scholarly journals Regulated expression of a chimeric histone gene introduced into mouse fibroblasts.

1985 ◽  
Vol 5 (9) ◽  
pp. 2316-2324 ◽  
Author(s):  
R B Alterman ◽  
C Sprecher ◽  
R Graves ◽  
W F Marzluff ◽  
A I Skoultchi
Keyword(s):  
Histone Gene ◽  
Chimeric Gene ◽  
Regulatory Sequences ◽  
Mrna Levels ◽  
Histone Genes ◽  
Mouse Fibroblasts ◽  
Cis Acting ◽  
Regulated Expression ◽  
Chimeric Rna ◽  
The Stability

The regulated expression of a mouse histone gene was studied by DNA-mediated gene transfer. A chimeric H3 histone gene was constructed by fusing the 5' and 3' portions of two different mouse H3 histone genes. Transfection of the chimeric gene into mouse fibroblasts resulted in the production of chimeric mRNA at levels nearly equal to that of the total endogenous H3 histone mRNAs. Most chimeric RNA transcripts had correct 5' and 3' termini, and the chimeric mRNA was translated into an H3.1 protein that accumulated in the nucleus of the transfected cells. Expression of the chimeric gene was studied under several conditions in which the rate of transcription and the stability of endogenous H3 transcripts change. Chimeric mRNA levels were regulated in parallel with endogenous H3 mRNAs, suggesting that cis-acting regulatory sequences lie within or near individual histone genes. In addition to correctly initiated and terminated chimeric mRNA, we also detected a novel H3 transcript containing an additional 250 bases at the 3' end. Surprisingly, the longer transcript is polyadenylated and accumulates in the cytoplasm.

Regulated expression of a chimeric histone gene introduced into mouse fibroblasts

1985 ◽  
Vol 5 (9) ◽  
pp. 2316-2324
Author(s):  
R B Alterman ◽  
C Sprecher ◽  
R Graves ◽  
W F Marzluff ◽  
A I Skoultchi
Keyword(s):  
Histone Gene ◽  
Chimeric Gene ◽  
Regulatory Sequences ◽  
Mrna Levels ◽  
Histone Genes ◽  
Mouse Fibroblasts ◽  
Cis Acting ◽  
Regulated Expression ◽  
Chimeric Rna ◽  
The Stability

The regulated expression of a mouse histone gene was studied by DNA-mediated gene transfer. A chimeric H3 histone gene was constructed by fusing the 5' and 3' portions of two different mouse H3 histone genes. Transfection of the chimeric gene into mouse fibroblasts resulted in the production of chimeric mRNA at levels nearly equal to that of the total endogenous H3 histone mRNAs. Most chimeric RNA transcripts had correct 5' and 3' termini, and the chimeric mRNA was translated into an H3.1 protein that accumulated in the nucleus of the transfected cells. Expression of the chimeric gene was studied under several conditions in which the rate of transcription and the stability of endogenous H3 transcripts change. Chimeric mRNA levels were regulated in parallel with endogenous H3 mRNAs, suggesting that cis-acting regulatory sequences lie within or near individual histone genes. In addition to correctly initiated and terminated chimeric mRNA, we also detected a novel H3 transcript containing an additional 250 bases at the 3' end. Surprisingly, the longer transcript is polyadenylated and accumulates in the cytoplasm.

DNA-mediated transformation in Dictyostelium discoideum: regulated expression of an actin gene fusion

1984 ◽  
Vol 4 (12) ◽  
pp. 2890-2898
Author(s):  
W Nellen ◽  
C Silan ◽  
R A Firtel
Keyword(s):  
Dictyostelium Discoideum ◽  
Gene Fusion ◽  
Regulatory Sequences ◽  
Control Of Gene Expression ◽  
Cis Acting ◽  
Regulated Expression ◽  
Promoter Gene ◽  
Flanking Region ◽  
Average Copy ◽  
Flanking Regions

