Showing papers on "Transcription (biology) published in 1988"
TL;DR: These procedures, which can circumvent the need for large-scale phage or plasmid growths, preparative gel-electrophoresis and the screening of molecular clones, can facilitate the rapid study of sequence-specific interactions of proteins and DNA.
Abstract: Specific, end-labeled DNA fragments can be simply and rapidly prepared using the polymerase chain reaction (PCR). Such fragments are suitable for use in DNase I protection footprint assays, chemical sequencing reactions, and for the production and analysis of paused RNA polymerase transcription complexes. Moreover, a general means of introducing a specific mutation at any position along the length of such PCR-generated fragments is described. These procedures, which can circumvent the need for large-scale phage or plasmid growths, preparative gel-electrophoresis and the screening of molecular clones, can facilitate the rapid study of sequence-specific interactions of proteins and DNA. A rapid means of removing excess oligonucleotide primers from completed PCRs is also described.
2,471 citations
TL;DR: It is shown that the hybrid protein (GAL4-VP16) activates transcription unusually efficiently in mammalian cells when bound close to, or at large distances from the gene, and suggested that the activating region of VP16 may be near-maximally potent.
Abstract: Recent work has defined a class of transcriptional activators1–5, members of which activate transcription in yeast, plant, insect and mammalian cells6–9. These proteins contain two parts: one directs DNA binding and the other, called the activating region, presumably interacts with some component of the transcriptional machinery. Activating regions are typically acidic and require some poorly-understood aspect of structure, probably at least in part an α-helix1–5,10. Here we describe a new member of this class, formed by fusing a DNA-binding fragment of the yeast activator GAL4 to a highly acidic portion of the herpes simplex virus protein VP16 (ref. 11; also called Vmw65). VP16 activates transcription of immediate early viral genes by using its amino-terminal sequences to attach to one or more host-encoded proteins that recognise DNA sequences in their promoters11–15. We show that the hybrid protein (GAL4-VP16) activates transcription unusually efficiently in mammalian cells when bound close to, or at large distances from the gene. We suggest that the activating region of VP16 may be near-maximally potent and that it is not coincidental that such a strong activator is encoded by a virus.
1,312 citations
TL;DR: Drosophila tissue culture cells provide an Sp1-deficient background and have been used in a complementation assay to identify functional domains of human transcription factor Sp1, and it is proposed that these glutamine-rich domains represent a novel structural motif for transcriptional activation.
1,295 citations
TL;DR: In this paper, the human transcription factor Jun/AP-1 was found to be responsible for increased transcription of different cellular genes in response to tumor promoters, such as TPA, and serum factors.
1,095 citations
TL;DR: L'AMPc coordonne differentes voies metaboliques allant de la degradation du glycogene dans le foie des mammiferes a la synthese d'enzymes specifiques impliquees dansLe metabolisme energetique d'Escherichia coli.
971 citations
TL;DR: A cDNA clone of SRF expressed in vitro generates protein that forms complexes indistinguishable from those formed with HeLa cell SRF, as judged by DNA binding specificity and the ability to promote SRE-dependent in vitro transcription.
862 citations
TL;DR: The experimental results reported here indicate that the JUN oncoprotein is a sequence-specific transcriptional activator similar to AP-1, the product of the c-jun proto-oncogene.
Abstract: Proto-oncogenes encode proteins with three main sites of action: the cell-surface membrane, the cytoplasm and the nucleus. Although the exact biochemical function of most proto-oncogene products is not understood, several of them are known to be involved in signal transduction. A role in gene regulation through DNA binding has been suggested for a recently isolated member of the group of oncogenes acting at the nucleus, v-jun. The C-terminus of the putative v-jun-encoded protein is similar in sequence to the C-terminus of the yeast transcriptional activator GCN4 (refs 8, 9), which forms its minimal DNA-binding domain. GCN4 binds to specific sites whose consensus sequence is highly similar to the recognition sequence of the mammalian transcriptional activator AP-1 (refs 12, 13). Like GCN4, AP-1 binds to promoter elements of specific genes and activates their transcription. Because of the similarity between the recognition sites for GCN4 and AP-1, we examined the possibility that AP-1 could be the product of the c-jun proto-oncogene. The experimental results reported here indicate that the JUN oncoprotein is a sequence-specific transcriptional activator similar to AP-1.
