scholarly journals Expression of NF-L and NF-M in fibroblasts reveals coassembly of neurofilament and vimentin subunits.

1989 ◽  
Vol 108 (2) ◽  
pp. 579-593 ◽  
Author(s):  
M J Monteiro ◽  
D W Cleveland
Keyword(s):  
Transcription Initiation ◽  
Initiation Site ◽  
Cellular Protein ◽  
Cell Protein ◽  
Coding Region ◽  
Animal Cells ◽  
Murine Sarcoma Virus ◽  
Sarcoma Virus ◽  
Specific Factors

We have used transient and stable DNA transfection to force synthesis of the mouse NF-L and NF-M genes in nonneuronal cultured animal cells. When the authentic NF-L gene (containing 1.7 kb of sequences 5' to the transcription initiation site) was transfected into L cells, correctly initiated NF-L mRNA was produced from the transfected gene but not the endogenous NF-L genes. Therefore, the normal restriction of NF-L expression to neurons cannot derive exclusively from absence in nonneuronal cells of neuron-specific transcription factors. When the NF-L coding region was linked to the strong promoter from Moloney Murine Sarcoma virus, we obtained high levels of synthesis of NF-L subunits (accumulating to as much as 9% of cell protein in stable cell lines). Although NF-L and NF-M polypeptides are normally expressed exclusively in postmitotic neurons, NF-L or NF-M polypeptides expressed in fibroblasts were efficiently assembled into intermediate filament arrays, thus demonstrating the competence of both NF-L and NF-M to assemble in vivo in the absence of additional neuron-specific factors. As judged by immunofluorescence localization and by the alteration in the solubility of the endogenous vimentin filaments, filaments containing NF-L appeared to be copolymers with vimentin. Neither the alteration in the properties of the vimentin array nor the accumulation of NF-L to a level that made it the second most abundant cellular protein (after actin) had any observable effect on cell viability or growth rate.

scholarly journals L1 Antisense Promoter Drives Tissue-Specific Transcription of Human Genes

2006 ◽  
Vol 2006 ◽  
pp. 1-16 ◽  
Author(s):  
Kert Mätlik ◽  
Kaja Redik ◽  
Mart Speek
Keyword(s):  
Transcription Initiation ◽  
Regulatory Elements ◽  
Initiation Site ◽  
Coding Region ◽  
Tissue Specific ◽  
Multiple Loci ◽  
Complex Interactions ◽  
Chimeric Transcripts ◽  
Human Genes

Transcription of transposable elements interspersed in the genome is controlled by complex interactions between their regulatory elements and host factors. However, the same regulatory elements may be occasionally used for the transcription of host genes. One such example is the human L1 retrotransposon, which contains an antisense promoter (ASP) driving transcription into adjacent genes yielding chimeric transcripts. We have characterized 49 chimeric mRNAs corresponding to sense and antisense strands of human genes. Here we show that L1 ASP is capable of functioning as an alternative promoter, giving rise to a chimeric transcript whose coding region is identical to the ORF of mRNA of the following genes:KIAA1797,CLCN5, andSLCO1A2. Furthermore, in these cases the activity of L1 ASP is tissue-specific and may expand the expression pattern of the respective gene. The activity of L1 ASP is tissue-specific also in cases where L1 ASP produces antisense RNAs complementary toCOL11A1andBOLLmRNAs. Simultaneous assessment of the activity of L1 ASPs in multiple loci revealed the presence of L1 ASP-derived transcripts in all human tissues examined. We also demonstrate that L1 ASP can act as a promoter in vivo and predict that it has a heterogeneous transcription initiation site. Our data suggest that L1 ASP-driven transcription may increase the transcriptional flexibility of several human genes.

scholarly journals Structure and transcriptional regulation of the Nat2 gene encoding for the drug-metabolizing enzyme arylamine N-acetyltransferase type 2 in mice

2003 ◽  
Vol 375 (3) ◽  
pp. 593-602 ◽  
Author(s):  
Sotiria BOUKOUVALA ◽  
Naomi PRICE ◽  
Kathryn E. PLANT ◽  
Edith SIM
Keyword(s):  
Transcriptional Regulation ◽  
Transcription Initiation ◽  
Consensus Sequence ◽  
Simian Virus 40 ◽  
Initiation Site ◽  
Cellular Protein ◽  
Transcription Initiation Site ◽  
Gene Promoters ◽  
Coding Region ◽  
Nat2 Gene

