The stress protein ERp57/GRP58 binds specific DNA sequences in HeLa cells

2006 ◽  
Vol 210 (2) ◽  
pp. 343-351 ◽  
Author(s):  
Silvia Chichiarelli ◽  
Anna Ferraro ◽  
Fabio Altieri ◽  
Margherita Eufemi ◽  
Sabina Coppari ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Hela Cells ◽  
Stress Protein

scholarly journals Specific growth inhibitory sequences in genomic DNA from quiescent human embryo fibroblasts.

1987 ◽  
Vol 7 (5) ◽  
pp. 1894-1899 ◽  
Author(s):  
R Padmanabhan ◽  
T H Howard ◽  
B H Howard
Keyword(s):  
Dna Sequences ◽  
Hela Cells ◽  
Human Embryo ◽  
Inhibitory Activity ◽  
Genomic Dna ◽  
Molecular Mechanisms ◽  
Growth Inhibitory ◽  
Growth Inhibitory Activity ◽  
Embryo Fibroblasts

We used HeLa cells as recipients in a gene transfer assay to characterize DNA sequences that negatively regulate mammalian cell growth. In this assay, genomic DNA from quiescent human embryo fibroblasts was more inhibitory for HeLa replication than was DNA from either Escherichia coli or HeLa cells. Surprisingly, growth inhibitory activity depended on the growth state of the cells from which genomic DNA was prepared; it was strongest in DNA prepared from serum-deprived, quiescent embryo fibroblasts. This latter observation implies a role for DNA modification(s) in regulating the activity of the inhibitory sequences detected in our assay. The level of the observed growth inhibitory activity was sometimes high, suggesting that the relevant sequences may be abundantly represented in the mammalian genome. We speculate that these findings may provide new insights into the molecular mechanisms involved in cellular quiescence and in vitro senescence.

scholarly journals Expression of amplified DNA sequences for ornithine transcarbamylase in HeLa cells: arginine residues may be required for mitochondrial import of enzyme precursor.

1985 ◽  
Vol 100 (5) ◽  
pp. 1515-1521 ◽  
Author(s):  
A L Horwich ◽  
W A Fenton ◽  
F A Firgaira ◽  
J E Fox ◽  
D Kolansky ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Hela Cells ◽  
Regulatory Elements ◽  
Ornithine Transcarbamylase ◽  
Mitochondrial Localization ◽  
Mitochondrial Import ◽  
Cdna Sequences ◽  
Cell Extracts ◽  
Arginine Residues ◽  
Sequence Encoding

Expression of ornithine transcarbamylase (OTC), a nuclear-coded mitochondrial enzyme, was programmed in HeLa cells by the use of a strategy of gene co-amplification. HeLa cells, ordinarily devoid of OTC activity, were transfected with a plasmid containing viral regulatory elements joined with two cDNA sequences, one encoding the human OTC precursor and a second encoding a mutant mouse dihydrofolate reductase. After transfection and selection in increasing concentrations of methotrexate, several hundred copies per cell of the sequence encoding OTC were detected by blot analysis. Immunoprecipitation of extracts of radiolabeled cells with anti-OTC antiserum revealed newly synthesized mature OTC subunits. Furthermore, OTC enzymatic activity in cell extracts was comparable to that of control human liver, and mitochondrial localization of OTC was demonstrated by immunofluorescence. When we incubated transfected HeLa cells with dinitrophenol, a known inhibitor of mitochondrial import, the only form of newly synthesized OTC detected was the precursor. We estimated the rate of import of precursor by performing an inhibitor-free chase; precursor was converted to mature subunit with a half-life of less than two minutes. When a HeLa transformant was incubated with the arginine analogue canavanine, the major form of newly synthesized OTC detected was a species migrating slightly more slowly than the normal precursor; little mature-sized subunit was recovered. This indicates that substitution of the analogue for arginine in the OTC precursor interferes with mitochondrial import and processing. Thus, arginine residues in the OTC precursor--most likely the four residues contained in its NH2-terminal leader sequence--probably play an important role in mitochondrial import and/or processing.

Amplification of DNA sequences coding for the Na,K-ATPase alpha-subunit in ouabain-resistant C+ cells

1986 ◽  
Vol 6 (7) ◽  
pp. 2476-2481
Author(s):  
J R Emanuel ◽  
S Garetz ◽  
J Schneider ◽  
J F Ash ◽  
E J Benz ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Hela Cells ◽  
Restriction Analysis ◽  
Mrna Levels ◽  
C Cells ◽  
Human Dna ◽  
Gene Coding ◽  
Atpase Gene ◽  
Marked Decline ◽  
Membrane Bound