We have constructed a new vector for transformation that carries a fusion of the Dictyostelium discoideum actin 6 promoter gene and 5' flanking region with the bacterial Tn5 NeoR (KanR) gene which can confer resistance to the aminoglycoside G418. This vector can be used to transform D. discoideum cells. Approximately 200 to 2,000 transformants were obtained per 10(7) cells. Transformed cell populations carried vector DNA at an average copy number of ca. 5 per cell, and the DNA was stable for more than 40 generations in the absence of selection. We have shown that transformed cells synthesize functional kanamycin phosphotransferase and that initiation of transcription of the actin 6-NeoR gene fusion occurs at the actin 6 cap site. Moreover, analysis of RNA isolated from transformed and untransformed cells during vegetative growth and during development indicated that the actin 6-NeoR gene fusion was regulated in parallel with the endogenous actin 6 gene, suggesting that the upstream flanking regions of actin 6 contain the cis-acting regulatory sequences sufficient for differential regulation of this gene during D. discoideum development. These results indicate that this system can be used to examine control of gene expression during D. discoideum development.

scholarly journals DNA-mediated transformation in Dictyostelium discoideum: regulated expression of an actin gene fusion.

1984 ◽  
Vol 4 (12) ◽  
pp. 2890-2898 ◽  
Author(s):  
W Nellen ◽  
C Silan ◽  
R A Firtel
Keyword(s):  
Dictyostelium Discoideum ◽  
Gene Fusion ◽  
Regulatory Sequences ◽  
Control Of Gene Expression ◽  
Cis Acting ◽  
Regulated Expression ◽  
Promoter Gene ◽  
Flanking Region ◽  
Average Copy ◽  
Flanking Regions

We have constructed a new vector for transformation that carries a fusion of the Dictyostelium discoideum actin 6 promoter gene and 5' flanking region with the bacterial Tn5 NeoR (KanR) gene which can confer resistance to the aminoglycoside G418. This vector can be used to transform D. discoideum cells. Approximately 200 to 2,000 transformants were obtained per 10(7) cells. Transformed cell populations carried vector DNA at an average copy number of ca. 5 per cell, and the DNA was stable for more than 40 generations in the absence of selection. We have shown that transformed cells synthesize functional kanamycin phosphotransferase and that initiation of transcription of the actin 6-NeoR gene fusion occurs at the actin 6 cap site. Moreover, analysis of RNA isolated from transformed and untransformed cells during vegetative growth and during development indicated that the actin 6-NeoR gene fusion was regulated in parallel with the endogenous actin 6 gene, suggesting that the upstream flanking regions of actin 6 contain the cis-acting regulatory sequences sufficient for differential regulation of this gene during D. discoideum development. These results indicate that this system can be used to examine control of gene expression during D. discoideum development.

scholarly journals Regulation of expression of the human fructose transporter (GLUT5) by cyclic AMP

1994 ◽  
Vol 301 (1) ◽  
pp. 169-175 ◽  
Author(s):  
L Mahraoui ◽  
J Takeda ◽  
J Mesonero ◽  
I Chantret ◽  
E Dussaulx ◽  
...  
Keyword(s):  
Cyclic Amp ◽  
Reporter Gene ◽  
Cancer Cell Line ◽  
Human Colon ◽  
Colon Cancer Cell Line ◽  
Regulatory Sequences ◽  
Mrna Levels ◽  
Regulation Of Expression ◽  
Human Colon Cancer ◽  
Cis Acting

The effect of cyclic AMP on the expression of the fructose transporter, GLUT5, was studied in Caco-2 cells, a human colon cancer cell line that differentiates spontaneously in culture into cells with the properties of small intestine enterocytes. Treatment of differentiated Caco-2 cells with 50 microM forskolin, which stimulates adenylate cyclase and raises intracellular cyclic AMP levels, increased fructose uptake 2-fold and raised GLUT5 protein and mRNA levels 5- and 7-fold respectively. The increased GLUT5 mRNA levels in forskolin-treated cells are a result of stabilization of GLUT5 mRNA in these cells and increased transcription. The effect of cyclic AMP on GLUT5 transcription was assessed by measuring the activity of human GLUT5 promoter-reporter gene constructs in forskolin-treated differentiated Caco-2 cells. The results showed that forskolin stimulated the activity of the GLUT5-reporter gene constructs and this stimulatory effect was mediated by cis-acting regulatory sequences.