Arylamine N-acetyltransferases (NATs) are polymorphic enzymes, well-known for their role in the metabolism of drugs and carcinogens. Mice have three NAT isoenzymes, of which NAT2 is postulated to be involved in endogenous, as well as xenobiotic, metabolism. To understand expression of the murine Nat2 gene, we have analysed its structure and transcriptional regulation. We have accurately mapped the transcription initiation site 6.5 kb upstream of the coding region of the gene, adjacent to a recently described non-coding exon. Transcription was demonstrated to start from this region in embryonic and adult liver, spleen, submaxillary gland, kidney, brain, thymus, lung and placenta, but not in the heart. Database searches and analyses of cDNA by PCR suggested alternative splicing of the single 6.2 kb intron of Nat2, and determined the position of the polyadenylation signal at 0.44 kb downstream of the coding region of the gene. Examination of the 13 kb sequence flanking the coding and non-coding exons of Nat2 revealed a single promoter, located close to the transcription-initiation site, and indicated regions likely to harbour control elements. The Nat2 promoter consists of an atypical TATA box and a Sp1 [SV40 (simian virus 40) protein 1] box identical with that found in many housekeeping gene promoters. Activity of the Nat2 promoter was severely reduced by deletion or mutation of either of these two elements, whereas the region of the Sp1 box bound cellular protein and resisted DNase I digestion. Finally, the ability of the promoter region to bind cellular protein was reduced by competition with oligonucleotides bearing the Sp1 consensus sequence.

scholarly journals Nucleotide sequence of the two rat cellular rasH genes.

1986 ◽  
Vol 6 (5) ◽  
pp. 1706-1710 ◽  
Author(s):  
M Ruta ◽  
R Wolford ◽  
R Dhar ◽  
D Defeo-Jones ◽  
R W Ellis ◽  
...  
Keyword(s):  
Amino Acids ◽  
Nucleotide Sequence ◽  
Coding Region ◽  
3T3 Cells ◽  
Ras Gene ◽  
P21 Protein ◽  
Murine Sarcoma Virus ◽  
Sarcoma Virus ◽  
Nih 3T3 ◽  
Murine Sarcoma

We present the nucleotide sequence of the coding region of the rat c-rasH-1 gene and a partial sequence analysis of the rat c-rasH-2 gene. By comparing these sequences with the Harvey murine sarcoma virus ras gene, we predict that the p21 protein encoded by the Harvey virus differs from the cellular c-rasH-1-encoded p21 at only two amino acids; those at positions 12 and 59. Alterations at each of these positions may play a role in activating the viral p21 protein. The c-rasH-2 gene is likely to be a nonfunctional pseudogene because it lacks introns, cannot be activated to transform NIH 3T3 cells, and differs in sequence from both c-rasH-1 and v-rasH at several base pair positions.

Design of a Retrovirus-Derived Vector for Expression and Transduction of Exogenous Genes in Mammalian Cells

1983 ◽  
Vol 3 (6) ◽  
pp. 1123-1132
Author(s):  
Archibald S. Perkins ◽  
Paul T. Kirschmeier ◽  
Sebastiano Gattoni-Celli ◽  
I. Bernard Weinstein
Keyword(s):  
Mammalian Cells ◽  
High Efficiency ◽  
Leukemia Virus ◽  
Restriction Enzymes ◽  
Virus Genome ◽  
Opposite Polarity ◽  
High Yield ◽  
Animal Cells ◽  
Murine Sarcoma Virus ◽  
Sarcoma Virus

We have developed a transfection vector for animal cells that contains long terminal repeat (LTR) sequences to promote expression. Plasmid p101/101, a derivative of plasmid pBR322 containing the complete Moloney murine sarcoma virus genome, was cut with restriction enzymes and religated so that both the 5′ and 3′ LTRs were retained and all but about 700 base pairs of the intervening viral sequences were removed. To test this vector, the Escherichia coli gene gpt was cloned into a unique Pst I site, between the two LTRs, with guanine and cytosine tailing, a method that can be generalized for insertion of any DNA segment into this vector. When DNA from recombinant plasmids in which the gpt gene was inserted in the same transcriptional polarity as the LTR sequences was transfected onto murine or rat fibroblast cultures, we obtained a high yield of Gpt + colonies. However, plasmid constructs with the gpt gene in the opposite polarity were virtually devoid of activity. With gpt in the proper orientation, restriction enzyme cuts within the LTRs or between the 5′ LTR and the gpt gene reduced transfection by more than 98%, whereas a cut between the gpt gene and the 3′ LTR gave an 80% reduction in activity. Thus, both 5′ and 3′ LTR sequences are essential for optimal gpt expression, although the 5′ LTR appears to play a more important role. When the LTR- gpt plasmid was transfected onto murine leukemia virus-infected mouse fibroblasts, we obtained evidence that RNA copies became pseudotyped into viral particles which could transfer the Gpt + phenotype into rodent cells with extremely high efficiency. This vector should prove useful for high-efficiency transduction of a variety of genes in mammalian cells.

