Mouse-hamster Hybrid Cells Research Articles

Different pathways for chromosomal integration of transfected circular pSVneo plasmids in normal and established rodent cells

The chromosomal integration of transfected circular plasmid pSV2neo molecules was investigated in normal and established mammalian fibroblasts, including two secondary cultures of normal mouse fibroblast lines, one established mouse-hamster hybrid cell line, R44, and the human fibrosarcoma line, HT1080. The physical organization of the integrated molecules was studied by restriction analysis. The results showed that whereas the normal fibroblasts predominantly integrated one head-to-tail partial dimeric molecule, the established cells predominantly integrated distinctly different molecular forms including deleted monomeres (HT1080) and various complex concatemeric molecules (R44), and frequently at more than one chromosomal site (R44). We also constructed a head-to-tail dimeric version of the plasmid, which in the case of the normal fibroblasts again integrated as a partial dimeric molecule in at least 50% of these cells. This result excluded the possibility that the normal mouse transfectants were selected for the integration of two functional neo genes. Thus, it is concluded that the distinctly different molecular forms integrated in normal and established cells demonstrate the operation of different integration pathways, the possible nature of which is discussed.

Rapid analysis of mouse-hamster hybrid cell lines by in situ hybridization

In situ htbridization techniques for analyzing the murine DNA complement of mouse-hamster hybrid cells are described. Total genomic mouse DNA is labeled with biotin and hybridized without suppression to metaphase spreads from a mouse-hamster hybrid line containing the mouse fusion chromosome X 12. Detection via fluorochrome-conjugated avidin reveals mouse chromosomal DNA with high sensitivity and permits the identification of both normal and aberrant murine chromosomes. Conversely, biotinylated total genomic DNA from a hybrid line can be used as a probe on normal mouse metaphase spreads if suppression techniques are employed, facilitating the analysis of mouse chromosomes present in the hybrid line.

The mouse smooth muscle ? actin gene is on chromosome 6

Smooth muscle gamma actin (Actg) is expressed in smooth muscle and in haploid male germ cells. In order to further characterize the Actg gene, a 60-nucleotide-long isotype-specific probe was synthesized. Single bands of DNA were detected when this oligonucleotide was used to probe blots of mouse genomic DNA digested with PstI, EcoRI, KpnI, or XbaI. These results suggest Actg is a single-copy gene with no detectable pseudogenes. The Actg gene was mapped to mouse chromosome 6 by Southern blot analysis of DNA isolated from 15 mouse-hamster hybrid cell lines.

Sequences homologous to glutamic acid decarboxylase cDNA are present on mouse chromosomes 2 and 10

The chromosomal locations of mouse DNA sequences homologous to a feline cDNA clone encoding glutamic acid decarboxylase (GAD) were determined. Although cats and humans are thought to have only one gene for GAD, GAD cDNA sequences hybridize to two distinct chromosomal loci in the mouse, chromosomes 2 and 10. The chromosomal assignment of sequences homologous to GAD cDNA was determined by Southern hybridization analysis using DNA from mousehamster hybrid cells. Mouse genomic sequences homologous to GAD cDNA were isolated and used to determine that GAD is encoded by a locus on mouse chromosome 2 ( Gad-1) and that an apparent pseudogene locus is on chromosome 10 ( Gad-1ps). An interspecific backcross and recombinant inbred strain sets were used to map these two loci relative to other loci on their respective chromosomes. The Gad-1 locus is part of a conserved homology between mouse chromosome 2 and the long arm of human chromosome 2.

Chromosomal mapping of the prolactin/growth hormone gene family in the mouse.

The chromosomal assignments of genes in the PRL/GH family in the mouse have been determine in mouse-hamster hybrid cell lines. Mouse GH (mGH) appears to be encoded by a single copy gene located on chromosome 11 and is part of a highly conserved region between mouse chromosome 11 and human chromosome 17. All of the other genes in this hormone family, including those encoding mPRL, mouse placental lactogens I and II, and mouse proliferin and proliferin-related protein, map to chromosome 13.

Three functional ribosomal protein genes are unlinked in mouse genome.

The mouse chromosomes bearing three functional ribosomal protein (rp) genes were identified by Southern blot analysis of DNA from a panel of mouse-hamster hybrid cell lines. Unique sequence intron probes were used to distinguish the functional rp genes from their multiple processed pseudogene counterparts. The genes specifying ribosomal proteins S16, L30, and L32 were found to be on chromosomes 7, 15, and 6, respectively. Since these functional rp genes are widely dispersed in the mouse genome, coordinate regulation of their transcriptional activity cannot be accomplished by a structural alteration of a single chromosomal region. Rather, it would have to involve interactions with sequences or structural motifs that are common to all three genes.