846 citations
TL;DR: The above experiments support a relatively simple model to explain the role of DNA methylation in vivo and provide a general mechanism for transcriptional repression which may operate independent of the requirement for interactions between cis-acting regulatory elements and tissue-specific factors.
830 citations
TL;DR: It is discussed that triplex formation (site-specific RNA binding to a DNA duplex) could serve as the basis for an alternative program of gene control in vivo.
Abstract: A 27-base-long DNA oligonucleotide was designed that binds to duplex DNA at a single site within the 5' end of the human c-myc gene, 115 base pairs upstream from the transcription origin P1. On the basis of the physical properties of its bound complex, it was concluded that the oligonucleotide forms a colinear triplex with the duplex binding site. By means of an in vitro assay system, it was possible to show a correlation between triplex formation at -115 base pairs and repression of c-myc transcription. The possibility is discussed that triplex formation (site-specific RNA binding to a DNA duplex) could serve as the basis for an alternative program of gene control in vivo.
762 citations
TL;DR: Comparisons of cDNAs encoding potential finger proteins indicate the existence of at least two classes of proteins with specific nucleic acid recognition capabilities, characterized according to the number and position of the cysteine and histidine residues available for zinc coordination.
680 citations
TL;DR: The 3' untranslated region of the mRNA for the human TfR was shown to be necessary and sufficient for iron-dependent control of mRNA levels and an mRNA element has been implicated in the mediation of distinct regulatory phenomena dependent on the context of the element within the transcript.
Abstract: The biosynthetic rates for both the transferrin receptor (TfR) and ferritin are regulated by iron. An iron-responsive element (IRE) in the 5' untranslated portion of the ferritin messenger RNA (mRNA) mediates iron-dependent control of its translation. In this report the 3' untranslated region of the mRNA for the human TfR was shown to be necessary and sufficient for iron-dependent control of mRNA levels. Deletion studies identified a 678-nucleotide fragment of the TfR complementary DNA that is critical for this iron regulation. Five potential stem-loops that resemble the ferritin IRE are contained within the region critical for TfR regulation. Each of two of the five TfR elements was independently inserted into the 5' untranslated region of an indicator gene transcript. In this location they conferred iron regulation of translation. Thus, an mRNA element has been implicated in the mediation of distinct regulatory phenomena dependent on the context of the element within the transcript.
TL;DR: The results indicate that the state of supercoiling of bacterial DNA is strongly modulated by transcription, and that DNA topoisomerases are normally involved in the elongation step of transcription.
TL;DR: It is suggested that this inhibition, which the authors call squelching, reflects titration of a transcription factor by the activating region of GAL4, and that more efficient activators inhibit more strongly and that inhibition does not depend on the DNA-binding domain.
Abstract: The yeast transcriptional activator GAL4 binds specific sites on DNA to activate transcription of adjacent genes. The distinct activating regions of GAL4 are rich in acidic residues and it has been suggested that these regions interact with another protein component of the transcriptional machinery (such as the TATA-binding protein or RNA polymerase II) while the DNA-binding region serves to position the activating region near the gene. Here we show that various GAL4 derivatives, when expressed at high levels in yeast, inhibit transcription of certain genes lacking GAL4 binding sites, that more efficient activators inhibit more strongly and that inhibition does not depend on the DNA-binding domain. We suggest that this inhibition, which we call squelching, reflects titration of a transcription factor by the activating region of GAL4.
TL;DR: It is shown that a promoter-associated RNA polymerase II molecule is transcriptionally engaged and has formed a nascent RNA chain, but is apparently arrested at that point and unable to penetrate further into the hsp70 gene without heat induction.
TL;DR: The findings that the products of nuclear proto-oncogenes jun and erbA are transcriptional factors supports the notion of the role of the nuclear oncoproteins in the regulation of gene expression.
