On the nature of hereditable variation in cultured somatic cells

Siminovitch, Louis

doi:10.1016/0092-8674(76)90249-x

Cited by 484 publications

(172 citation statements)

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“…During adaptation to tissue culture conditions, many chromosomal changes took place in this cell line resulting in its current karyotype of 21-22 nonidentical chromosomes (Deaven and Petersen, 1973). In this process, many genes were lost or became haploid (Siminovitch, 1976). In addition, we have shown that CHO cells produce a surfeit of tubulin and can lose half of their ␤1-tubulin (35% of total ␤-tubulin) with no apparent effect on growth of the cells (Boggs and Cabral, 1987).…”

Section: Discussionmentioning

confidence: 96%

A Ubiquitous β-tubulin Disrupts Microtubule Assembly and Inhibits Cell Proliferation

Bhattacharya

Cabral

2004

MBoC

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Vertebrate tubulin is encoded by a multigene family that produces distinct gene products, or isotypes, of both the ␣-and ␤-tubulin subunits. The isotype sequences are conserved across species supporting the hypothesis that different isotypes subserve different functions. To date, however, most studies have demonstrated that tubulin isotypes are freely interchangeable and coassemble into all classes of microtubules. We now report that, in contrast to other isotypes, overexpression of a mouse class V ␤-tubulin cDNA in mammalian cells produces a strong, dose-dependent disruption of microtubule organization, increased microtubule fragmentation, and a concomitant reduction in cellular microtubule polymer levels. These changes also disrupt mitotic spindle assembly and block cell proliferation. Consistent with diminished microtubule assembly, there is an increased tolerance for the microtubule stabilizing drug, paclitaxel, which is able to reverse many of the effects of class V ␤-tubulin overexpression. Moreover, transfected cells selected in paclitaxel exhibit increased expression of class V ␤-tubulin, indicating that this isotype is responsible for the drug resistance. The results show that class V ␤-tubulin is functionally distinct from other tubulin isotypes and imparts unique properties on the microtubules into which it incorporates. INTRODUCTIONMicrotubules are essential filamentous structures in eukaryotic cells where they are responsible for the directed movement of vesicles, the organization of the endoplasmic reticulum and Golgi apparatus in the cytoplasm, and the equipartitioning of chromosomes before cell division. The organelles are assembled from heterodimers of ␣-and ␤-tubulin, which polymerize in a head-to-tail manner to form linear protofilaments, and these associate laterally into tubular structures that normally consist of 13 protofilaments. Vertebrate ␣-and ␤-tubulins are each encoded by a 6-to 7-member multigene family that produces highly homologous and conserved gene products that differ most radically in their last 10 -15 amino acids (Sullivan, 1988;Luduena, 1998). These carboxyl-terminal sequences have been used to assign ␤-tubulin gene products to seven distinct classes (Lopata and Cleveland, 1987). Each of these classes (hereafter referred to as ␤1, ␤2, ␤3, ␤4a, ␤4b, ␤5, and ␤6) defines a ␤-tubulin isotype that differs significantly from other isotypes within the same organism, but differs very little from the same isotype in other vertebrate species.With the discovery that tubulin proteins in an organism are heterogeneous, a hypothesis was formulated suggesting that different tubulin proteins might perform different functions in the cell (Fulton and Simpson, 1976). In the intervening years, however, most of the experimental and genetic evidence has argued that different ␤-tubulins coassemble freely into all cellular microtubules (see Joshi and Cleveland, 1990; Luduena, 1998 for reviews). In gene replacement experiments using the fungal organism, Aspergillus nidulans, for example, May demonst...

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Section: Discussionmentioning

confidence: 96%

A Ubiquitous β-tubulin Disrupts Microtubule Assembly and Inhibits Cell Proliferation

Bhattacharya

Cabral

2004

MBoC

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“…The data indicate that the quasi-diploi CHO cells appear only as functionally hemizygous as would be expected of a slightly hypodiploid cell line derived from an organism in which the haploid number is 11L The ease of production of large numbers of phenotypically recessive mutations (auxotrophic, drug-resistant, temperature-sensitive; see ref. 1 for listing) in Chinese hamster ovary cells (CHO) remains a major paradox in cell biology. That the total DNA content in CHO cells is close to that of diploid normal Chinese hamster cells and that most of the chromosomal bands can be identified (2) lead to the expectation that most autosomal loci are represented twice.…”

mentioning

confidence: 99%

Electrophoretic shift mutants in Chinese hamster ovary cells: evidence for genetic diploidy.

Siciliano¹,

Siciliano²,

Humphrey³

1978

Proc. Natl. Acad. Sci. U.S.A.

