scholarly journals Isolation and characterization of the rat huntingtin promoter

1998 ◽  
Vol 336 (1) ◽  
pp. 227-234 ◽  
Author(s):  
Carsten HOLZMANN ◽  
Winfried MÄUELER ◽  
Dirk PETERSOHN ◽  
Thorsten SCHMIDT ◽  
Gerald THIEL ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Binding Sites ◽  
Neurodegenerative Disorder ◽  
Core Promoter ◽  
Chinese Hamster ◽  
Gc Content ◽  
Ovary Cell ◽  
Mobility Shift ◽  
Isolation And Characterization ◽  
Hd Gene

Huntington's disease (HD) is a neurodegenerative disorder caused by a (CAG)>37 repeat expansion in a novel gene of unknown function. Although the huntingtin gene is expressed in neuronal and non-neuronal tissues, the disease affects nerve cells of selected regional areas of the central nervous system. To gain insight into the regulation of the HD gene we analysed 1348 bp of the rat huntingtin promoter region. This region lacks a TATA and a CAAT box, is rich in GC content and has several consensus sequences for binding sites for SP1, PEA3, Sif and H2A. The stretch between nucleotides -56 and -206 relative to the first ATG is highly conserved between human and rodents and it harbours several potential binding sites for transcription factors. We analysed deletion mutants fused with the chloramphenicol acetyltransferase reporter gene in transfected, HD-expressing neuronal (NS20Y, NG108-15) and non-neuronal Chinese hamster ovary cell lines. Hence these cells should contain the required trans-acting factors necessary for HD gene expression. Partial deletion of the evolutionarily conserved part of the promoter significantly decreases the activity in both neuronal and non-neuronal cells, indicating that the core promoter activity is located between nucleotides -332 and -15. DNase I footprinting and electrophoretic mobility-shift assays were used to define the nucleotide positions and binding affinity of DNA–protein interactions.

Isolation and characterization of Chinese hamster ovary cell mutants with altered sensitivity to high doses of tunicamycin

1981 ◽  
Vol 7 (5) ◽  
pp. 507-521 ◽  
Author(s):  
Michihiko Kuwano ◽  
Toshiko Tabuki ◽  
Shin-ichi Akiyama ◽  
Kumato Mifune ◽  
Akira Takatsuki ◽  
...  
Keyword(s):  
Chinese Hamster Ovary Cell ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Isolation And Characterization ◽  
High Doses ◽  
Altered Sensitivity

scholarly journals Isolation and characterization of Chinese hamster ovary cell variants defective in adhesion to fibronectin-coated collagen.

1980 ◽  
Vol 87 (3) ◽  
pp. 755-763 ◽  
Author(s):  
P A Harper ◽  
R L Juliano
Keyword(s):  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Divalent Cations ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Human Diploid Fibroblasts ◽  
Isolation And Characterization ◽  
A Cell ◽  
Cold Insoluble Globulin

Variant clones of Chinese hamster ovary (CHO) cells were selected for reduced adhesion to serum-coated tissue culture plates. These clones also displayed reduced adhesion to substrata composed of collagen layers coated with bovine serum or with fibronectin (cold-insoluble globulin). Wild-type (WT) and adhesion variant (ADv) cells grew at comparable rates in suspension culture, but the adhesion variants could not be grown in monolayer culture because of their inability to attach to the substratum. The adhesion deficit in these cells was not corrected by raising the concentration of divalent cations or of serum to levels 10-fold greater than those normally utilized in cell culture. However, both WT and ADv clones could adhere, spread, and attain a normal CHO morphology on substrata coated with concanavalin A or poly-L-lysine. In addition, the adhesion variants could attach to substrata coated with "footpad" material (substratum-attached material) derived from monolayers of human diploid fibroblasts or WT CHO cells. These observations suggest that the variant clones may have a cell surface defect that prevents them from utilizing exogeneous fibronectin as an adhesion-promoting ligand; however the variants seem to have normal cytoskeletal and metabolic capacities that allow them to attach and spread on substrata coated with alternative ligands. These variants should be extremely useful in studying the molecular basis of cell adhesion.

scholarly journals Isolation and characterization of Chinese hamster ovary cell variants deficient in the expression of fibronectin receptor.