Expression of an amylase - alcohol dehydrogenase chimeric gene in transgenic strains of Drosophila melanogaster

Genome ◽  
1993 ◽  
Vol 36 (5) ◽  
pp. 954-961 ◽  
Author(s):  
Allan A. Grunder ◽  
Ada Loverre-Chyurlia ◽  
Donal A. Hickey
Keyword(s):  
Drosophila Melanogaster ◽  
Alcohol Dehydrogenase ◽  
Dna Analysis ◽  
Glucose Repression ◽  
Chimeric Gene ◽  
P Element ◽  
Regulatory Sequences ◽  
Mrna Levels ◽  
Promoter Sequences ◽  
Adh Gene

A chimeric gene, consisting of 428 bp of the promoter sequences of the α-amylase gene of Drosophila melanogaster, fused to the transcribed region of the alcohol dehydrogenase (Adh) gene, was introduced into the genome of an Adhnull stock of Drosophila via P element mediated transformation. DNA analysis (Southern blotting) of three transformant strains confirmed the insertion of either one or two copies of the chimeric gene per strain. A histochemical study of ADH enzyme activity in dissected tissues of the transgenic larvae revealed that the chimeric Amy–Adh gene was expressed only in the posterior larval midgut and that this expression was repressed by dietary glucose, thus representing an expression pattern characteristic of the Amy gene. This indicates that the Amy upstream promoter sequences contain signals mediating both tissue specificity and glucose repression of the Adh structural gene in the transgenic larvae. The level of ADH activity expressed in transgenic flies was relatively low. This was paralleled by a low level of Adh mRNA, indicating a reduction in the transcriptional rate of the chimeric gene.Key words: Drosophila, germline transformation, chimeric gene, cis-regulatory sequences, α-amylase, alcohol dehydrogenase, tissue-specific expression, glucose repression, mRNA levels.

scholarly journals A Review of Methods to Monitor the Modulation of mRNA Stability

2010 ◽  
Vol 15 (6) ◽  
pp. 609-622 ◽  
Author(s):  
Dominique Cheneval ◽  
Tania Kastelic ◽  
Peter Fuerst ◽  
Christian N. Parker
Keyword(s):  
Mrna Stability ◽  
Posttranscriptional Regulation ◽  
Regulation Of Gene Expression ◽  
Translational Efficiency ◽  
Mrna Levels ◽  
Cis Acting ◽  
Novel Approach ◽  
Specific Mrnas ◽  
Steady State Levels ◽  
The Stability

Posttranscriptional regulation of gene expression is an elaborate and intricate process, constituting an important mechanism for the control of protein expression. During its existence, mRNA is escorted by proteins and other RNAs, which control the maturation, transportation, localization, translational efficiency, and ultimately its degradation. Without changes at the transcription level, mRNA steady-state levels can vary dramatically by just small changes in mRNA stability. By influencing the metabolism of specific mRNAs, the abundance of specific mRNAs can be controlled in organisms from bacteria to mammals. In eukaryotic cells, the control of mRNA stability is exerted through specific cis-acting elements (sequence-specific control elements) and trans-acting factors (mRNA binding proteins and some miRNAs). mRNA stability appears to be a key regulator in controlling the expression of many proteins. Dysregulation of mRNA stability has been associated with human diseases, including cancer, inflammatory disease, and Alzheimer’s. These observations suggest that modulating the stability of specific mRNAs may represent a viable strategy for pharmaceutical intervention. The literature already describes several compounds that influence mRNA stability. Measuring mRNA stability by conventional methods is labor intensive and time-consuming. However, several systems have been described that can be used to screen for modulators of mRNA levels in a high-throughput format. Thus, these assay systems offer a novel approach for screening targets that at present appear to be poorly “drugable.” This review describes the utility of mRNA stability as a novel approach to drug discovery, focusing on assay methods and tool compounds available to monitor mRNA stability. The authors describe mRNA stability assays and issues related to this approach.

