Showing papers on "Transcription (biology) published in 1993"
TL;DR: It is shown that, when expressed in Saccharomyces cerevisiae, human MDM2 inhibits human p53's ability to stimulate transcription by binding to a region that nearly coincides with the p53 acidic activation domain.
Abstract: THE tumour-suppressor gene p53 is inactivated in most human malignancies1 either by missense mutations1 or by binding to oncogenic proteins2–4. In human soft tissue sarcomas, inactivation apparently results from MDM2 gene amplification4. MDM2 is an oncogene product5,6 that may function by binding to p53 and inhibiting its ability to activate transcription3. Here we show that, when expressed in Saccharomyces cerevisiae, human MDM2 inhibits human p53's ability to stimulate transcription by binding to a region that nearly coincides with the p53 acidic activation domain. The isolated p53 activation domain fused to another DNA-binding protein is also inactivated by MDM2, confirming that MDM2 can inhibit p53 function by concealing the activation domain of p53 from the cellular transcription machinery.
1,502 citations
1,409 citations
TL;DR: It is demonstrated that Hif-1 DNA binding activity is also induced by hypoxia in a variety of mammalian cell lines in which the EPO gene is not transcribed, providing evidence that HIF-1 and its recognition sequence are common components of a general mammalian cellular response to Hypoxia.
Abstract: Transcription of the human erythropoietin (EPO) gene is activated in Hep3B cells exposed to hypoxia. Hypoxia-inducible factor 1 (HIF-1) is a nuclear factor whose DNA binding activity is induced by hypoxia in Hep3B cells, and HIF-1 binds at a site in the EPO gene enhancer that is required for hypoxic activation of transcription. In this paper, we demonstrate that HIF-1 DNA binding activity is also induced by hypoxia in a variety of mammalian cell lines in which the EPO gene is not transcribed. The composition of the HIF-1 DNA binding complex and its isolated DNA binding subunit and the mechanism of HIF-1 activation appear to be similar or identical in EPO-producing and non-EPO-producing cells. Transcription of reporter genes containing the EPO gene enhancer is induced by hypoxia in non-EPO-producing cells and mutations that eliminate HIF-1 binding eliminate inducibility. These results provide evidence that HIF-1 and its recognition sequence are common components of a general mammalian cellular response to hypoxia.
1,405 citations
TL;DR: Delineation of these two cooperative regions explains at the level of transcription how IFN-gamma and LPS act in concert to induce maximally the mac-NOS gene and, furthermore, howIFN-Gamma augments the inflammatory response to LPS.
Abstract: The promoter region of the mouse gene for macrophage-inducible nitric oxide synthase (mac-NOS; EC 1.14.13.39) has been characterized. A putative TATA box is 30 base pairs upstream of the transcription start site. Computer analysis reveals numerous potential binding sites for transcription factors, many of them associated with stimuli that induce mac-NOS expression. To localize functionally important portions of the regulatory region, we constructed deletion mutants of the mac-NOS 5' flanking region and placed them upstream of a luciferase reporter gene. The macrophage cell line RAW 264.7, when transfected with a minimal promoter construct, expresses little luciferase activity when stimulated by lipopolysaccharide (LPS), interferon gamma (IFN-gamma), or both. Maximal expression depends on two discrete regulatory regions upstream of the putative TATA box. Region I (position -48 to -209) increases luciferase activity approximately 75-fold over the minimal promoter construct. Region I contains LPS-related responsive elements, including a binding site for nuclear factor interleukin 6 (NF-IL6) and the kappa B binding site for NF-kappa B, suggesting that this region regulates LPS-induced expression of the mac-NOS gene. Region II (position -913 to -1029) alone does not increase luciferase expression, but together with region I it causes an additional 10-fold increase in expression. Together the two regions increase expression 750-fold over activity obtained from a minimal promoter construct. Region II contains motifs for binding IFN-related transcription factors and thus probably is responsible for IFN-mediated regulation of LPS-induced mac-NOS. Delineation of these two cooperative regions explains at the level of transcription how IFN-gamma and LPS act in concert to induce maximally the mac-NOS gene and, furthermore, how IFN-gamma augments the inflammatory response to LPS.
