scholarly journals Stepwise activation of the mouse acetylcholine receptor delta- and gamma-subunit genes in clonal cell lines.

1988 ◽  
Vol 8 (12) ◽  
pp. 5257-5267 ◽  
Author(s):  
C M Crowder ◽  
J P Merlie
Keyword(s):  
Acetylcholine Receptor ◽  
Cell Lines ◽  
Subunit Gene ◽  
Rnase Protection ◽  
Gamma Subunit ◽  
Clonal Cell ◽  
Hypersensitive Sites ◽  
Clonal Cell Lines ◽  
Gamma Locus ◽  
Delta Subunit

We used the DNase I-hypersensitive sites around the mouse acetylcholine receptor delta-subunit gene as a guide toward the cloning and sequencing of delta and gamma transcriptional regulatory regions and as a means to assess chromatin structural activation of the delta- and gamma-subunit genes during myogenesis. Genomic cloning of hypersensitive sites downstream of the delta-subunit gene revealed the presence of the gamma-subunit gene approximately 5 kilobases away; the hypersensitive sites mapped to the 5' end of the gamma-subunit gene. Sequence comparison of restriction fragments containing hypersensitive sites in analogous locations at the 5' ends of the delta- and gamma-subunit genes uncovered little overall homology between the two genomic fragments; however, an 11- of 13-base-pair match between the two sequences was found. Homologs to this sequence were also found to be present in the upstream regions of the chicken alpha- and mouse beta-subunit genes. By RNase protection and primer extension analyses, the delta-subunit gene transcription start site was mapped to 56 base pairs upstream of the initiator ATG codon. Clonal cell lines with various potentials to differentiate to the skeletal muscle phenotype were examined for their hypersensitive site pattern within the delta-gamma locus. Only remote hypersensitive sites flanking the locus appear in pluripotential mesodermal cells. A cell line of determined but inducible myoblasts expressed only one more intergenic site, while in permissively differentiating myoblasts hypersensitive sites were already present at the 5' ends of the delta and gamma genes prior to differentiation. Terminal differentiation resulted in an identical pattern of hypersensitive sites in all muscle cell lines examined so far, with an intergenic site near the gamma-subunit gene being the only site specific to the differentiated muscle phenotype.

Stepwise activation of the mouse acetylcholine receptor delta- and gamma-subunit genes in clonal cell lines

1988 ◽  
Vol 8 (12) ◽  
pp. 5257-5267
Author(s):  
C M Crowder ◽  
J P Merlie
Keyword(s):  
Acetylcholine Receptor ◽  
Cell Lines ◽  
Subunit Gene ◽  
Rnase Protection ◽  
Gamma Subunit ◽  
Clonal Cell ◽  
Hypersensitive Sites ◽  
Clonal Cell Lines ◽  
Gamma Locus ◽  
Delta Subunit

We used the DNase I-hypersensitive sites around the mouse acetylcholine receptor delta-subunit gene as a guide toward the cloning and sequencing of delta and gamma transcriptional regulatory regions and as a means to assess chromatin structural activation of the delta- and gamma-subunit genes during myogenesis. Genomic cloning of hypersensitive sites downstream of the delta-subunit gene revealed the presence of the gamma-subunit gene approximately 5 kilobases away; the hypersensitive sites mapped to the 5' end of the gamma-subunit gene. Sequence comparison of restriction fragments containing hypersensitive sites in analogous locations at the 5' ends of the delta- and gamma-subunit genes uncovered little overall homology between the two genomic fragments; however, an 11- of 13-base-pair match between the two sequences was found. Homologs to this sequence were also found to be present in the upstream regions of the chicken alpha- and mouse beta-subunit genes. By RNase protection and primer extension analyses, the delta-subunit gene transcription start site was mapped to 56 base pairs upstream of the initiator ATG codon. Clonal cell lines with various potentials to differentiate to the skeletal muscle phenotype were examined for their hypersensitive site pattern within the delta-gamma locus. Only remote hypersensitive sites flanking the locus appear in pluripotential mesodermal cells. A cell line of determined but inducible myoblasts expressed only one more intergenic site, while in permissively differentiating myoblasts hypersensitive sites were already present at the 5' ends of the delta and gamma genes prior to differentiation. Terminal differentiation resulted in an identical pattern of hypersensitive sites in all muscle cell lines examined so far, with an intergenic site near the gamma-subunit gene being the only site specific to the differentiated muscle phenotype.

scholarly journals Reduction of Target Gene Expression by a Modified U1 snRNA

2001 ◽  
Vol 21 (8) ◽  
pp. 2815-2825 ◽  
Author(s):  
S. A. Beckley ◽  
P. Liu ◽  
M. L. Stover ◽  
S. I. Gunderson ◽  
A. C. Lichtler ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Target Gene ◽  
Fluorescent Protein ◽  
Stable Transfection ◽  
Rnase Protection ◽  
Intact Cells ◽  
Clonal Cell ◽  
U1 Snrna ◽  
Clonal Cell Lines

