Somatic Cell Genetics Research Articles

Cell cycle parameters in Aedes albopictus mosquito cells.

We have analyzed cell cycle parameters for the Aedes albopictus C7-10 mosquito cell line, which has been systematically developed for somatic cell genetics, expression of transfected genes, and synthesis of hormone-inducible proteins. In rapidly cycling cells, we measured a generation time of 10-12 h. The duration of mitosis (M) was < or = 1 h, and the DNA synthesis phase (S) required 6 h. Unlike Drosophila melanogaster Kc cells, in which the G2 gap is substantially longer than G1, in C7-10 cells G1 and G2 each lasted approximately 2 h. In these cells, the duration of both S and G2 was independent of the population doubling time, and the increase in population doubling time as cells approached confluency was due to prolongation of G1. When treated with the insect steroid hormone, 20-hydroxyecdysone, C7-10 mosquito cells complete the cycle in progress before undergoing a reversible arrest.

The cells of the body: a history of somatic cell genetics

The cells of the body: a history of somatic cell genetics

Genomic localization of the human gene encoding Dr1, a negative modulator of transcription of class II and class III genes.

Dr1 is a nuclear protein of 19 kDa that exists in the nucleoplasm as a homotetramer. By binding to TBP (the DNA-binding subunit of TFIID, and also a subunit of SL1 and TFIIIB), the protein blocks class II and class III preinitiation complex assembly, thus repressing the activity of the corresponding promoters. Since transcription of class I genes is unaffected by Dr1. it has been proposed that the protein may coordinate the expression of class I, class II and class III genes. By somatic cell genetics and fluorescence in situ hybridization, we have localized the gene (DR1), present in the genome of higher eukaryotes as a single copy, to human chromosome region 1p21-->p13. The nucleotide sequence conservation of the coding segment of the gene, as determined by Noah's ark blot analysis, and its ubiquitous transcription suggest that Dr1 has an important biological role, which could be related to the negative control of cell proliferation.

Characterization and Chromosomal Localization of a New Protein Disulfide Isomerase, PDIp, Highly Expressed in Human Pancreas

Protein disulfide isomerase (PDI) catalyzes protein folding and thiol-disulfide interchange reactions. The enzyme is localized in the lumen of endoplasmic reticulum (ER) and is abundant in secretory cells of various tissues. In this study we describe the isolation and characterization from human pancreas of a new protein, PDIp, that is structurally and functionally related to PDIs. PDIp cDNA is 1,659 bp in length and predicts a protein with an open reading frame of 511 amino acids. PDIp amino acid sequence shows 46% identity and 66% similarity to that of human PDI. PDIp possesses two thioredoxin-like active sites (WCGHCQ and WCTHCK) and an endoplasmic reticulum retention signal sequence, KEEL, at the carboxyl terminus. Northern analysis of normal human tissues and various human tumor cell lines revealed PDIp mRNA (2.0 kb) expression only in the normal pancreas. Recombinant PDIp protein catalyzed reductive cleavage of insulin and renaturation of reduced RNaseA. Somatic cell genetics and fluorescence in situ hybridization localized the PDIp gene to the short arm of human chromosome 16. It is concluded that PDIp is a new member of the PDI family and is highly expressed in human pancreas.

FISH localization of the soluble thymidine kinase gene (TK1) to human 17q25, a region of chromosomal loss in sporadic breast tumors

Soluble thymidine kinase (TK1) is an important 17q marker in somatic cell genetics. Its activity is increased in many malignancies, including breast cancer. Through somatic cell hybrid and fluorescence in situ hybridization studies, we mapped TK1 to 17q25.2-->25.3, in region demonstrating loss of heterozygosity in primary breast tumors. It lies near D17S836 and is proximal to the avian erythroblastic leukemia viral oncogene homolog 2-like gene (ERBA2L).

Functional Studies to Identify Tumor Suppressor Genes

Techniques in somatic cell genetics have proven to be very important in the elucidation of genetic mechanisms in cancer and, in particular, the functional definition of tumor suppressor genes. Genetic complementation studies using microcell fusion have allowed the identification of specific chromosomes and particular chromosome regions that contain functional tumor suppressor genes for a variety of human cancers. In this article, the basic strategy for insertion of dominant selectable markers into human chromosomes is described, as are the methods for microcell fusion of tagged chromosomes into malignant cell lines. Molecular cytogenetic characterization protocols for microcell hybrids generated by such strategies are outlined. Finally, novel approaches to the isolation of dominantly tagged subchromosomal fragments for functional assays as well as to the isolation of new tumor suppressor genes are also described.

Irradiation and fusion gene transfer

Irradiation and fusion gene transfer (IFGT) is a technique that spans the gap between the limitations of molecular methods and somatic-cell genetics, allowing the separation of DNA fragments between 0.25 and 30 Mb in size. In conjunction with genetic linkage analysis and physical mapping techniques, IFGT provides a very useful addition to methods for cloning disease loci, and mapping chromosomes and entire genomes.