1,083 citations
TL;DR: DNA footprinting studies suggest that in the absence of coinducer many LTTRs bind to regulated promoters via a 15-bp dyadic sequence with a common structure and position (near -65).
Abstract: The LysR family is composed of > 50 similar-sized, autoregulatory transcriptional regulators (LTTRs) that apparently evolved from a distant ancestor into subfamilies found in diverse prokaryotic genera. In response to different coinducers, LTTRs activate divergent transcription of linked target genes or unlinked regulons encoding extremely diverse functions. Mutational studies and amino acid sequence similarities of LTTRs identify: (a) a DNA-binding domain employing a helix-turn-helix motif (residues 1-65), (b) domains involved in coinducer recognition and/or response (residues 100-173 and 196-206), (c) a domain required for both DNA binding and coinducer response (residues 227-253). DNA footprinting studies suggest that in the absence of coinducer many LTTRs bind to regulated promoters via a 15-bp dyadic sequence with a common structure and position (near -65). Coinducer causes additional interactions of LTTRs with sequences near the -35 RNA polymerase binding site and/or DNA bending that results in transcription activation.
1,079 citations
TL;DR: It is shown that the cloned RNAIII determinant restores both positive and negative regulatory functions of agr to an agr‐null strain and that the RNA itself, rather than any protein, is the effector molecule.
Abstract: The production of most toxins and other exoproteins in Staphylococcus aureus is controlled globally by a complex polycistronic regulatory locus, agr. Secretory proteins are up-regulated by agr whereas surface proteins are down-regulated. agr contains two divergent promoters, one of which directs the synthesis of a 514 nucleotide (nt) transcript, RNAIII. In this report, we show that the cloned RNAIII determinant restores both positive and negative regulatory functions of agr to an agr-null strain and that the RNA itself, rather than any protein, is the effector molecule. RNAIII acts primarily on the initiation of transcription and, secondarily in some cases, at the level of translation. In these cases, translation and transcription are regulated independently. RNAIII probably regulates translation directly by interacting with target gene transcripts and transcription indirectly by means of intermediary protein factors.
1,048 citations
TL;DR: It is demonstrated that overexpression of the E2F1 complementary DNA can activate DNA synthesis in cells that would otherwise growth-arrest, with an efficiency that is similar to that achieved by the expression of the adenovirus El A gene.
Abstract: SEVERAL lines of evidence implicate the E2F transcription factor as an important component of cell proliferation control. First, E2F binding sites are found in the promoters of genes responsive to proliferation signals and the level of E2F binding activity increases at a time when many of these genes are activated1–3. Second, the tumour suppressor protein Rb, as well as the related p107 protein, complexes with E2F2–7, resulting in an inhibition of E2F transcriptional activity3,8–12. Third, oncogenic products of the DNA tumour viruses can dissociate these E2F complexes4,13. We provide here direct evidence that E2F is involved in cellular proliferation control. Specifically, we demonstrate that overexpression of the E2F1 complementary DNA14,15 can activate DNA synthesis in cells that would otherwise growth-arrest, with an efficiency that is similar to that achieved by the expression of the adenovirus El A gene. Moreover, microinjection of the E2F1 cDNA into quiescent cells can induce S-phase entry, whereas two E2F1 mutants, which are unable to transactivate the DHFR and TK promoters, are unable to induce S phase. We conclude that the E2F transcription factor plays an important role in progression into S phase and that this probably coincides with its capacity to stimulate transcription.
978 citations
TL;DR: It is demonstrated that induction of both Hif-1 and erythropoietin RNA is inhibited by the protein kinase inhibitor 2-aminopurine, consistent with the proposed function of HIF-1 as a physiologic regulator of gene expression that responds to changes in cellular oxygen tension.
875 citations
TL;DR: The use of a previously described 17-mer phosphorothioate for structure-function analysis of 2'-sugar modifications and the results demonstrate the importance of target affinity in the action of antisense oligonucleotides and of RNase H as a mechanism by which these compounds exert their effects.