Characterization and mapping of DNA sequence homologous to mouse U1a1 snRNA: localization on chromosome 11 near the Dlb-1 and Re loci.

A phage clone which contained a functional U1a1 snRNA gene was isolated from a mouse genomic library. A single copy fragment was isolated from the 3' flanking region of the U1a1 gene and used as a hybridization probe for Southern blotted DNAs from recombinant inbred strains of mice, mouse-hamster hybrid cells, and the offspring from backcrosses between BALB/c mice and mice which were heterozygous for the Rex (Re) marker. The results of these experiments prove that the U1a1 gene is located on chromosome 11 near the Delb-1 and Re loci.

Use of a cDNA recombinant for the gamma-subunit of mouse nerve growth factor to localize members of this multigene family near the TAM-1 locus on chromosome 7.

The gamma-subunit of mouse 7S nerve growth factor (gamma-NGF) is a member of a family of closely related serine proteases that includes kallikreins and tamases. We have isolated from a DBA/2J male submaxillary gland cDNA library a clone, pSM676, which codes for gamma-NGF. Sequence analysis of the clone shows that it codes for the C-terminal 138 amino acids of the protein plus 23 bases of the 3'-nontranslated portion of the message. The predicted amino acid sequence agrees with that determined by Thomas et al. (1) for the gamma-subunit of nerve growth factor from Swiss Webster mice except for the single, conservative substitution of glutamate for aspartate at amino acid 175. When used as a probe for Southern blot analysis, pSM676 hybridizes to at least twelve fragments of restricted mouse genomic DNA which correspond to several different serine protease genes. Using mouse-hamster hybrid cell lines and recombinant inbred strains of mice, we have demonstrated that all of the genes which show homology to pSM676 are located on mouse chromosome 7, clustered at or near the Tam-1 locus.

Regulation of ribosomal RNA and proteins in mouse-hamster hybrid cells.

Regulation of ribosomal RNA and proteins in mouse-hamster hybrid cells.

Chromosomal distribution of ribosomal protein genes in the mouse

The chromosomal distributions of five families of mouse r-protein genes (S16, L18, L19, L30 and L32/33) were studied by Southern blot analysis of DNA from a panel of mouse-hamster hybrid cells containing various complements of mouse chromosomes. Our results indicated that members of a particular family are often located on more than one chromosome, that extensive clustering of many r-protein gene families on a few chromosomes is unlikely, and that there is no obligatory linkage of r-protein and rRNA genes.

Heterogenetic, interspecific nature of ribosomal proteins in mammals

The nature of the differences between ribosomal proteins of mammalian species (man, mouse, rat, hamster), separated by polyacrylamide gel electrophoresis, has been investigated in cell lines and inter‐specific somatic hybrids cultured in vitro, and in livers of the corresponding animals. The proteins of the large and small ribosomal subunits have only a few bands with the same electrophoretic mobilities. Comparing, for each kind of subunit, the ribosomal proteins in the four species, two categories of proteins are described: (a) proteins which are always present, qualitatively and quantitatively constant; (b) proteins which exhibit qualitative or quantitative differences. One qualitative difference, possibly due to a mutation slightly altering the electrophoretic mobility of one protein of the large subunit of the syrian hamster has been observed. The mouse‐hamster hybrid cells possess on their large ribosomal subunits, the proteins from each “parent”. Several quantitative differences, affecting only certain proteins of the subunits, are described. They seem to be the main source of the heterogeneity of ribosomal proteins observed between mammalian species. These findings are discussed in relation to the expression of the genome, and to the structure and function of the ribosomes during the process of speciation and differentiation.

Two types of ribosome in mouse-hamster hybrid cells.

ONLY mouse 28S ribosomal RNA (rRNA) can be detected in mouse-human hybrid cells1. Because these cells contain as many as thirty-five human chromosomes, including those believed to have ribosomal RNA genes, it has been suggested that the transcription of human ribosomal RNA genes may be repressed. Similar studies have not been performed with mouse-hamster hybrid cells because hamster and mouse 28S rRNAs have only a very small difference in their electro-phoretic mobilities2. We have now made use of the observation that free ribosomes (not engaged in translation) from hamster cells form dimers in conditions where mouse free ribosomes remain as monomers3 to determine whether mouse-hamster hybrid cells contain free ribosomes as monomers, dimers or as both.