Location of rRNA genes in three inbred strains of rat and suppression of rat rRNA activity in rat-human somatic cell hybrids

Nucleolus organizer regions (NORs) of rat chromosomes were stained by the Ag-AS method. The Ag-NORs were found on chromosomes 3, 11 and 12 in the ACI, Wistar Brown and Wistar Lewis inbred strains of rat. The size of the Ag-NOR on each pair of chromosomes varied from strain to strain. Rat-human somatic hybrid cells that retained human and lost some of the rat chromosomes had no Ag-NOR on rat chromosomes 3, 11 or 12. Since NORs can be Ag-stained only if their 18 + 28S rRNA genes are active, the activity of the rat rRNA genes must have been suppressed in the hybrid cells.     

Loss of Human Genetic Markers in Man-Chinese Hamster Somatic Cell Hybrids

MAN-MOUSE somatic cell hybrids are useful for the study of genetic linkage in man because the human chromosomes are preferentially lost and most murine and human forms of homologous isozymes are clearly distinguishable^1,2. There are, however, certain limitations in this system which call for the introduction of other interspecific somatic cell hybrids. (1) Not all the enzyme phenotypes of man and mouse are easily distinguishable by conventional electrophoretic procedures. (2) Groups of human chromosomes may be preferentially retained or lost in the man-mouse hybrids³. We do not know whether such groups form regular patterns and are constant for a particular hybrid type. (3) The frequency and types of chromosomal rearrangements, which has been reported in man-mouse hybrids, might be different in other human interspecific hybrids.     

Arginine-rich histones do not exchange between human and mouse chromosomes in hybrid cells

Following division of HeLa-3T3 heterokaryons, human and mouse chromosomes occupy distinct regions within the resulting hybrid nuclei. This favorable orientation of genomes has allowed us to determine whether histones exchange between chromosomes in vivo. Acrylamide gel electrophoresis of the proteins from HeLa cells labeled with ³H-arginine during S phase showed that the core histones were labeled preferentially, constituting 30% of the total cellular tritium and 50% of the label in a crude nuclear fraction. Autoradiographic analysis of cells formed by fusion of ³H-arginine-labeled HeLa cells and 3T3-4E cells showed that ³H-arginine-labeled proteins did not migrate between nuclei in heterokaryons; hybrid cells formed from such heterokaryons contained nuclei in which ³H proteins occupied a sector within the nucleus; “sectored nuclei” could persist for at least 4 days; and the unequal distribution of ³H proteins did not change during DNA synthesis. Electron microscopic examination of hybrid nuclei failed to reveal a physical partition between human and mouse chromosome sets. Sectored nuclei were also observed in synkaryons derived from ³H-arginine-labeled HeLa and unlabeled HeLa cells, indicating that the unequal distribution of ³H-arginine-labeled proteins in HeLa-3T3 hybrid cells did not result from species-specific binding of proteins and DNA. The persistent unequal distribution of ³H-arginine-labeled proteins within hybrid nuclei in the apparent absence of a barrier between mouse and human chromosomes indicates that histones, the principal ³H-arginine-labeled proteins, do not dissociate from DNA in vivo.     

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.     

Somatic cell genetic analysis of the interferon system.

Current advances in the use of somatic cell hybrid systems have enhanced the value of these systems for studying eukaryotic cell functions. We have reviewed the use of somatic cells to investigate the human interferon system. It has been shown that interspecific heterokaryons and hybrid cells can produce interferon(s) of both parental types and may be protected from viral challenge by interferon(s) from either parent. Using mouse-human hybrid cells we have assigned a human gene(s) responsible for regulating interferon to chromosome 21 and genes involved in the production of human interferon to chromosomes 2 and 5. Our data also suggest possible assignment of a locus involved in control of interferon production to chromosome 16. Suggested further uses of the somatic cell system for interferon studies include study of the subunit structure of interferons and the development of hybrid lines that produce human interferon at high levels (interferon/somatic cell hybrids/human gene assignment.     