Analysis of Saccharomyces cerevisiae his3 transcription in vitro: biochemical support for multiple mechanisms of transcription

1990 ◽  
Vol 10 (6) ◽  
pp. 2832-2839
Author(s):  
A S Ponticelli ◽  
K Struhl
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcriptional Activation ◽  
Transcription Initiation ◽  
Molecular Mechanisms ◽  
Initiation Site ◽  
Activator Proteins ◽  
Nuclear Extracts ◽  
Transcription In Vitro

The promoter region of the Saccharomyces cerevisiae his3 gene contains two TATA elements, TC and TR, that direct transcription initiation to two sites designated +1 and +13. On the basis of differences between their nucleotide sequences and their responsiveness to upstream promoter elements, it has previously been proposed that TC and TR promote transcription by different molecular mechanisms. To begin a study of his3 transcription in vitro, we used S. cerevisiae nuclear extracts together with various DNA templates and transcriptional activator proteins that have been characterized in vivo. We demonstrated accurate transcription initiation in vitro at the sites used in vivo, transcriptional activation by GCN4, and activation by a GAL4 derivative on various gal-his3 hybrid promoters. In all cases, transcription stimulation was dependent on the presence of an acidic activation region in the activator protein. In addition, analysis of promoters containing a variety of TR derivatives indicated that the level of transcription in vitro was directly related to the level achieved in vivo. The results demonstrated that the in vitro system accurately reproduced all known aspects of in vivo his3 transcription that depend on the TR element. However, in striking contrast to his3 transcription in vivo, transcription in vitro yielded approximately 20 times more of the +13 transcript than the +1 transcript. This result was not due to inability of the +1 initiation site to be efficiently utilized in vitro, but rather it reflects the lack of TC function in vitro. The results support the idea that TC and TR mediate transcription from the wild-type promoter by distinct mechanisms.

Direct identification of palmitic acid as the lipid attached to p21ras

1986 ◽  
Vol 6 (1) ◽  
pp. 116-122
Author(s):  
J E Buss ◽  
B M Sefton
Keyword(s):  
Palmitic Acid ◽  
Sodium Dodecyl ◽  
Transformed Cells ◽  
Direct Identification ◽  
Ras Protein ◽  
Murine Sarcoma Virus ◽  
Sarcoma Virus ◽  
Layer Chromatography ◽  
Murine Sarcoma

p21v-H-ras, the transforming protein of Harvey murine sarcoma virus, contains a covalently attached lipid. Using thin-layer chromatography, we identified the acyl group as the 16-carbon saturated fatty acid palmitic acid. No myristic acid was detected in fatty acids released from in vivo-labeled p21v-H-ras. The p21v-K-ras protein encoded by Kirsten sarcoma virus was also palmitylated. The processing and acylation of p21v-K-ras however differed from that of p21v-H-ras. Three forms of [3H]palmitic acid-labeled p21ras proteins were detected in Kirsten sarcoma virus-transformed cells. This contrasted with Harvey sarcoma virus, in which two forms of p21v-H-ras contained palmitic acid. Analysis by partial proteolysis of p21v-H-ras labeled with [3H]palmitic acid suggested that all of the lipid found in intact p21v-H-ras was located in the C-terminal region. On sodium dodecyl sulfate-polyacrylamide gels, p21v-H-ras labeled with [3H]palmitic acid migrated slightly ahead of the majority of p21v-H-ras. Of the mature forms of p21v-H-ras, apparently only a subpopulation contains palmitic acid.

scholarly journals The same normal cell protein is phosphorylated after transformation by avian sarcoma viruses with unrelated transforming genes.

1981 ◽  
Vol 1 (1) ◽  
pp. 43-50 ◽  
Author(s):  
E Erikson ◽  
R Cook ◽  
G J Miller ◽  
R L Erikson
Keyword(s):  
Rous Sarcoma Virus ◽  
Direct Consequence ◽  
Cellular Protein ◽  
Cell Protein ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Transforming Proteins ◽  
Avian Sarcoma ◽  
Transforming Genes ◽  
Chicken Embryo Fibroblasts

The phosphorylation of a normal cellular protein of molecular weight 34,000 (34K) is enhanced in Rous sarcoma virus-transformed chicken embryo fibroblasts apparently as a direct consequence of the phosphotransferase activity of the Rous sarcoma virus-transforming protein pp60src. We have prepared anti-34K serum by using 34K purified from normal fibroblasts to confirm that the transformation-specific phosphorylation described previously occurs on a normal cellular protein and to further characterize the nature of the protein. In this communication, we also show that the phosphorylation of 34K is also increased in cells transformed by either Fujinami or PRCII sarcoma virus, two recently characterized avian sarcoma viruses whose transforming proteins, although distinct from pp60src, are also associated with phosphotransferase activity. Moreover, comparative fingerprinting of tryptic phosphopeptides shows that the major site of phosphorylation of 34K is the same in all three cases.