Reconstitution of Human DNA Repair Excision Nuclease in a Highly Defined System

Xeroderma pigmentosum is a hereditary disease caused by defective DNA repair. Somatic cell genetics and biochemical studies with cell-free extracts indicate that at least 16 polypeptides are required to carry out the repair reaction proper, i.e. the removal of the lesion from the DNA by the dual incisions of the damaged strand. To find out if these proteins are necessary and sufficient for excision repair, they were obtained at a high level of purity in five fractions. The mixture of these five fractions reconstituted the excision nuclease (excinuclease) activity. Using the reconstituted excinuclease, we found that the excised fragment remains associated with the post-incision DNA-protein complex, suggesting that accessory proteins are needed to release the excised oligomer.

A genetic and physical map of bovine chromosome 3.

This paper reports a map of nine polymorphic microsatellite markers previously assigned to bovine chromosome 3 (BTA3) by somatic cell genetics. The linkage group covers 101 cM on the chromosome with an average intermarker distance of 13.9 cM. One marker (INRA200) was isolated from a peak of flow sorted chromosomes 2 and 3. Another marker (INRA197) was derived from a cosmid. The localization of the cosmid by in situ hybridization enabled the orientation of the linkage group on BTA3. Markers were relatively evenly spaced and consequently can be used to complement other mapping data about this chromosome. This establishes a framework of polymorphic markers that can be used to search for quantitative trait loci (QTL).

Germline and somatic mutation at the APRT locus of mice and man.

Somatic cell genetics has its origins in the notion that it is possible to understand gene expression in mammalian cells by in vitro manipulations similar to those used with bacterial cultures. For example, cultures can be propagated in logarithmic fashion and single cell phenotypic variants can be subsequently isolated as clones. It was further assumed that phenotypic variation in cultured mammalian cells would arise by mechanisms similar to those proposed for such events in vivo. Thus it was anticipated that epigenetic variation similar to that observed in organismal ontogeny would be common. However, most early reports described phenomena that have now been demonstrated to be the result of mutations rather than epigenetic changes.

Assignment of bovine synteny group U2 to chromosome 9.

One cosmid containing a microsatellite (INRA144, D9S14) was assigned to bovine synteny group U2 by somatic cell genetics and localized to bovine chromosome 9q25 by fluorescent in situ hybridization. These results permitted the assignment of one more synteny group to a bovine chromosome. There are now 22 out of 31 bovine synteny groups which are related to a chromosome. The mapping data have been entered in the BovMap database, Jouy-en-Josas, France.

How variable is an spontaneous mutation rate in cultured mammalian cells?

The Luria-Delbrück fluctuation analysis provides a method to estimate mutation rates and is commonly applied in somatic cell genetics and in cancer biology. We developed an assay for a Luria-Delbrück fluctuation analysis using the mouse lymphoma cell line, GRSL13. As these cells grow in suspension, one can handle hundreds of parallel cultures using multiwell dishes and dispensers. This assay thereby allows not only an accurate determination of the mutation rate per cell generation but also makes it possible to determine at which time after seeding mutations take place. Using approx. 8000 parallel cultures it has been possible to test whether the mutation rate is constant during the assay. It has been found that the spontaneous mutation rate of GRSL13 cells decreases in the course of a fluctuation test from 2 × 10 −6 to about 2 × 10 −7/cell/generation. It was shown that this increased replication fidelity may partly be caused by cell density: maintenance of cells at high cell density resulted in a spontaneous mutation rate of 0.7 ± 4.0 × 10 −7 compared to 4.0 ± 3.1 × 10 −7 for the standard protocol. In contrast, growing the cells at extremely low cell density resulted in an enhanced mutation rate of 7.7 ± 1.3 × 10 −7. Thus altogether the mutation rate can vary from 2 × 10 −6 to 0.7 × 10 −7 (approx. 30-fold). These results show that the spontaneous mutation rate is not constant, but highly dependent on experimental conditions. As incomplete expression and metabolic cooperation cannot explain the findings, the data suggest that the fidelity of DNA replication is not fixed but open to variation. Hence, determination of replication infidelity in cultured cells needs rigorous standardization or/and application of controlled variation in culture conditions.

Polypeptide signalling to the nucleus through tyrosine phosphorylation of Jak and Stat proteins.

Binding of interferons IFN-alpha and IFN-gamma to their cell surface receptors promptly induces tyrosine phosphorylation of latent cytoplasmic transcriptional activators (or Stat proteins, for signal transducers and activators of transcription). Interferon-alpha activates both Stat91 (M(r) 91,000; ref. 1) and Stat113 (M(r) 113,000; ref. 2) whereas IFN-gamma activates only Stat91 (refs 3, 4). The activated proteins then move into the nucleus and directly activate genes induced by IFN-alpha and IFN-gamma. Somatic cell genetics experiments have demonstrated a requirement for tyrosine kinase-2 (Tyk2) in the IFN-alpha response pathway and for Jak2 (ref. 6), a kinase with similar sequence, in the IFN-gamma response pathway. Here we investigate the tyrosine phosphorylation events on Stat and Jak proteins after treatment of cells with IFNs alpha and gamma and with epidermal growth factor (EGF). Stat91 is phosphorylated on Tyr701 after cells are treated with IFN-alpha and EGF, as it was after treatment with IFN-gamma (ref. 8). We find that Jak1 also becomes phosphorylated on tyrosine after cells are treated with these same three ligands, although each ligand is shown to activate at least one other different kinase. Jak1 may therefore be the enzyme that phosphorylates Tyr 701 in Stat91.