ABSTRACT Although the primary function of U1 snRNA is to define the 5′ donor site of an intron, it can also block the accumulation of a specific RNA transcript when it binds to a donor sequence within its terminal exon. This work was initiated to investigate if this property of U1 snRNA could be exploited as an effective method for inactivating any target gene. The initial 10-bp segment of U1 snRNA, which is complementary to the 5′ donor sequence, was modified to recognize various target mRNAs (chloramphenicol acetyltransferase [CAT], β-galactosidase, or green fluorescent protein [GFP]). Transient cotransfection of reporter genes and appropriate U1 antitarget vectors resulted in >90% reduction of transgene expression. Numerous sites within the CAT transcript were suitable for targeting. The inhibitory effect of the U1 antitarget vector is directly related to the hybrid formed between the U1 vector and target transcripts and is dependent on an intact 70,000-molecular-weight binding domain within the U1 gene. The effect is long lasting when the target (CAT or GFP) and U1 antitarget construct are inserted into fibroblasts by stable transfection. Clonal cell lines derived from stable transfection with a pOB4GFP target construct and subsequently stably transfected with the U1 anti-GFP construct were selected. The degree to which GFP fluorescence was inhibited by U1 anti-GFP in the various clonal cell lines was assessed by fluorescence-activated cell sorter analysis. RNA analysis demonstrated reduction of the GFP mRNA in the nuclear and cytoplasmic compartment and proper 3′ cleavage of the GFP residual transcript. An RNase protection strategy demonstrated that the transfected U1 antitarget RNA level varied between 1 to 8% of the endogenous U1 snRNA level. U1 antitarget vectors were demonstrated to have potential as effective inhibitors of gene expression in intact cells.

scholarly journals Regulation of acetylcholine receptor transcript expression during development in Xenopus laevis

1988 ◽  
Vol 106 (2) ◽  
pp. 469-478 ◽  
Author(s):  
TJ Baldwin ◽  
CM Yoshihara ◽  
K Blackmer ◽  
CR Kintner ◽  
SJ Burden
Keyword(s):  
Xenopus Laevis ◽  
Acetylcholine Receptor ◽  
Alpha Subunit ◽  
Cdna Libraries ◽  
Rnase Protection ◽  
Gamma Subunit ◽  
Transcript Levels ◽  
Actin Mrna ◽  
Regulatory Controls ◽  
Delta Subunit

The level of transcripts encoding the skeletal muscle acetylcholine receptor (AChR) was determined during embryonic development in Xenopus laevis. cDNAs encoding the alpha, gamma, and delta subunits of the Xenopus AChR were isolated from Xenopus embryo cDNA libraries using Torpedo AChR cDNAs as probes. The Xenopus AChR cDNAs have greater than 60% amino acid sequence homology to their Torpedo homologues and hybridize to transcripts that are restricted to the somites of developing embryos. Northern blot analysis demonstrates that a 2.3-kb transcript hybridizes to the alpha subunit cDNA, a 2.4-kb transcript hybridizes to the gamma subunit cDNA, and that two transcripts, of 1.9 and 2.5 kb, hybridize to the delta subunit cDNA. RNase protection assays demonstrate that transcripts encoding alpha, gamma, and delta subunits are coordinately expressed at late gastrula and that the amount of each transcript increases in parallel with muscle-specific actin mRNA during the ensuing 12 h. After the onset of muscle activity the level of actin mRNA per somite remains relatively constant, whereas the level of alpha subunit and delta subunit transcripts decrease fourfold per somite and the level of gamma subunit transcript decreases greater than 50-fold per somite. The decrease in amount of AChR transcripts per somite, however, occurs when embryos are paralyzed with local anaesthetic during their development. These results demonstrate that AChR transcripts in Xenopus are initially expressed coordinately, but that gamma subunit transcript levels are regulated differently than alpha and delta at later stages. Moreover, these results demonstrate that AChR transcript levels in Xenopus myotomal muscle cells are not responsive to electrical activity and suggest that AChR transcript levels are influenced by other regulatory controls.

ESTABLISHMENT AND CHARACTERIZATION OF CLONAL CELL LINES FROM THE VAGINA OF p53-DEFICIENT YOUNG MICE

2002 ◽  
Vol 38 (10) ◽  
pp. 547 ◽  
Author(s):  
KAYO TANAHASHI ◽  
SHINOBU SHIBAHARA ◽  
MINAKO OGAWA ◽  
MAKOTO HANAZONO ◽  
SHINICHI AIZAWA ◽  
...  
Keyword(s):  
Cell Lines ◽  
Clonal Cell ◽  
Clonal Cell Lines ◽  
Young Mice

Implantation of c-mycERTAMImmortalized Human Mesencephalic-Derived Clonal Cell Lines Ameliorates Behavior Dysfunction in a Rat Model of Parkinson’s Disease

2009 ◽  
Vol 18 (2) ◽  
pp. 307-320 ◽  
Author(s):  
Erik A. Miljan ◽  
Susan J. Hines ◽  
Priyadarshini Pande ◽  
Randolph L. Corteling ◽  
Caroline Hicks ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Cell Lines ◽  
Rat Model ◽  
Clonal Cell ◽  
Clonal Cell Lines
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