Suppression of replication of SV40 and polyoma virus in mouse-human hybrids.

Mouse-human heterokaryons are permissive for the replication of both SV40 virus and polyoma virus. If the hybrids which develop from these heterokaryons segregate human chromosomes (mouse greater than human hybrids), the hybrids are permissive for replication of polyoma virus but not for replication of SV40 virus. If the subsequent hybrids segregate mouse chromosomes (human greater than mouse hybrids), such hybrids support the replication of SV40 virus but not the replication of polyoma virus, even when the hybrids contain at least one copy of each mouse chromosome. This indicates that during the transition from heterokaryon to hybrid cell, suppression of expression of species-specific function(s) required for the replication of these species-specific viruses occurs in parallel with the direction of chromosome loss and suppression of nucleolus organizer activity.     

Painting of human chromosomes with probes generated from hybrid cell lines by PCR with Alu and L1 primers

Summary Specific amplification of human sequences of up to several kb length has recently been accomplished in man-hamster and man-mouse somatic hybrid cell DNA by IRS-PCR (interspersed repetitive sequence — polymerase chain reaction). This approach is based on oligonucleotide primers that anneal specifically to human Alu- or L1-sequences and allows the amplification of any human sequences located between adequately spaced, inverted Alu- or L1-blocks. Here, we demonstrate that probe pools generated from two somatic hybrid cell lines by Alu- and L1-PCR can be used for chromosome painting in normal human lymphocyte metaphase spreads by chromosomal in situ suppression (CISS-) hybridization. The painted chromosomes and chromosome subregions directly represent the content of normal and deleted human chromosomes in the two somatic hybrid cell lines. The combination of IRS-PCR and CISS-hybridization will facilitate and improve the cytogenetic analysis of somatic hybrid cell panels, in particular, in cases where structurally aberrant human chromosomes or human chromosome segments involved in interspecies translocations cannot be unequivocally identified by classical banding techniques. Moreover, this new approach will help to generate probe pools for the specific delineation of human chromosome subregions for use in cytogenetic diagnostics and research without the necessity of cloning.     

The frequency and distribution of sister chromatid exchanges in human chromosomes

Summary A detailed procedure is described for a rapid detection of phosphoglucomutase-2 (=phosphopentomutase; PGM-2) on Cellogel following electrophoresis of extracts of human red blood cells and other tissues, including cultured fibroblasts and various types of primate-rodent somatic hybrid cells.The present study indicated that there is only one locus for phosphopentomutase in man. The data from a selected panel of 20 independent clones of man-mouse somatic cell hybrids, investigated for the presence of human chromosomes and for the presence or absence of human PGM-2 favored the assignment of the human PGM-2 locus to chromosome 4.     

Autoradiographic studies of nicotinic acid utilization in human-mouse heterokaryons and inhibition of utilization in newly-formed hybrid cells

Although most mammalian cell lines can utilize either nicotinic acid or nicotinamide for the biosynthesis of nicotinamide adenine dinucleotide (NAD), thymidine kinase-deficient, mouse 3T3–4F cells are unable to utilize nicotinic acid. When 3T3–4E cells were fused with human D98/AH2 cells, autoradiography showed that the resultant heterokaryons synthesized NAD from nicotinic acid at rates comparable to the human parental cell. The rate of nicotinic acid utilization in heterokaryons remained unchanged over the fourday period of study following cell fusion. In contrast to the results observed with heterokaryons, nicotinic acid utilization was markedly reduced in hybrid cells. Of 100 hybrid clones examined at four or five days following cell fusion, 60 utilized nicotinic acid at rates less than one tenth that of the parental human cell. Similar results were observed in hybrid clones at nine or ten days following fusion. Uniformly high rates of NAD biosynthesis were observed in hybrid clones with nicotinamide as the precursor. This excludes the possibility that the reduction in nicotinic acid utilization in hybrid cells is due to a general metabolic dysfunction. The biochemical mechanism by which nicotinic acid utilization is markedly reduced has not been determined with certainty, however, several observations suggest genetic suppression.     