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Electrophoretic shift mutants induced in Chinese hamster ovary (CHO) cells indicate that these cells are not extensively functionally hemizygotic. Therefore, effective haploidy is unsatisfactory as a general theory to explain the frequency of recessive mutants in this cell line.CHO cells were screened for electrophoretic shift variants of enzymes coded by -40 genetic loci. Clones isolated after exposure to ultraviolet radiation were examined by starch gel and Cellogel electrophoresis. Shift variants were recovered for enzymes representing 11 different loci. Variant clones were subcloned to demonstrate the heritability of the variations.Mutants at nine loci produced multiple-banded patterns consistent with the patterns expected of genes at loci represented twice (diploid). Chromosome localization of these diploid loci in other mammalian species where they have been mapped, suggests that they represent a random sample of CHO genes. This question can now be readily addressed as a result of our studies on the induction of electrophoretic shift variants at a large number of specific enzyme loci in CHO cells (5). An electrophoretic shift variant may be interpreted as resulting from a nucleotide substitution in a structural gene, causing the substitution of an amino acid residue carrying a different charge (6). If such a mutation were to take place in a cell with two copies of the structural gene (diploid), both mutant and wildtype polypeptide chains of the enzyme would be produced in the same cell. After electrophoresis and histochemical staining for the enzyme, one would see the products of both the modified gene and the unaffected allele. One would also see the expected number of intermediate hybrid molecules according to the subunit structure of the enzyme: none if the enzyme is a monomer, one if a dimer, two if a trimer, and three if a tetramer (7). However, if the shift variant was induced at a functionally haploid locus, only the modified polypeptide would then be produced, resulting in a single band of activity located in a different position than the wild-type enzyme.Over the last 3 years we have seen a sufficient number of shift mutants to state that most CHO cell loci appear to be functionally diploid and the amount of effective hemizygosity appears to be no more than would be expected for a slightly hypodiploid cell line such as CHO. MATERIALS AND METHODSCell Culture. CHO cells (8) were grown in McCoy's 5a medium (9) supplemented with 20% fetal calf serum (GIBCO, Inc.) at 370 in a 5% C02/95% air, water-saturated environment to maintain a pH of 7.0-7.2. The cell line was supplied to us by S. C. Barranco (University of Texas Medical Branch, Galveston, TX) who received it from L. Deaven (Los Alamos National Laboratories, Los Alamos, NM). The line has a modal chromosome number of 21 (8), an average generation time of 12 hr, and a plating efficiency of 70-90% (10).Mutagen Treatment. Cells (1 X 106) were plated into a 60-mm2 petri dish and incubated overnight. The medium was then removed, the cells wer...

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“…Besides their usefulness for cell hybridization, marked marsupial cell strains are potentially valuable for studies of biochemistry and heritable phenotypic variation of marsupial somatic cells. It would be interesting to compare the results of such investigations with the information available from studies of eutherian cells (Puck 1974;Siminovitch 1976).…”

Section: Differences In Adhesion and Growthmentioning

confidence: 99%

“…However, we do not know if they result from an alteration in a specific gene product, nor whether they can be assigned to a specific locus on a chromosome. Independently derived strains may be heterogeneous with respect to their genetic lesions, and may include structural gene mutants, deletions, changes of a regulatory nature, or epigenetic changes (Siminovitch 1976).…”

Section: Differences In Adhesion and Growthmentioning

confidence: 99%

Fusion and Hybridization of Marsupial and Eutherian Cells V. Development of Selective Systems

Hope¹,

Graves²

1978

Aust. Jnl. Of Bio. Sci.

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Aust. J. BioI. Sci., 1978, 31, 293-301 The availability of systems which permit the selective elimination of marsupial cells from fused cultures is an essential requirement for the production of marsupial x eutherian somatic cell hybrids. Such hybrids have particular advantages for genetic studies of mammalian cells. We describe the isolation and characterization of several drug-resistant marsupial cell strains. We have selected strains resistant to concentrations of 10 p,g/ml of the purine analogues 8-azaguanine and 6-thioguanine. Several of these strains were found to be deficient in the enzyme hypoxanthine phosphoribosyltransferase and consequently sensitive to hypoxanthine--aminopterin-thymidine (HAT) selective medium. We have also isolated marsupial cell strains resistant to concentrations of 22p,g/ml of the thymidine analogue 5-bromodeoxyuridine. These strains were thymidine kinase deficient and HAT sensitive. Drug resistance was a stable characteristic maintained for many generations in the absence of the drug. However, inhibition of growth of these drug-resistant strains was strongly density dependent, a factor that caused difficulties in the selection of hybrids. We have also developed selective systems which exploit differences between marsupial and eutherian cells in sensitivity to growth in ouabain, and in adhesiveness and other growth properties. Marsupial cells were found to be naturally much more sensitive to ouabain than rodent cells, a phenomenon that should be useful in the selection of marsupial x rodent cellular hybrids. We discuss a number of difficulties associated with the derivation and use of variant marsupial cell strains.

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On the nature of hereditable variation in cultured somatic cells

Cited by 484 publications

References 70 publications

A Ubiquitous β-tubulin Disrupts Microtubule Assembly and Inhibits Cell Proliferation

A Ubiquitous β-tubulin Disrupts Microtubule Assembly and Inhibits Cell Proliferation

Electrophoretic shift mutants in Chinese hamster ovary cells: evidence for genetic diploidy.

Fusion and Hybridization of Marsupial and Eutherian Cells V. Development of Selective Systems

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