounced after seven days of incubation with LPS and could be observed by light and electron microscopy. An LPS-resistant cell population was obtained by intermittent in vitro exposure to high levels of LPS; these variant cells or clones derived from them failed to display growth inhibition in the presence of LPS. A clone from the LPS-resistant variant population showed altered cell properties compared to the parental cell line which included changes in cell morphology, adhesion, and endocytosis. Parental cells were markedly density-inhibited, whereas the variant clone exhibited considerable growth after confluency. The LPS-resistant variant cells showed a more elongated morphology than the parental line. No significant differences were observed between rates of detachment of parental and variant cells when sparse cultures of either line were removed from tissue culture dishes by ethylenediaminetetracetate (EDTA). However, at confluency approximately 100% of the variant cells versus 35% of the parental cells were removed by EDTA in one hour. Measurements of ¹²⁵I-ferritin uptake by parental and variant cells showed approximately twenty-fold and twofold increases, respectively, in uptake induced by LPS when compared to untreated control cultures.     

Supermelanotic hybrids between mouse teratocarcinoma and mouse melanoma cells

Each of four kinds of teratocarcinoma cells, OTT6050P, PCC4, PSA1 and LT, derived from 129 or LT mouse strain, was fused with B16-CAPr melanoma cells derived from C57BL/6J by using Sendai virus. The resultant hybrids were morphologically melanotic melanoma cells which were larger and more heavily pigmented than the parental B16-CAPr melanoma cells. The chromosome analysis and GPI electrophoresis demonstrated that all hybrids were products of fusion between a single teratocarcinoma cell and a single melanoma cell. The pigmentation in the hybrids between a 129 teratocarcinoma cell and a melanoma cell was much stronger than that in hybrids between an LT teratocarcinoma cell and a melanoma cell. This phenomenon was consistent with the difference of coat color between 129 and LT mouse strain. From these results, it was suggested that the genes of teratocarcinoma cells involved in the pigmentation are activated in the hybrids with B16-CAPr melanoma cells.     

Analysis of the expression of chicken and rat gene products in myoblast × myoblast cell hybrids

Hybrid cells were isolated by fusing primary chicken myoblasts to HPRT-deficient rat L6 myoblasts and incubating the cells in medium containing HAT and ouabain. All hybrid clones contained both rat and chicken chromosomes and expressed a number of gene products characteristic of both species. Although all clones were capable of fusing spontaneously to form myofibers, immunofluorescence and isoenzyme analysis revealed only the rat forms of skeletal muscle myosin and MM-creatine kinase. No differentiated gene products of chicken origin were detected. Analysis of the expression of chicken HPRT revealed that some hybrid clones were capable of modulating this enzyme activity when switched from HAT medium into thioguanine medium and back into HAT, even though HPRT is normally a constitutively expressed enzyme. Parental control cells were incapable of this modulation phenomenon.     

Characterization of hybrids between bovine (MDBK) and mouse (L-cell) cell lines

Hypoxanthine-guanine phosphoribosyltransferase (HGPRT)-deficient mutants of a bovine kidney cell line (MDBK) were selected following mutagenesis with ethylmethane sulfonate or ICR-170G. MDBK mutants were hybridized to thymidine kinase-deficient L cells and selected in HAT medium. Parental and hybrid cells were characterized for isozyme patterns of lactic dehydrogenase, malate dehydrogenase, glucose-6-phosphate dehydrogenase, and glutamate oxalate transaminase. Chromosomes of MDBK can be distinguished from mouse L cells by configuration and by fluorescent staining with Hoechst 33–258 stain. Hybrid cells contained both MDBK and L-cell chromosomes and had elevated DNA content. MDBK cells are normally restrictive for mengovirus replication. Both permissive and restrictive hybrids were found. Our data indicate that there was preferential loss of MDBK chromosomes in the hybrid cell lines.This work was supported by a grant from the Institute for General Medicine [GM18924 (M. W. T.)]. V. G. C. was a predoctoral fellow on Genetic Training Grant No. GM12 from the National Institute for General Medicine.     

Somatic cell hybrids between totipotent mouse teratocarcinoma and rat hepatoma cells.

We have produced somatic cell hybrids between totipotent mouse teratocarcinoma cells and rat hepatoma cells. These hybrids were tested for the expression of liver specific functions expressed in the hepatoma cell parent and for their ability to differentiate when injected into nude mice. The results of this study indicate that hybrid cell clones do not resemble either of the parental cells, since they do not produce albumin and tyrosine aminotransferase that are expressed in the rat hepatoma parent, and are incapable of forming either teratocarcinomas or hepatomas when injected in experimental animals.     

Epstein-Barr virus in somatic cell hybrids between mouse cells and human nasopharyngeal carcinoma cells.

Somatic cell hybrids between mouse cells and cells derived directly from NPC biopsies were produced in order to study the association of the Epstein-Barr virus (EBV) genome and the expression of Epstein-Barr nuclear antigen (EBNA) with the human chromosome(s). All attempts to correlate the presence of EBV-DNA and the expression of EBNA with the presence of a particular human chromosome(s) showed that the segregation of EBV-DNA or of EBNA and human chromosomes was dysconcordant. The data, therefore, suggest that in the hybrids studied the presence of EBA-DNA is not determined by the presence of a specific human chromosome.     

Chromosome replication in somatic hybrids of mouse and temperature sensitive Chinese hamster cells

Hybrid clones were obtained between a mouse cell line (3TP) and a temperature-sensitive Chinese hamster cell line (K12) unable to grow at 40° because of a ts defect apparently located at the G1/S transition. The great majority of hybrid clones grew at 40°, showing the ts defect to be “recessive.” Chromosome DNA replication was analyzed in some detail in three hybrid clones with balanced complements. Although the S period of these hybrids was longer than that of K12, DNA replication in mouse and hamster chromosomes started and ended synchronously. Upon prolonged culture, mouse chromosomes were lost as they are in hybrids involving a non ts Chinese hamster partner, in which case asynchronous chromosome replication appears to be the rule. It seems therefore that asynchronous replication is not the determining factor in chromosome loss from cell hybrids.     

Chromosome loss is responsible for segregation at the HPRT locus in Chinese hamster cell hybrids

The phenomenon of segregation of gene expression has been examined in intraspecific somatic cell hybrids. Specifically, segregation at the hypoxanthine guanine phosphoribosyltransferase (HPRT) locus has been studied in hybrids of Chinese hamster cell lines. The role of chromosome segregation, or other chromosomal events has been assessed by detailed comparison of karyotypes in the 6-thioguanine resistant segregants with those of the parental hybrid lines. The results clearly demonstrate that loss of an entire X chromosome is the primary event responsible for segregation at the HPRT locus, while deletion of a portion of the short arm of an X chromosome was also a frequent event. The results provide the first direct evidence for the assignment of the mapping of this locus to the distal region of the short arm. Analysis of chromosome number distributions in the hybrids and segregants suggests that in selecting chromosomal segregants one may also select for hybrid lines with reduced chromosome stability.