Abstract: The response of a cell to mitogens and differentiation agents involves the transcriptional induction of several cellular genes. Prominent among these so-called 'immediate early' or 'competence' genes are the nuclear oncogenes fos and mycl–7. Although the precise function of these early response genes in growth control is not understood, it is likely that many of them are involved in the transition from G0 to G1 in the cell cycle. The findings that the products of nuclear proto-oncogenes jun and erbA are transcriptional factors supports the notion of the role of the nuclear oncoproteins in the regulation of gene expression8–11. Recently, it has been reported that the FOS protein is associated in transcriptional complexes with the product of the jun oncogene, the transcription factor AP-1 (refs 12–15). As the fos gene is induced in response to mitogens during initiation of cell growth, we investigated whether expression of the nuclear transcription factor AP-1 is also inducible. We report that mouse c-jun gene transcription is rapidly induced by serum and phorbol-ester 12-o-tetradecanoyl phorbol 13-acetate (TPA). Furthermore, induction is transient and the mRNA is superinduced by inhibitors of protein synthesis.
TL;DR: Using DNA transfection, it is shown that TGF-beta stimulates the activity of the mouse alpha 2(l) collagen promoter 5- to 10-fold in mouse NIH 3T3 and rat osteosarcoma cells.
TL;DR: A recombinant plasmid was constructed with six synthetic DNA oligomers such that the DNA sequence corresponding to yeast tRNA(Phe) is flanked by a T7 promoter and a BstNI restriction site to give an unmodified tRNA of the expected sequence having correct 5' and 3' termini.
Abstract: A recombinant plasmid was constructed with six synthetic DNA oligomers such that the DNA sequence corresponding to yeast tRNA(Phe) is flanked by a T7 promoter and a BstNI restriction site. Runoff transcription of the BstNI-digested plasmid with T7 RNA polymerase gives an unmodified tRNA of the expected sequence having correct 5' and 3' termini. This tRNA(Phe) transcript can be specifically aminoacylated by yeast phenylalanyl-tRNA synthetase and has a Km only 4-fold higher than that of the native yeast tRNA(Phe). The Km is independent of Mg2+ concentration, whereas the Vmax is very dependent on Mg2+ concentration. Comparison of the melting profiles of the native and the unmodified tRNA(Phe) at different Mg2+ concentrations suggests that the unmodified tRNA(Phe) has a less stable tertiary structure. Using one additional DNA oligomer, a mutant plasmid was constructed having a guanosine to thymidine change at position 20 in the tRNA gene. A decrease in Vmax/Km by a factor of 14 for aminoacylation of the mutant tRNA(Phe) transcript is observed.
TL;DR: The design of the method and careful optimization of first strand synthesis have permitted cloning of several large cDNAs, and Transcription of essentially full-length clones with phage SP6 RNA polymerase produces RNAs that are efficiently translated in vitro to give complete, unfused products, thus permitting rapid characterization of the clones via the encoded polypeptides.
TL;DR: Analysis of the changes in transcription and mRNA levels after serum stimulation demonstrated that the kinetics and extent of the induction vary dramatically between the different genes, and showed that induction of proliferation is accompanied by the onset of a complex genetic program.
Abstract: Genes whose expression is growth factor regulated are likely to be important components in the mechanisms controlling cell proliferation and differentiation. With the aim of identifying some of those genes, a lambda cDNA library was prepared with poly(A)+ RNA from quiescent NIH 3T3 cells stimulated with serum for 4 h in the presence of cycloheximide. Differential screening of approximately 200,000 recombinant phage plaques revealed 2,540 clones that cross hybridized preferentially with [32P]cDNA derived from RNA of stimulated cells rather than with cDNA derived from nonstimulated cells. Cross hybridization of these clones identified 82 independent sequences, including c-fos and c-myc. Seventy-one clones were further studied. Analysis of the changes in transcription and mRNA levels after serum stimulation demonstrated that the kinetics and extent of the induction vary dramatically between the different genes. Cycloheximide in all cases superinduced the mRNA levels by two mechanisms, inhibiting the shutoff of transcription and prolonging the half-lives of the mRNAs. Our results showed that induction of proliferation is accompanied by the onset of a complex genetic program.