Endosome maturation: Insights from somatic cell genetics and cell-free analysis

Endosome maturation: Insights from somatic cell genetics and cell-free analysis

Resistance of human squamous carcinoma cells to transforming growth factor beta 1 is a recessive trait.

Because most human squamous carcinoma cell lines of the aerodigestive and genital tracts are refractory to the antiproliferative action of transforming growth factor beta 1 (TGF beta 1) in vitro, we have begun to identify the causes for resistance of squamous carcinoma cell lines to TGF beta 1 by using somatic cell genetics. Two stable hybrid cell lines (FaDu-HKc.1 and FaDu-HKc.2) were obtained by fusing a TGF beta 1-resistant human squamous carcinoma cell line, FaDu-HygR, with a human papilloma virus 16-immortalized, TGF beta 1-sensitive, human foreskin keratinocyte cell line, HKc-neoR. Whereas TGF beta 1 did not inhibit DNA synthesis in parental FaDu-HygR cells, it reduced DNA synthetic activity of HKc-neoR, FaDu-HKc.1, and FaDu-HKc.2 cells by 75-85% (IC50, 2-5 pM). Although squamous carcinoma cells express lower than normal levels of TGF beta 1 type II receptors on their cell surface, TGF beta 1 type II receptor mRNA was detected in all four cell lines. Recessive genes involved in TGF beta 1 signaling may be localized to the distal portion of chromosome 18q, as this was the sole chromosomal region of homozygous deletion in parental FaDu-HygR cells. Furthermore, our previous observation that mutant p53 decreases sensitivity of keratinocytes to TGF beta 1 was supported by the finding that the level of the mutant p53 protein expressed by the hybrid cell lines was greatly reduced. In summary, TGF beta 1 resistance of FaDu cells appears to be recessive and is presumably due to the loss of one or more post-receptor elements of the signaling pathway.

The Human Urokinase-Plasminogen Activator Gene (PLAU) Is Located on Chromosome 10q24 Centromeric to the HOX11 Gene

Urokinase-plasminogen activator is one of two soluble serine proteases that are produced by humans and that convert plasminogen, an inactive proenzyme present in plasma and other extracellular fluids, to plasmin, a protease with broad substrate specificities. Its activity is involved in processes requiring localized extracellular proteolysis such as fibrinolysis, tissue remodeling, and cell migration. Increased production of urokinase has been associated with cancer metastases. The gene for urokinase-plasminogen activator, PLAU, was mapped to chromosome 10q24-qter. By employing somatic cell genetics, polymerase chain reaction (PCR), and Southern blot analysis, the authors assign PLAU to chromosome 10q24. Human chromosome segment 10q23-q25 contains the genes for terminal deoxynucleotidyltransferase, cytochrome P450IIC, glutamic-oxaloacetic transaminase, and plasma retinol binding protein, which form a syntenic group on murine chromosome 19. It is therfore of interest that PLAU and glutamate dehydrogenase, which are on murine chromosome 14, also map in or close to this region of human chromosome 10.

A fresh look at the somatic cell genetics of hepatoma differentiation.

A fresh look at the somatic cell genetics of hepatoma differentiation.

Somatic cell mapping of omega and trophoblast interferon genes to bovine syntenic group U18 and in situ localization to chromosome 8.

Bovine omega (IFNW) and trophoblast (IFNT) interferon genes were assigned to bovine syntenic group U18 by somatic cell genetics. Fluorescent in situ hybridization subsequently localized these genes to bovine chromosome 8 band 15. This assignment conflicts with a previous assignment of U17 genes to the same chromosome.

Elevation of sister chromatid exchange frequency in transformed human fibroblasts following exposure to widely used aminoglycosides.

Aminoglycosides are a class of antibiotics that interfere with protein translation. Geneticin and hygromycin are two such agents, which have been shown to exhibit highly toxic effects in mammalian cells. Cloned bacterial genes, which inactivate these antibiotics, have facilitated the establishment of dominant selection systems, which are widely used in eukaryotic molecular genetics. We have examined the effect of aminoglycosides on the sister chromatid exchange (SCE) frequency in transformed human fibroblast cell lines. Geneticin and hygromycin were both found to increase SCE frequency in all cell lines examined, including a cell line derived from a patient with Bloom syndrome, a disorder exhibiting an elevated spontaneous SCE frequency. Induction was seen to occur in a dose-responsive manner and was also observed in cells expressing the resistance genes that inactivate the cellular toxicity of these antibiotics. The implications of these findings for somatic cell genetics and for human gene therapy protocols are discussed.

Roots: Contributions of boris, ephrussi to the development of somatic cell genetics

Roots: Contributions of boris, ephrussi to the development of somatic cell genetics