Histone gene expression and chromatin structure in mammalian cell hybrids

DNA isolated from mammalian cell nuclear reveals discrete size patterns when partially digested with micrococcal nuclease. The DNA repeat lengths from different tissues within a species or from different species may vary. These differences have been attributed to the presence of different species of histone H1. To examine the nature of regulation of DNA repeat lengths and their possible relationship to histone H1, we have selected several mouse and human cell lines that differ in their DNA repeat lengths and examined them and their cell hybrids. 24 mouse X human and five mouse X mouse hybrid cell lines were analyzed. All the interspecific hybrids exhibited the repeat pattern characteristic of the murine parent. The mouse intraspecific hybrids had a repeat pattern of only one of the parents. We conclude that the partial human chromosome complements retained in the hybrids assume the repeat lengths exhibited by the mouse cells. Because H1 histones have been implicated in the determination of DNA repeat lengths, we also investigated the regulation of H1 histone expression in these cell hybrids. Purified H1 histones were radioactively labeled in vitro, and individual subfractions were subjected to proteolysis followed by gel electrophoresis. The resulting partial peptide maps off H1 histone subfractions A and B were distinguishable from one another and from different cell lines. In the mouse X human hybrids analyzed, only the mouse H1 histones were detected. These observations were extended to H2b by analysis of the hybrid cell histone by Triton-acid-urea gels. Neither the DNA repeat length nor histone expression is affected by the presence of any specific human chromosome. The fact that human genes are expressed in these hybrids suggests that the H1 histones of one species is able to interact with the chromatin of another species in a biologically funtional conformation. Analysis of the intraspecific PG19 X B82 (mouse X mouse) hybrids reveals the presence of H1 histone subfractions of the B82 mouse cells. Because these hybrids exhibit the nucleosome repeat length only of the PG19 cells, it appears that if histone H1 plays a role in determining the repeat length it does so in consort with other nonhistone chromosomal proteins.     

Mitochondrial DNA analysis of mouse-rat hybrid cells: effect of chloramphenicol selection on the relative amounts of parental mitochondrial DNAs

Several mouse-rat somatic hybrid cell lines were isolated by fusing chloramphenicol-resistant (CAPr) and CAP-sensitive (CAPs) parent cells, and propagation of the parent mitochondrial DNA (mtDNA) species in the hybrid cells was studied. The restriction endonucleases EcoRI, HpaII, and HaeIII were used for identification of mtDNA species. Both mouse and rat mtDNAs were propagated in all the hybrid cells examined and maintained during long-term cultivation and repeated cell division. Moreover, in CAPr mouse-rat hybrid cells, selection and successive cultivation in the presence of CAP did not increase the relative amount of mtDNA species of CAPr parent cell origin, and when CAP was removed from the culture medium, mtDNA species of CAPr parent cell origin did not decrease appreciably. The amount of mouse mtDNAs was consistently 1-4 times that of rat mtDNAs inthe mouse-rat hybrid cells regardless of the species of parent cells from which the CAP resistance was derived. Thus mouse-rat hybrid cells have a stable mtDNA population in which the amount of mouse mtDNAs is larger than that of rat mtDNAs without any influence of CAP selection.     

Patterns of chromosome segregation in chinese hamster × mouse cell hybrids between permanent cell lines and thymus cells

Hybrids between a tumorigenic Chinese hamster cell line (DC3F-aza) and normal mouse thymus cells very rapidly lost most of their mouse chromosomes, whereas hybrids between tumorigenic mouse cell lines (either Cl.1D of L cell line origin, or PCC4-aza1 teratocarcinoma cells) and normal Chinese hamster thymus cells lost most of their hamster chromosomes. From three such fusion experiments, 20 cell lines were developed which all followed the same evolution, namely, the elimination of the majority of the chromosomes contributed by the normal thymus cell. In some hybrids, the elimination process resulted in the total absence of intact chromosomes contributed by the thymus cell parent. Such hybrids were distinguished from revertant parental cells growing in the selective hybrids were distinguished from revertant parental cells growing in the selective medium by the presence of at least one enzyme in their cell extracts which displayed the electrophoretic mobility of the enzyme of the thymus cell parent. These observations, together with data from other reports, suggest that, as a rule, interspecific cell hybrids which develop upon fusion between normal diploid cells and tumorigenic cell lines maintain the chromosomes of the latter and eliminate preferentially many or most of the chromosomes contributed by the normal cell parents, independent of the respective species of the parental cells.     