TL;DR: It is shown that there is a dramatic increase in the FSE2 binding complex when Fos levels are induced with serum, benzodiazepine, and nerve growth factor or are expressed from a v-fos gene, suggesting a functional relationship between Fos and AP-1.
TL;DR: Nine P-element vectors that can be used to study gene regulation and function in Drosophila are described, designed for use in germline transformation and cell culture transfection assays.
01 Jan 1988
TL;DR: A He La cell nuclear transcription extract that is approximately 20 times more efficient than standard HeLa cell transcription extracts was developed, and exhibits an increase of approximately twofold on transcription of a closed circular, as opposed to a linear, major late promoter template.
Abstract: A HeLa cell nuclear transcription extract that is approximately 20 times more efficient than standard HeLa cell transcription extracts was developed. Transcription of the strong adenovirus II major late promoter by this extract results in the synthesis of 1.5-4 molecules of product RNA per molecule of template, indicating that the extract is capable of multiple rounds of initiation. Standard HeLa cell nuclear extracts transcribe closed circular and linear adenovirus major late promoter templates with equal efficiency. In contrast, the new extract exhibits an increase of approximately twofold on transcription of a closed circular, as opposed to a linear, major late promoter template.
TL;DR: These results establish a key role for fos in signal transduction and implicate the fos protein as a trans-activating and -repressing molecule.
TL;DR: Transfection of cDNA clones into Drosophila cells indicates that the AP-2 gene product can also activate gene expression in vivo in a DNA template-dependent manner and may be a transcription factor involved in the control of developmentally regulated gene expression.
Abstract: Human AP-2 is a sequence-specific DNA-binding protein that interacts with inducible viral and cellular enhancer elements to stimulate transcription of selected genes. Here, we report the isolation and characterization of a human cDNA clone containing the entire protein-coding region of AP-2. The deduced primary amino acid sequence of AP-2 does not contain a domain resembling any previously identified DNA binding motif. However, an interesting feature of the AP-2 protein is a clustered arrangement of proline and glutamine residues that have been found recently within the activation domains of other transcription factors. Expression of the AP-2 clone in bacteria yields a protein that binds to DNA and activates transcription in vitro in a comparable manner to native human AP-2. Transfection of cDNA clones into Drosophila cells indicates that the AP-2 gene product can also activate gene expression in vivo in a DNA template-dependent manner. Expression of endogenous AP-2 is repressed in a hepatoma cell line and stimulated following retinoic-acid-induced differentiation of a human teratocarcinoma cell line. This indicates that AP-2 may be a transcription factor involved in the control of developmentally regulated gene expression.
TL;DR: Two classes of IFN-inducible nuclear factors were found that bind to the ISRE, and the most rapidly induced factor appeared without new protein synthesis, whereas a second factor required active protein synthesis for its appearance and maintenance.
Abstract: Human alpha- and beta-interferons (IFNs) stimulate rapid but transient increases in transcription from a set of previously quiescent genes. Protein synthesis is not required for initial stimulation, but duration of the response is limited to a few hours by a process requiring synthesis of new proteins. An IFN-stimulated response element (ISRE) was identified 5' to an inducible gene by deletion analysis and point mutagenesis, and sequence comparisons with other promoters defined the consensus element YAGTTTC(A/T)YTTTYCC. Two classes of IFN-inducible nuclear factors were found that bind to the ISRE. The most rapidly induced factor appeared without new protein synthesis, whereas a second factor required active protein synthesis for its appearance and maintenance. The kinetics of appearance and loss of these binding activities correlate with the activation and repression of IFN-stimulated genes. These different IFN-activated or induced factors may bind sequentially to the same essential promoter element to first increase and then repress transcription.
TL;DR: It is demonstrated that in quiescent fibroblasts c-jun transcription is rapidly induced during the G0 to G1 transition, and the nucleotide sequence of a mouse cDNA clone coding for a 334 residue protein which shows 80% similarity with v-JUN17 and more than 98% similarities with the human c-Jund sequence is presented.