scholarly journals DBC1 (Deleted in Breast Cancer 1) modulates the stability and function of the nuclear receptor Rev-erbα

2013 ◽  
Vol 451 (3) ◽  
pp. 453-461 ◽  
Author(s):  
Claudia C. S. Chini ◽  
Carlos Escande ◽  
Veronica Nin ◽  
Eduardo N. Chini
Keyword(s):  
Breast Cancer ◽  
Nuclear Receptor ◽  
Clock Genes ◽  
Mrna Levels ◽  
Protein Levels ◽  
The Stability ◽  
And Function ◽  
Interfering Rna ◽  
In Cells

The nuclear receptor Rev-erbα has been implicated as a major regulator of the circadian clock and integrates circadian rhythm and metabolism. Rev-erbα controls circadian oscillations of several clock genes and Rev-erbα protein degradation is important for maintenance of the circadian oscillations and also for adipocyte differentiation. Elucidating the mechanisms that regulate Rev-erbα stability is essential for our understanding of these processes. In the present paper, we report that the protein DBC1 (Deleted in Breast Cancer 1) is a novel regulator of Rev-erbα. Rev-erbα and DBC1 interact in cells and in vivo, and DBC1 modulates the Rev-erbα repressor function. Depletion of DBC1 by siRNA (small interfering RNA) in cells or in DBC1-KO (knockout) mice produced a marked decrease in Rev-erbα protein levels, but not in mRNA levels. In contrast, DBC1 overexpression significantly enhanced Rev-erbα protein stability by preventing its ubiquitination and degradation. The regulation of Rev-erbα protein levels and function by DBC1 depends on both the N-terminal and C-terminal domains of DBC1. More importantly, in cells depleted of DBC1, there was a dramatic decrease in circadian oscillations of both Rev-erbα and BMAL1. In summary, our data identify DBC1 as an important regulator of the circadian receptor Rev-erbα and proposes that Rev-erbα could be involved in mediating some of the physiological effects of DBC1.

Replication-independent cis-acting element of a maize histone gene promoter

Plant Science ◽  
1993 ◽  
Vol 89 (2) ◽  
pp. 177-184 ◽  
Author(s):  
Marc Lepetit ◽  
Martine Ehling ◽  
Rossitza Atanassova ◽  
Nicole Chaubet ◽  
Claude Gigot
Keyword(s):  
Gene Promoter ◽  
Histone Gene ◽  
Cis Acting

Developmentally regulated expression of a human "finger"-containing gene encoded by the 5' half of the ret transforming gene

1988 ◽  
Vol 8 (4) ◽  
pp. 1853-1856
Author(s):  
M Takahashi ◽  
Y Inaguma ◽  
H Hiai ◽  
F Hirose
Keyword(s):  
Human Gene ◽  
Mrna Levels ◽  
Nucleic Acid Binding ◽  
Developmentally Regulated ◽  
Unique Transcript ◽  
Binding Domains ◽  
Regulated Expression ◽  
Pachytene Spermatocytes ◽  
Human Finger ◽  
Transforming Gene

We isolated and sequenced a cDNA clone of the human gene encoded by the 5' half of the ret transforming gene. The nucleotide sequence indicates that it encodes a protein with "finger" structures which represent putative metal- and nucleic acid-binding domains. Transcription of this gene was detected at high levels in a variety of human and rodent tumor cell lines, mouse testis, and embryos. In addition, a unique transcript was observed in testis RNA. When the expression of the unique transcript was examined at different stages of spermatogenesis, a striking increase in mRNA levels accompanied progression from meiotic prophase pachytene spermatocytes to postmeiotic round spermatids. This finger-containing gene may thus function in male germ cell development.

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