1989 ◽  
Vol 109 (6) ◽  
pp. 3157-3167 ◽  
Author(s):  
C L Schreiner ◽  
J S Bauer ◽  
Y N Danilov ◽  
S Hussein ◽  
M M Sczekan ◽  
...  
Keyword(s):  
Chinese Hamster Ovary Cell ◽  
Chinese Hamster Ovary ◽  
Plasma Membranes ◽  
Cdna Probe ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Wild Type ◽  
Fibronectin Receptor ◽  
Alpha Chain ◽  
Isolation And Characterization

Chinese hamster ovary cell populations were enriched for cells displaying low surface expression of the 140-kD integrin fibronectin receptor (FnR) by means of fluorescence-activated cell sorting using monoclonal anti-FnR antibodies. Selected cells were cloned by limiting dilution, and the resulting clones were screened for low cell surface FnR expression by ELISA. Two multiply sorted populations gave rise to variant clones possessing approximately 20 or 2% FnR expression, respectively, compared with wild-type cells. Growth rates of the "20%" and "2%" clones on serum-coated plastic dishes were similar to that of wild-type cells. Variant cells expressing 20% FnR could attach and spread on substrata coated with purified fibronectin, although somewhat more slowly than wild-type cells, while cells expressing 2% FnR could not attach or spread. Cells from all variant clones attached normally to vitronectin substrata, but some of the 2% clones displayed altered morphology on this type of substratum. Motility assays in blind well chambers showed a correlation of movement with level of expression of FnR. The number of cells migrating in response to fibronectin was greatly reduced compared with wild-type cells for the 20% FnR variant clones, while variant clones with 2% FnR showed virtually no migratory activity. Surface labeling with 125I and immunoaffinity purification of FnR showed reduced levels of intact FnR on the plasma membranes of variants with 20% FnR, while none was detected in variants expressing 2% FnR. Nevertheless, beta subunits were detected on the surfaces of all variant clones. Immunoblots of cell lysates from wild-type cells and from both types of variant clones showed substantial amounts of FnR beta chain as well as enhanced amounts of a pre-beta moiety in the variants. alpha chain was markedly reduced in the 20% variants and essentially absent in the 2% variants, indicating that failure to assemble intact FnR in these variants was due to deficiencies of alpha chain production. Dot blots of total mRNA from a representative clone expressing 20% FnR showed reduced levels of material hybridizing to an 0.97-kb hamster FnR alpha chain cDNA probe as compared with wild type, while mRNA from a representative clone expressing 2% FnR had no detectable hybridizable RNA; this seems to agree well with the results obtained by immunoblotting. Thus, the defect in the variant clones seems to be due to reduced levels of alpha chain mRNA leading to a deficit of mature FnR and consequent alterations in cell adhesion and motility on fibronectin substrata.

scholarly journals Isolation and characterization of Chinese hamster ovary cell mutants defective in intracellular low density lipoprotein-cholesterol trafficking.

1990 ◽  
Vol 110 (2) ◽  
pp. 295-308 ◽  
Author(s):  
K M Cadigan ◽  
D M Spillane ◽  
T Y Chang
Keyword(s):  
Cholesteryl Ester ◽  
Low Density Lipoprotein ◽  
Chinese Hamster ◽  
Density Lipoprotein ◽  
Ovary Cell ◽  
Ester Synthesis ◽  
Cholesterol Trafficking ◽  
Isolation And Characterization ◽  
Mutant Cells

This paper reports the isolation and characterization of Chinese hamster ovary cell mutants defective in low density lipoprotein (LDL)-cholesterol trafficking. The parental cell line was 25-RA, which possesses LDL receptors and various cholesterogenic enzyme activities that are partially resistant to down regulation by exogenous sterols (Chang, T. Y., and J. S. Limanek. 1980. J. Biol. Chem. 255:7787-7795). Because these cells accumulate a large amount of intracellular cholesteryl ester when grown in medium containing 10% fetal calf serum, mutagenized populations of 25-RA cells were grown in the presence of a specific inhibitor of acyl-coenzyme A: cholesterol acyltransferase (ACAT), which depleted their cholesteryl ester stores. Without this cholesterol ester storage, 99% of 25-RA cells die after 5-d growth in cholesterol starvation medium, while the mutant cells, which accumulate free cholesterol intracellularly, survived. In two mutant clones chosen for characterization, activation of cholesteryl ester synthesis by LDL was markedly reduced in the mutant cells compared with 25-RA cells. This lack of activation of cholesterol ester synthesis in the mutant cells could not be explained by defective uptake and/or processing of LDL or by a decreased amount of ACAT, as determined by in vitro enzyme activity. Mutant cells grown in the presence of LDL contain numerous cytosolic particles that stain intensely with the fluorescent compound acridine orange, suggesting that they are acidic. The particles are also stained with filipin, a cholesterol-specific fluorescent dye. Indirect immunofluorescence with a monoclonal antibody specific for a lysosomal/endosomal fraction revealed a staining pattern that colocalized with the filipin signal. The mutant phenotype was recessive. The available evidence indicates that the mutant cells can take up and process LDL normally, but the hydrolyzed cholesterol accumulates in an acidic compartment, probably the lysosomes, where it can not be transported to its normal intracellular destinations.

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