Abstract: Before quiescent cells can respond to mitogens and progress through the G1 phase of cell growth, new messenger RNA synthesis is required1 The G1 phase seems to be a critical point of control in the cell cycle, where normal cells deprived of growth factors halt cycling while transformed cells do not, suggesting that regulatory genes, uncontrolled in the neoplastic phenotype, are expressed during the G0 to G1 transition Some of these may code for nuclear proteins that participate in the transactivation of genes required for the progression through G1 The observed changes in expression of the proto-oncogenes c-fos and c-myc, following stimulation of fibroblasts with growth factors2–7, support this notion as recent evidence suggests that c-FOS and c-MYC proteins can function as transactivating factors8–12 Moreover, the rapid induction of several genes in fibroblasts coding for putative transacting factors during the G0 to G1 transition has been recently reported13–16 Here we present the nucleotide sequence of a mouse cDNA clone coding for a 334 residue protein which shows 80% similarity with v-JUN17 and more than 98% similarity with the human c-JUN sequence18,19 We have demonstrated that in quiescent fibroblasts c-jun transcription is rapidly induced during the G0 to G1 transition
TL;DR: Inhibition of the formation and effects IL-1 is a mechanism by which glucocorticoids can exert antiinflammatory and immunosuppressive effects and kinetic analyses and pulse-labeling of mRNAs showed that glucoc Corticoids selectively decrease the stability of IL- 1 beta mRNA, without affecting the stabilityof beta-actin and FOS mRN as.
Abstract: Transcription of the interleukin 1 beta (IL-1 beta) gene was studied by mRNA hybridization with a cDNA probe in the human promonocytic cell line U-937. Phorbol ester and lipopolysaccharide increased the steady-state level of IL-1 beta mRNA. Glucocorticoids markedly decreased IL-1 beta mRNA levels by two mechanisms. Transcription of the IL-1 gene was inhibited, as shown by in vitro transcription assays with nuclei isolated from glucocorticoid-treated cells. Moreover, kinetic analyses and pulse-labeling of mRNAs showed that glucocorticoids selectively decrease the stability of IL-1 beta mRNA, without affecting the stability of beta-actin and FOS mRNAs. Inhibition of the formation and effects IL-1 is a mechanism by which glucocorticoids can exert antiinflammatory and immunosuppressive effects.
TL;DR: It is shown that GAL4, when expressed in particular tissues of Drosophila larvae, stimulates tissue-specific transcription of a Dosophila promoter linked to GAL 4 binding sites.
Abstract: GAL4 is a yeast regulatory protein that binds to specific sites within a DNA sequence called UASG (galactose upstream activating sequence) and activates transcription of linked genes1–6. This activation requires two functions of the protein7,8: a DNA binding domain located near the amino terminus8, and one or more 'activating regions'7–11. The 'activating regions' are highly acidic9–11 (see also ref. 12) and can be replaced, for example, by a short peptide designed to form a negatively charged, amphipathic α-helix13. GAL4, as well as deletion derivatives bearing one or more 'activating regions' attached to the DNA binding domain, activates transcription in cultured mammalian cells from mammalian promoters linked to a UASG (refs 14,15). Here we show that GAL4, when expressed in particular tissues of Drosophila larvae, stimulates tissue-specific transcription of a Drosophila promoter linked to GAL4 binding sites.
TL;DR: A steroid responsive unit can be composed of several modules that, if positioned correctly, act synergistically, according to the degree of synergism of the GRE.
Abstract: Progesterone (PRE) or glucocorticoid receptor (GRE) DNA binding sites are often found clustered with binding sites for other transcription factors. Individual protein binding sites were tested without the influence of adjacent factors by analyzing isolated combinations of several transcription factor binding sites with PREs or GREs. All show strong synergistic effects on steroid induction. The degree of synergism is inversely related to the strength of the GRE. Thus, a steroid responsive unit can be composed of several modules that, if positioned correctly, act synergistically.
TL;DR: It is shown that one of the fos-associated proteins, p39, is immunologically and structurally related to nuclear factor AP-1, and the sequence TGACTCA, which is the consensus minimal binding site of AP- 1, is also recognized by gel-purified p39.