Fibroblast interferon in man is coded by two loci on separate chromosomes.

We have examined viral and poly(rl):poly(rC) induction of interferon synthesis in several human, mouse and Chinese hamster cell lines, and in hybrids derived from the fusion of such cells. We observed species and cell-type differences in inducer effectiveness and in the kinetics of interferon production. In some cases, parental characteristics are preserved in somatic cell hybrids, and in other cases, the expression of the donor phenotype is modulated by the epigenetic state of the recipient cell. Mapping studies in human/mouse and human/Chinese hamster hybrids indicate that there are at least two structural genes for human fibroblast interferon. Chromosomes 2 and 5 each contain genetic information for the synthesis of fibroblast interferon. Gene dosage experiments indicate that one gene is on the long arm of chromosome 2 and another is on the short arm of chromosome 5. Leukocyte interferon genes could not be mapped to these chromosomes, but this negative result could be influenced by the epigenetic state of the hybrid cells.     

THE RIBOSOMAL RNA OF HAMSTER-MOUSE HYBRID CELLS

The ribosomal RNA (rRNA) of a series of hamster-mouse somatic cell hybrids was studied. Mouse 28S rRNA was separated from its hamster counterpart by a two-step procedure involving sucrose gradient centrifugation of ribosomes and polyacrylamide gel electrophoresis of rRNA. Both hamster and mouse types of rRNA were synthesized in the 11 hybrids tested, including hybrids containing only about one-half the haploid number of either mouse or hamster chromosomes. It appears that, for both hamster and mouse rRNA, when the chromosomes of one species constituted the majority of the chromosomes of a hybrid, a disproportionately higher percentage of rRNA of that species was present in the hybrid. Some hybrid clones, having a majority of mouse chromosomes, had a mouse rRNA cell concentration approximately four to five times higher than the concentration expected from linear extrapolation of the value found for the mouse parental cell line.     

Differential screening of a cDNA library with cDNA probes amplified in a heterologous host: isolation of murine GRP78 (BiP) and other serum-regulated low-abundance mRNAs

A novel method was used to screen differentially a cDNA library for clones representing serum-regulated mRNA species of low abundance. To increase the amount of probe available for screening, the cDNA probe was cloned and amplified. Two separate cDNA 'probe' libraries were constructed in the Escherichia coli plasmid vector pDE613, using poly(A)+mRNA from murine cells at 0 and 16 h after stimulation of a G0 population. Radiolabelled plasmid DNA from each library was hybridized sequentially to colony blots of the third 'target' library, constructed with mRNA from serum-stimulated cells in the Bacillus subtilis vector pBD214. Differential screening of the target cDNA library with the two probe libraries identified novel murine cDNA clones, some representing cytoplasmic poly(A)+RNA species of low (0.01%) abundance, accumulating after serum stimulation of a quiescent mouse embryo fibroblast population. One cDNA clone was found to correspond to mitochondrial 16S rRNA and a second was identified as the murine equivalent of previously described cDNA clones for the hamster 78-kDa glucose-regulated protein (GRP78) and the rat immunoglobulin heavy-chain-binding protein. GRP78 mRNA has not previously been recognized as a serum-inducible message.     

cDNA sequence of the small subunit of the hamster ribonucleotide reductase.