We have studied the mechanism of cellular resistance to cardiac glycosides in C+ cells. C+ cells were resistant to ouabain and overproduced plasma membrane-bound Na,K-ATPase relative to parental HeLa cells. Overexpression of Na,K-ATPase in C+ cells correlated with increased ATPase mRNA levels and amplification (approximately 100 times) of the ATPase gene. Growth of C+ cells in ouabain-free medium resulted in a marked decline in ATPase mRNA and DNA levels. However, when cells were reexposed to ouabain, they proliferated and ATPase mRNA and DNA sequences were reamplified. Restriction analysis of C+ and other human DNA samples revealed the occurrence of rearrangements in the region of the Na,K-ATPase gene in C+ cells. Furthermore, C+ cells expressed an ATPase mRNA species not found in HeLa cells. These results suggest that amplification of the gene coding for Na,K-ATPase results in overproduction of Na,K-ATPase polypeptides. Amplification of the ATPase gene or the expression of new ATPase mRNA sequences or both may also be responsible for acquisition of the ouabain-resistant phenotype.

Mouse p53 represses the rat brain creatine kinase gene but activates the rat muscle creatine kinase gene

1994 ◽  
Vol 14 (12) ◽  
pp. 8483-8492
Author(s):  
J Zhao ◽  
F I Schmieg ◽  
D T Simmons ◽  
G R Molloy
Keyword(s):  
Creatine Kinase ◽  
Dna Sequences ◽  
Hela Cells ◽  
Lung Tumor ◽  
P53 Protein ◽  
Wild Type Mouse ◽  
High Energy ◽  
Wild Type ◽  
Kinase Gene

The creatine kinases (CK) regenerate ATP for cellular reactions with a high energy expenditure. While muscle CK (CKM) is expressed almost exclusively in adult skeletal and cardiac muscle, brain CK (CKB) expression is more widespread and is highest in brain glial cells. CKB expression is also high in human lung tumor cells, many of which contain mutations in p53 alleles. We have recently detected high levels of CKB mRNA in HeLa cells and, in this study, have tested whether this may be due to the extremely low amounts of p53 protein present in HeLa cells. Transient transfection experiments showed that wild-type mouse p53 severely repressed the rat CKB promoter in HeLa but not CV-1 monkey kidney cells, suggesting that, in HeLa but not CV-1 cells, p53 either associates with a required corepressor or undergoes a posttranslational modification necessary for CKB repression. Conversely, mouse wild-type p53 strongly activated the rat CKM promoter in CV-1 cells but not in HeLa cells, suggesting that, in CV-1 cells, p53 may associate with a required coactivator or is modified in a manner necessary for CKM activation. The DNA sequences required for p53-mediated modulations were found to be within bp -195 to +5 of the CKB promoter and within bp -168 to -97 of the CKM promoter. Moreover, a 112-bp fragment from the proximal rat CKM promoter (bp -168 to -57), which contained five degenerate p53-binding elements, was capable of conferring p53-mediated activation on a heterologous promoter in CV-1 cells. Also, this novel p53 sequence, when situated in the native 168-bp rat CKM promoter, conferred p53-mediated activation equal to or greater than that of the originally characterized far-upstream (bp -3160) mouse CKM p53 element. Therefore, CKB and CKM may be among the few cellular genes which could be targets of p53 in vivo. In addition, we analyzed a series of missense mutants with alterations in conserved region II of p53. Mutations affected p53 transrepression and transactivation activities differently, indicating that these activities in p53 are separable. The ability of p53 mutants to transactivate correlated well with their ability to inhibit transformation of rat embryonic fibroblasts by adenovirus E1a and activated Ras.

The human prointerleukin 1 beta gene requires DNA sequences both proximal and distal to the transcription start site for tissue-specific induction

1993 ◽  
Vol 13 (3) ◽  
pp. 1332-1344 ◽  
Author(s):  
F Shirakawa ◽  
K Saito ◽  
C A Bonagura ◽  
D L Galson ◽  
M J Fenton ◽  
...  
Keyword(s):  
Transcription Start Site ◽  
Phorbol Myristate Acetate ◽  
Dna Sequences ◽  
Hela Cells ◽  
Start Site ◽  
Myristate Acetate ◽  
Binding Studies ◽  
Transcription Start ◽  
Specific Induction ◽  
Beta Gene