804 citations
804 citations
TL;DR: The interaction between the lymphoid-specific factor NFATp and the ubiquitous transcription factors Fos and Jun provides a novel mechanism for combinatorial regulation of interleukin-2 gene transcription, which integrates the calcium-dependent and the protein-kinase C-dependent pathways of T-cell activation.
Abstract: TRANSCRIPTION of lymphokine genes in activated T cells is inhibited by the immunosuppressive agents cyclosporin A and FK506, which act by blocking the phosphatase activity of calcineurin1–3. NFAT, a DNA-binding protein required for interleukin-2 gene transcription, is a potential target for calcineurin, cyclosporin A and FK5064–11. NFAT contains a subunit (NFATp) which is present in unstimulated T cells and which forms a complex with Fos and Jun proteins in the nucleus of activated T cells9,11. Here we report that NFATp is a DNA-binding phosphoprotein of relative molecular mass ∼ 120,000 and is a substrate for calcineurin in vitro. Purified NFATp forms DNA–protein complexes with recombinant Jun homodimers or Jun–Fos heterodimers; the DNA-binding domains of Fos and Jun are essential for the formation of the NFATp–Fos–Jun–DNA complex. The interaction between the lymphoid-specific factor NFATp and the ubiquitous transcription factors Fos and Jun provides a novel mechanism for combinatorial regulation of interleukin-2 gene transcription, which integrates the calcium-dependent and the protein-kinase C-dependent pathways of T-cell activation.
TL;DR: The results show that the SWI2 family DNA-dependent ATPase domain has functional con-servation between yeast and humans and suggest that a SWI/SNF protein complex is required for the activation of selective mammalian genes.
Abstract: SEQUENCE-SPECIFIC DNA binding activators of gene transcription may be assisted by SWI2(SNF2)1,2, which contains a DNA-depen-dent ATPase domain3. We have isolated a human complementary DNA encoding a 205K nuclear protein, BRG1, that contains extensive homology to SWI2 and Drosophila brahma4,5. We report here that a SWI2/BRG1 chimaera with the DNA-dependent ATPase domain replaced by corresponding human sequence restored normal mitotic growth and capacity for transcriptional activation to swi2& minus; yeast cells. Point mutation of the conserved ATP binding site lysine abolished this complementation. This mutation in SW12 exerted a dominant negative effect on transcription in yeast. A lysine to arginine substitution at the corresponding residue of BRG1 also generated a transcriptional dominant negative in human cells. BRG1 is exclusively nuclear and present in a high Mr complex of about 2 & times; 106. These results show that the SWI2 family DNA-dependent ATPase domain has functional con-servation between yeast and humans and suggest that a SWI/SNF protein complex is required for the activation of selective mammalian genes.
TL;DR: Both nucleolar and extra‐nucleolar foci remain after nucleolytic removal of approximately 90% chromatin, suggesting an underlying structure probably organizes groups of transcription units into ‘factories’ where transcripts are both synthesized and processed.
Abstract: HeLa cells were encapsulated in agarose microbeads, permeabilized and incubated with Br-UTP in a 'physiological' buffer; then sites of RNA synthesis were immunolabelled using an antibody that reacts with Br-RNA. After extending nascent RNA chains by < 400 nucleotides in vitro, approximately 300-500 focal synthetic sites can be seen in each nucleus by fluorescence microscopy. Most foci also contain a component of the splicing apparatus detected by an anti-Sm antibody. alpha-amanitin, an inhibitor of RNA polymerase II, prevents incorporation into these foci; then, using a slightly higher salt concentration, approximately 25 nucleolar foci became clearly visible. Both nucleolar and extra-nucleolar foci remain after nucleolytic removal of approximately 90% chromatin. An underlying structure probably organizes groups of transcription units into 'factories' where transcripts are both synthesized and processed.
TL;DR: It is demonstrated that progressive truncation of the TATA box results in a concomitant decrease in promoter strength as judged both by the loss of CAT activity in cell extracts and by a reduction in the steady-state level of cat mRNA.
TL;DR: Mfd appears to target the transcribed strand for repair by recognizing a stalledRNAP and actively recruiting the repair enzyme to the transcription blocking lesion as it dissociates the stalled RNAP.