v-src mutations outside the carboxyl-coding region are not sufficient to fully activate transformation by pp60c-src in NIH 3T3 cells

1988 ◽  
Vol 8 (2) ◽  
pp. 704-712
Author(s):  
S Reddy ◽  
P Yaciuk ◽  
T E Kmiecik ◽  
P M Coussens ◽  
D Shalloway
Keyword(s):  
Rous Sarcoma Virus ◽  
Terminal Region ◽  
Cell Protein ◽  
Coding Region ◽  
3T3 Cells ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Carboxyl Terminal ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

Previous studies have shown that carboxyl-terminal mutation of pp60c-src can activate its transforming ability. Conflicting results have been reported for the transforming ability of pp60c-src mutants having only mutations outside its carboxyl-terminal region. To clarify the effects of such mutations, we tested the activities of chimeric v(amino)- and c(carboxyl)-src (v/c-src) proteins at different dosages in NIH 3T3 cells. The focus-forming activity of Rous sarcoma virus long terminal repeat (LTR)-src expression plasmids was significantly reduced when the v-src 3' coding region was replaced with the corresponding c-src region. This difference was masked when the Rous sarcoma virus LTR was replaced with the Moloney murine leukemia virus LTR, which induced approximately 20-fold more protein expression, but even focus-selected lines expressing v/c-src proteins were unable to form large colonies in soft agarose or tumors in NFS mice. This suggests that pp60c-src is not equally sensitive to mutations in its different domains and that there are at least two distinguishable levels of regulation, the dominant one being associated with its carboxyl terminus. v/c-src chimeric proteins expressed with either LTR had high in vitro specific kinase activity equal to that of pp60v-src but, in contrast, were phosphorylated at both Tyr-527 and Tyr-416. Total cell protein phosphotyrosine was enhanced in cells incompletely transformed by v/c-src proteins to the same extent as in v-src-transformed cells, suggesting that the carboxyl-terminal region may affect substrate specificity in a manner that is important for transformation.

Effect of the Fv-1 Locus In Vivo: Host Range Pseudotypes of Murine Sarcoma Virus 2

1978 ◽  
Vol 60 (4) ◽  
pp. 875-880 ◽  
Author(s):  
James A. Otten ◽  
Fred E. Myer ◽  
Raymond W. Tennant ◽  
Arthur Brown
Keyword(s):  
Host Range ◽  
Murine Sarcoma Virus ◽  
Sarcoma Virus ◽  
Murine Sarcoma

scholarly journals Transcription of Drosophila Troponin I Gene Is Regulated by Two Conserved, Functionally Identical, Synergistic Elements

2004 ◽  
Vol 15 (3) ◽  
pp. 1185-1196 ◽  
Author(s):  
María-Cruz Marín ◽  
José-Rodrigo Rodríguez ◽  
Alberto Ferrús
Keyword(s):  
Transcription Initiation ◽  
Troponin I ◽  
Regulatory Element ◽  
Expression Patterns ◽  
In Silico Analysis ◽  
Initiation Site ◽  
Transcription Initiation Site ◽  
Upstream Regulatory Element ◽  
Transgenic Lines

The Drosophila wings-up A gene encodes Troponin I. Two regions, located upstream of the transcription initiation site (upstream regulatory element) and in the first intron (intron regulatory element), regulate gene expression in specific developmental and muscle type domains. Based on LacZ reporter expression in transgenic lines, upstream regulatory element and intron regulatory element yield identical expression patterns. Both elements are required for full expression levels in vivo as indicated by quantitative reverse transcription-polymerase chain reaction assays. Three myocyte enhancer factor-2 binding sites have been functionally characterized in each regulatory element. Using exon specific probes, we show that transvection is based on transcriptional changes in the homologous chromosome and that Zeste and Suppressor of Zeste 3 gene products act as repressors for wings-up A. Critical regions for transvection and for Zeste effects are defined near the transcription initiation site. After in silico analysis in insects (Anopheles and Drosophila pseudoobscura) and vertebrates (Ratus and Coturnix), the regulatory organization of Drosophila seems to be conserved. Troponin I (TnI) is expressed before muscle progenitors begin to fuse, and sarcomere morphogenesis is affected by TnI depletion as Z discs fail to form, revealing a novel developmental role for the protein or its transcripts. Also, abnormal stoichiometry among TnI isoforms, rather than their absolute levels, seems to cause the functional muscle defects.

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