Ribonucleotide reductase activity is markedly elevated in cell lines selected for resistance to hydroxyurea, a cytotoxic drug known specifically to inhibit ribonucleotide reductase. From a cDNA library constructed from a highly hydroxyurea-resistant hamster lung cell line, 600H in which the activity is elevated more than 80-fold, we have isolated a full length cDNA for the small subunit of the reductase. The cDNA is 3.48 kb long with an open reading frame of 1158 nucleotides and a long 3' flanking region of 2169 nucleotides from the termination codon. The derived polypeptide sequence is closely similar to the small subunit of the mouse, differing from it in 20 amino acid positions. Most of these replacements occur in the N-terminal segment of the protein. The hamster subunit does not contain 4 amino acid residues found in the mouse small subunit near the C-terminal end. RNA blots probed with the cDNA show two poly(A)+ RNA species which are elevated in hydroxyurea-resistant cells.     

Changes in the cell cycle during culture of mouse-Chinese hamster cell hybrids

Cell cycle studies, using PLM analysis, were carried out on a mouse-Chinese hamster cell hybrid and its derivatives which stably retained all parental chromosomes during the year of study. Parameter estimates were obtained from the PLM curves, using conjugate gradient curve fitting procedures. The hybrid initially grew very slowly, and all phases (especially G₁) were longer than those of either parent. During propagation, mean generation time decreased progressively, and the phase times approached those of the mouse parent (which had the longer G₁ and S). DNA replication could be scored separately in mouse and hamster chromosome sets; initially termination was highly asynchronous, but during growth asynchrony was progressively reduced as DNA synthesis in the hamster set was prolonged. We conclude that cell hybrids may undergo progressive modifications of the cell cycle, even in the absence of significant chromosome segregation, and suggest that such changes may at least partly account for the great variety of relationships between the growth rates and phase times of parent and hybrid cells which have been reported. Because of the complexity of these changes in the cycles of interspecific cell hybrids, we believe that somatic cell genetic analysis of the regulation of the cell cycle would be more usefully applied to intraspecific hybrids whose parents differ in only one specific cycle characteristic.     

Cellular titers and subcellular distributions of abundant polyadenylate-containing ribonucleic acid species during early development in the frog Xenopus laevis.

The distribution of cytoplasmic messenger ribonucleic acids (RNAs) in translationally active polysomes and inactive ribonucleoprotein particles changes during early development. Cellular levels and subcellular distributions have been determined for most messenger RNAs, but little is known about how individual sequences change. In this study, we used hybridization techniques with cloned sequences to measure the titers of 23 mitochondrial and non-mitochondrial polyadenylate-containing [poly(A)+]RNA species during early development in the frog Xenopus laevis. These RNA species were some of the most abundant cellular poly(A)+ RNA species in early embryos. The concentrations of most of the non-mitochondrial (cytoplasmic) RNAs remained constant in embryos during the first 10 h of development, although the concentrations of a few species increased. During neurulation, we detected several new poly(A)+ RNA sequences in polysomes, and with one possible exception the accumulation of these sequences was largely the result of new synthesis or de novo polyadenylation and not due to the recruitment of nonpolysomal (free ribonucleoprotein) poly(A)+ RNA. We measured the subcellular distributions of these RNA species in polysomes and free ribonucleoproteins during early development. In gastrulae, non-mitochondrial RNAs were distributed differentially between the two cell fractions; some RNA species were represented more in free ribonucleoproteins, and others were represented less. By the neurula stage this differential distribution in polysomes and free ribonucleoproteins was less pronounced, and we found species almost entirely in polysomes. Some poly(A)+ RNA species transcribed from the mitochondrial genome were localized within the mitochondria and were mapped to discrete fragments of the mitochondrial genome. Much of this poly(A)+ RNA was transcribed from the ribosomal locus. Nonribosomal mitochondrial poly(A)+ RNA species became enriched in polysome-like structures after fertilization, with time courses similar to the time course of mobilization of cytoplasmic poly(A)+ RNA.     