Abstract: Lesions in the transcribed strand block transcription and are repaired more rapidly than lesions in the nontranscribed (coding) strand which do not block RNA polymerase (RNAP). It has been shown previously that in Escherichia coli the mfd (mutation frequency decline) gene is necessary for strand-specific repair. The mfd gene was cloned and sequenced and the Mfd protein was purified and used to reconstitute strand-specific repair in a completely defined system. The mfd gene encodes a protein of 130 kilodaltons and contains the so-called "helicase motifs," a leucine zipper motif, and regions of sequence similarity to UvrB and RecG proteins. The Mfd protein was shown to (i) displace RNAP stalled at a lesion in an adenosine triphosphate-dependent reaction, (ii) bind to the damage recognition subunit (UvrA) of the excision nuclease, and (iii) stimulate the repair of the transcribed strand only when transcription is taking place. Thus, Mfd appears to target the transcribed strand for repair by recognizing a stalled RNAP and actively recruiting the repair enzyme to the transcription blocking lesion as it dissociates the stalled RNAP.
TL;DR: It is demonstrated that the bZIP regions of c‐Fos and c‐Jun are capable of physically interacting with NF‐kappa B p65 through the Rel homology domain, which supports a combinatorial mechanism of gene regulation involving the unexpected cross‐coupling of two different classes of transcription factors to form novel protein complexes exhibiting potentiated biological activity.
Abstract: NF-kappa B and AP-1 represent distinct mammalian transcription factors that target unique DNA enhancer elements. The heterodimeric NF-kappa B complex is typically composed of two DNA binding subunits, NF-kappa B p50 and NF-kappa B p65, which share structural homology with the c-rel proto-oncogene product. Similarly, the AP-1 transcription factor complex is comprised of dimers of the c-fos and c-jun proto-oncogene products or of closely related proteins. We now demonstrate that the bZIP regions of c-Fos and c-Jun are capable of physically interacting with NF-kappa B p65 through the Rel homology domain. This complex of NF-kappa B p65 and Jun or Fos exhibits enhanced DNA binding and biological function via both the kappa B and AP-1 response elements including synergistic activation of the 5' long terminal repeat of the human immunodeficiency virus type 1. These findings support a combinatorial mechanism of gene regulation involving the unexpected cross-coupling of two different classes of transcription factors to form novel protein complexes exhibiting potentiated biological activity.
TL;DR: A chronology of key events and events leading to the formation of the HJ-Uridine Incorporation, In Situ Nick Translation, and subsequent efforts to correct for these errors.
Abstract: DNA REPLICATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 276 TRANSCRIPTION AND PRE-mRNA PROCESSING .... . . ) ....... ,.... 279 Localization of Transcriptionally Active Nuclear Regions by [ HJ-Uridine Incorporation or In Situ Nick Translation . . . . . . . . . . . . . . . . 279 Pre-mRNA Splicing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 280 An Arginine/Serine-Rich Domain Targets Proteins to Nuclear Speckles . . . . . . 284 Effect of RNA Polymerase II Transcription on the Localization of Splicing Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ,.... 285 Distribution of hnRNP Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287 Localization of Splicing Factors in the Amphibian Germinal Vesicle . . . . . . . . 289 Localization of Splicing Factors in Yeast 290 Distribution of Poly(A)'t RNA . . . . . . ... . . . . ..... . ..... . . . . .... 291 Distribution of Specific Cellular Transcripts . . . . . . . . . . . . . . . . . . . . . . . 291 NUCLEOLAR ORGANIZATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293
TL;DR: The IL-1 beta gene may be considered as an important additional member of the family of cytokine genes regulated in part by the NF-kappa B/rel family of transcription factors.