Suppression of human nucleolus organizer activity in mouse-human somatic hybrid cells

Cells from four different mouse-human somatic cell hybrids were stained with quinacrine to identify each metaphase chromosome and with ammoniacal silver by the Ag-AS method to locate nucleolus organizer regions. Each of the hybrids contained human acrocentric chromosomes. None of these human acrocentric chromosomes was stained with silver in any hybrid cell. Diploid cells were available from the human parent of one of the hybrids. In these cells both copies of nos. 13 and 15 stained with silver; the same chromosomes in the hybrid cell were not stained. These results support earlier reports that the expression of human ribosomal RNA (rRNA) genes is suppressed in mouse-human hybrid cells. Further, they suggest that silver staining by the Ag-AS method reflects activity of rRNA genes rather than just the presence of these genes.     

Susceptibility of primate-mouse hybrid cells to SV40

Primate-mouse hybrid cells were challenged with SV40 DNA and monitored for their ability to produce virus. All of the hybrid cells had lost at least half of their primate chromosomes at the time of challenge. Only SV40 T-antigen-positive hybrid cells derived from an SV40-transformed human parental cell produced SV40. This finding suggests that the chromosome(s) necessary for SV40 replication are easily lost on fusion of mouse and primate cells unless the parental cells are already SV40-transformed.     

Heterokaryons in the analysis of genes and gene regulation.

Cytological and chemical analysis of heterokaryons, the immediate product of cell fusion, offer new possibilities for studying the factors responsible for genetic regulation in eukaryotic cells. In comparison with proliferating cell hybrids the heterokaryon state offers the important advantage that a heterokaryon contains two complete genomes since chromosome loss does not occur, but since segregation and recombination are absent, heterokaryons cannot be used for gene mapping in the same way as proliferating cell hybrids. However, if two cell types carrying different genetic defects are fused the analysis can be used for studies of gene complementation. The biological information obtained with heterokaryons has emphasized the role of the cytoplasm in the control of nuclear activity. When a G1 nucleus is brought into contact with the cytoplasm of an S phase cell the G1 nucleus is stimulated to synthesize DNA. If the nucleus is brought into a mitotic cell, the chromatin of the G1 nucleus is forced to condense into prematurely condensed chromosomes. Inactive nuclei such as the dormant chick erythrocyte nucleus will be stimulated to initiate RNA and DNA synthesis when brought into contact with an active cytoplasm by cell fusion. Specific nuclear proteins have been shown to be responsible for this process of reactivation. Other inactive nuclei such as the nuclei of macrophages and spermatozoa have likewise been shown to be reactivated by fusion with active cells. The degree of activation in all of these cases appears to be determined by the state of the active cell. Inactive nuclei are activated to the same level as the active nucleus but seldom beyond this level. If differentiated cells are fused with undifferentiated cells, usually the differentiated character is lost rapidly after fusion. This observation is in agreement with several studies on proliferating cell hybrids indicating some type of negative control of differentiated properties. In heterokaryons obtained by fusion of cells of a similar type of histotypic differentiation usually coexpression of the differentiated markers is observed.     

The irreversibility of subunit associations in glucose-6-phosphate dehydrogenase and a suggestion regarding an early step in cellular morphogenesis.

For the purpose of studying the stability of the association of subunits in the dimeric enzyme glucose-6-PO₄ dehydrogenase (G6PD), the formation of ‘hybrid’ G6PD in the presence or absence of translation inhibitors was studied in polyethylene glycol-induced heterokaryons of mouse and human fibroblasts. In heterokaryons cultured in the absence of translation inhibitors, hybrid G6PD made of one monomer each of the mouse and human types was detectable. In the presence of cycloheximide concentrations that inhibited over 90% of protein synthesis, however, no hybrid G6PD could be detected in heterokaryons, despite the presence in such heterokaryons of substantial amounts of the ‘parental’ mouse and human enzymes that had been produced prior to heterokaryon formation. Thus, we conclude that once the subunits form a dimeric G6PD, they remain irreversibly associated in the mature enzyme. This, we propose, may reflect an early step in the morphogenesis of relatively stable intracellular structures.     