In these studies, we have identified DNA sequences and specific protein interactions necessary for transcriptional regulation of the human prointerleukin 1 beta (proIL-1 beta) gene. A cell-type-independent lipopolysaccharide (LPS)-responsive enhancer element located between -3757 and -2729 bp upstream from the transcription start site (cap site) consisted of at least six discrete subregions which were essential to the maximal induction by LPS in transfected monocytes. The enhancer also appeared to mediate phorbol myristate acetate induction in monocytes and IL-1 responsiveness in fibroblasts. Deletion and base substitution mutations along with DNA binding studies demonstrated that the enhancer contained a minimum of three functional protein binding sequences, two of which appeared to be important for gene induction. One of the essential proteins which bound to the enhancer was similar or identical to members of the C/EBP family of transcription factors required for both IL-1- and LPS-specific induction of the IL-6 gene (i.e., the NF-IL6 proteins). When ligated to the proIL-1 beta cap site-proximal region (located between -131 to +12), both the proIL-1 beta and the simian virus 40 enhancer elements functioned more efficiently in monocytes than in HeLa cells, which are not normally competent for IL-1 beta expression. When ligated to the murine c-fos promoter, however, the proIL-1 beta enhancer was inducible in phorbol myristate acetate-stimulated HeLa cells, suggesting the existence of a proIL-1 beta promoter-proximal requirement for tissue specificity.

scholarly journals Cloning of plasmid DNA sequences involved in invasion of HeLa cells by Shigella flexneri.

1985 ◽  
Vol 49 (1) ◽  
pp. 164-171 ◽  
Author(s):  
A T Maurelli ◽  
B Baudry ◽  
H d'Hauteville ◽  
T L Hale ◽  
P J Sansonetti
Keyword(s):  
Dna Sequences ◽  
Hela Cells ◽  
Plasmid Dna ◽  
Shigella Flexneri

scholarly journals Random isolation of gene activator elements from the human genome.

1986 ◽  
Vol 6 (12) ◽  
pp. 4185-4194 ◽  
Author(s):  
H Hamada
Keyword(s):  
Human Genome ◽  
Dna Sequences ◽  
Hela Cells ◽  
Gene Activation ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Enhancer Trap ◽  
Cell Type Specificity ◽  
Transient Expression Assay ◽  
Enhancer Activity

Long-range-acting gene activator elements were randomly isolated from the human genome by functional selection. HeLa cells were transfected with an enhancer trap, a plasmid containing an enhancerless xanthine-guanosine phosphoribosyltransferase (gpt) gene transcribed from the simian virus 40 early promoter, and stably transformed GPT+ cells were selected. From several transformants, human DNA sequences flanking the enhancer trap were cloned. Two gene activators (GA1 and GA2) were found in the cloned human DNAs. GA1 and GA2 showed strong enhancer activity both in a stable transformation assay and in a transient expression assay. They had functional properties similar to those of other known enhancers: GA1 and GA2 activated the expression of a linked gene over distances of at least 5 kilobases both upstream and downstream in an orientation-independent fashion. GA1 may be required for the initial establishment of gene activation but was not essential for the maintenance of active expression. GA1 and GA2 were active not only in HeLa cells but also in other types of human cells, such as neuroblastoma cells. This indicates a limited but relatively broad cell type specificity. The HeLa genome contains multiple copies of GA1, while GA2 exists once in the genome.

scholarly journals The human prointerleukin 1 beta gene requires DNA sequences both proximal and distal to the transcription start site for tissue-specific induction.

1993 ◽  
Vol 13 (3) ◽  
pp. 1332-1344 ◽  
Author(s):  
F Shirakawa ◽  
K Saito ◽  
C A Bonagura ◽  
D L Galson ◽  
M J Fenton ◽  
...  
Keyword(s):  
Transcription Start Site ◽  
Phorbol Myristate Acetate ◽  
Dna Sequences ◽  
Hela Cells ◽  
Start Site ◽  
Myristate Acetate ◽  
Binding Studies ◽  
Transcription Start ◽  
Specific Induction ◽  
Beta Gene

In these studies, we have identified DNA sequences and specific protein interactions necessary for transcriptional regulation of the human prointerleukin 1 beta (proIL-1 beta) gene. A cell-type-independent lipopolysaccharide (LPS)-responsive enhancer element located between -3757 and -2729 bp upstream from the transcription start site (cap site) consisted of at least six discrete subregions which were essential to the maximal induction by LPS in transfected monocytes. The enhancer also appeared to mediate phorbol myristate acetate induction in monocytes and IL-1 responsiveness in fibroblasts. Deletion and base substitution mutations along with DNA binding studies demonstrated that the enhancer contained a minimum of three functional protein binding sequences, two of which appeared to be important for gene induction. One of the essential proteins which bound to the enhancer was similar or identical to members of the C/EBP family of transcription factors required for both IL-1- and LPS-specific induction of the IL-6 gene (i.e., the NF-IL6 proteins). When ligated to the proIL-1 beta cap site-proximal region (located between -131 to +12), both the proIL-1 beta and the simian virus 40 enhancer elements functioned more efficiently in monocytes than in HeLa cells, which are not normally competent for IL-1 beta expression. When ligated to the murine c-fos promoter, however, the proIL-1 beta enhancer was inducible in phorbol myristate acetate-stimulated HeLa cells, suggesting the existence of a proIL-1 beta promoter-proximal requirement for tissue specificity.

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