Characteristics of somatic cell hybrids (mouse X Chinese hamster) with different ratios of parental species chromosome sets. IV. Electrophoretic analysis of several enzymes of the dehydrogenase class]

Electrophoretic mobilities in polyacrylamide gel of five dehydrogenases: NADP-dependent malate dehydrogenase (NADP-MDH), 6-phosphogluconate dehydrogenase (6PGD), alcohol dehydrogenase (ADH), glucose-6-phosphate dehydrogenase (G6PD) and glutamate dehydrogenase (GDH) were investigated in a series of mouse X Chinese hamster somatic cell hybrids. Seven hybrid lines with different ratio of chromosome sets of hamster and mouse: 1:1, 2:1, 3:1 and 1:2 respectively were studied. NADP-MDH and 6PGD of both parental species and intermediate hybrid bands were present in all hybrids except two lines. These lines had only hamster MDH due to the elimination of mouse chromosomes. A correlation was found between the gene dose and the intensity of the expression of the MDH bands. The mouse type ADH was detected in all hybrids. The hamster ADH was found in one of the hybrid lines that lost all mouse chromosomes during cultivation. It is suggested that hamster ADH activity was suppressed in hybrids by the mouse genome. The species origin of GDH and G6PD could not be established due to similarity of electrophoretic mobilities of respective enzymes in parental cells.     

Characterization of mitochondrial DNA in chloramphenicol-resistant interspecific hybrids and a cybrid

We have examined the restriction endonuclease cleavage patterns exhibited by the mitochondrial DNAs (mtDNA) of four chloramphenicol-resistant (CAPR) human x mouse hybrids and one CAPR cybrid derived from CAPR HeLa cells and CAPS mouse RAG cells. Restriction fragments of mtDNAs were separated by electrophoresis and transferred by the Southern technique to diazobenzyloxymethyl paper. The covalently bound DNA fragments were hybridized initially with 32P-labeled complementary RNA (cRNA) prepared from human mtDNA and, after removal of the human probe, hybridized with mouse [32P]cRNA prepared from mouse mtDNA. Three hybrids which preferentially segregated human chromosomes and the cybrid exhibited mtDNA fragments indistinguishable from mouse cells. One hybrid, ROH8A, which exhibited "reverse" chromosome segregation, contained only human mtDNA. The pattern of chromosome and mtDNA segregation observed in these hybrids and the cybrid support the hypothesis that a complete set of human chromosomes must be retained if a human-mouse hybrid is to retain human mitochondrial DNA.     

Adaptation of the interspersed repetitive sequence polymerase chain reaction to the isolation of mouse DNA probes from somatic cell hybrids on a hamster background

A strategy for the rapid isolation of DNA probes from radiation-fusion Chinese hamster cell hybrids containing overlapping portions of the murine X chromosome based on the interspersed repetitive sequence polymerase chain reaction (IRS-PCR) previously used with human somatic cell hybrids has been developed. This specific amplification of mouse DNA on a hamster background depends on the use of primers directed to the B2 short interspersed repeat element family and the R repeat, from the long interspersed repeat element family, L1. Two sets of amplification conditions, which gave specific amplification of mouse DNA from either a mouse X-monochromosomal hybrid or irradiation-fusion hybrids having reduced X content, were defined. The mouse X-only chromosome hybrid yielded approximately 20 discrete reproducible bands, while the irradiation-fusion hybrids yielded between 1 and 10 discrete products. Comparison of different irradiation-fusion hybrids has allowed the definition of both specific and shared products corresponding to different regions within the overlapping X-chromosome fragments present within these hybrids. Use of such hybrids and the IRS-PCR technique has allowed the isolation of probes corresponding to the central region of the mouse X chromosome that contains the X-inactivation center. The method should be widely applicable to the isolation of mouse DNA sequences from mouse hybrid cell lines on either human or Chinese hamster backgrounds.     

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.