Unidirectional loss of human chromosomes in rat-human hybrids

Karyological analysis of 25 rat-human hybrid cell clones shows that only the human chromosomes are lost from the hybrids. Giemsa banding staining of the chromosomes of these hybrids allowed the identification of each human chromosome present in the hybrid cells.     

Complementation in heterokaryons derived from a thymidine kinase deficient cell line and senescent human diploid cells.

Incorporation of [3H]thymidine was observed in both parental nuclei in heterokaryons resulting from the fusion of post-mitotic, "senescent" human diploid cells and a thymidine kinase-deficient murine cell line (3T3der-4E). The senescent nuclei displayed a sudden increase of activity approximately 4--6 hours after fusion. A moderate increase of thymidine incorporation was observed in 3T3der-4E cells cocultivated with but not fused to senescent cells, consistent with metabolic cooperation. Chromosome preparations of cultures fixed approximately 24 hr after fusion revealed the presence of hybrid metaphase cells containing almost the entire human complement. All of the identifiable human chromosomes were bi-armed, suggesting that the senescent nuclei were stimulated to reinitiate replicative DNA synthesis rather than repair synthesis in these heterokaryons.     

Enucleation of mammalian cells by cytochalasin B. II. Formation of hybrid cells and heterokaryons by fusion of anucleate and nucleated cells

The development of methods for the formation of hybrid cells and heterokaryons by virus-induced fusion of chemically-enucleated cells and nucleated cells has been described. Heterokaryons and hybrid cells formed by fusion of anucleate mouse peritoneal macrophages (MPM) and nucleated mouse L and human HEp-2 cells were identified by mixed haemadsorption, by their sensitivity to trypsin and by their capacity to ingest antibody-coated sheep red blood cells. The expression of macrophage markers in these cells declined rapidly after fusion. Hybrid cell and heterokaryon formation was identified in mixed cultures of anucleate L cells and nucleated MPM, and was accompanied by the reactivation of DNA synthesis in the macrophage nuclei. Other hybrids and heterokaryons were formed by virus-induced fusion of anucleate MPM and nucleated chick embryo erythrocytes and anucleate L cells and nucleated HEp-2 cells. The value of anucleate-nucleate cell hybrids in the study of metabolic and genetic regulation in mammalian cells is discussed.     

In situ hybridization of DNA sequences in human metaphase chromosomes visualized by an indirect fluorescent immunocytochemical procedure

In situ hybridization and immunocytochemical procedures are described which allow identification and localization of specific DNA sequences in human chromosomes by fluorescence microscopy. With this method the genes coding for 18S and 28S ribosomal RNA (rRNA) were localized on human metaphase chromosomes by in situ hybridization of 18S or 28S rRNA followed by an immunocytochemical incubation with specific anti-RNA-DNA hybrid antiserum. Visualization of the immunocytochemically localized RNA-DNA hybrids was achieved by indirect immuno-fluorescence. The antiserum against RNA-DNA hybrid molecules was raised in a rabbit injected with poly(rA)-poly(dT). The specificity of the sera was determined using a model system of Sephadex beads to which various nucleic acids had been coupled. To obtain optimal specific fluorescence and very low aspecific background staining, several modifications of the in situ hybridization and the immunocytochemical procedures were investigated. The use of aminoalkylsilane-treated glass slides, removal of unbound fluorochrome molecules from the fluorochromelabelled antibody solutions and application of a proteinase K treatment during the hybridization procedure and the immunocytochemical procedure proved to be essential for optimal results.