Abstract: The -300 region of the interleukin 1 beta (IL-1 beta) promoter contains a functional NF-kappa B binding site composed of the decamer sequence 5'-GGGAAAATCC-3'. Probes representing the -300 region or the NF-kappa B site alone interacted with NF-kappa B proteins present in phorbol myristate acetate-, lipopolysaccharide-, or Sendai virus-induced myeloid cell extracts as well as recombinant NFKB1 (p50) and RelA (p65); furthermore, NF-kappa B protein-DNA complex formation was dissociated in vitro by the addition of recombinant I kappa B alpha. Mutation of the NF-kappa B site in the context of the IL-1 beta promoter reduced the responsiveness of the IL-1 beta promoter to various inducers, including phorbol ester, Sendai virus, poly(rI-rC), and IL-1 beta. A 4.4-kb IL-1 beta promoter fragment linked to a chloramphenicol acetyltransferase reporter gene was also preferentially inducible by coexpression of individual NF-kappa B subunits compared with a mutated IL-1 beta promoter fragment. When multiple copies of the IL-1 beta NF-kappa B site were linked to an enhancerless simian virus 40 promoter, this element was able to mediate phorbol ester- or lipopolysaccharide-inducible gene expression. In cotransfection experiments, RelA (p65) and c-Rel (p85) were identified as the main subunits responsible for the activation of the IL-1 beta NF-kappa B site; also, combinations of NFKB1 (p50) and RelA (p65) or c-Rel and RelA were strong transcriptional activators of reporter gene activity. The presence of a functional NF-kappa B binding site in the IL-1 beta promoter suggests that IL-1 positively autoregulates its own synthesis, since IL-1 is a strong inducer of NF-kappa B binding activity. Thus, the IL-1 beta gene may be considered as an important additional member of the family of cytokine genes regulated in part by the NF-kappa B/rel family of transcription factors.
TL;DR: The screening of an adipocyte cDNA expression library suggests that ADD1 plays a role in the regulation of determination- and differentiation-specific gene expression in adipocytes.
Abstract: DNA-binding proteins containing the basic helix-loop-helix (bHLH) domain have been implicated in lineage determination and the regulation of specific gene expression in a number of cell types. By oligonucleotide screening of an adipocyte cDNA expression library, we have identified a novel member of the bHLH-leucine zipper transcription factor family designated ADD1. ADD1 mRNA is expressed predominantly in brown adipose tissue in vivo and is regulated during both determination and differentiation of cultured adipocyte cell lines. ADD1 can function as a sequence-specific transcriptional activator in that it stimulates expression of a chloramphenicol acetyltransferase vector containing multiple ADD1 binding sequences but is unable to activate the myosin light-chain enhancer, which contains multiple binding sites for another bHLH factor, MyoD. ADD1 can also activate transcription through a binding site present in the 5'-flanking region of the fatty acid synthetase gene which is expressed in a differentiation-dependent manner in adipose cells. These data suggest that ADD1 plays a role in the regulation of determination- and differentiation-specific gene expression in adipocytes.
TL;DR: Differential ability of members of the kappa B/Rel family to bind to, and activate transcription from, the IL-8 promoter is demonstrated and provides direct evidence for the role of RelA in regulation ofIL-8 gene expression.
Abstract: Interleukin-8 (IL-8), a chemotactic cytokine for T lymphocytes and neutrophils, is induced in several cell types by a variety of stimuli including the inflammatory cytokines IL-1 and tumor necrosis factor alpha TNF-alpha. Several cis elements, including a binding site for the inducible transcription factor NF-kappa B, have been identified in the regulatory region of the IL-8 gene. We have examined the ability of various NF-kappa B subunits to bind to, and activate transcription from, the IL-8 promoter. A nuclear complex was induced in phorbol myristate acetate-treated Jurkat T cells which bound specifically to the kappa B site of the IL-8 promoter and was inhibited by addition of purified I kappa B alpha to the reaction mixture. Only antibody to RelA (p65), but not to NFKB1 (p50), NFKB2 (p50B), c-Rel, or RelB was able to abolish binding, suggesting that RelA is a major component in these kappa B binding complexes. Gel mobility shift analysis with in vitro-translated and purified proteins indicated that whereas the kappa B element in the human immunodeficiency virus type 1 long terminal repeat bound to all members of the kappa B/Rel family examined, the IL-8 kappa B site bound only to RelA and to c-Rel and NFKB2 homodimers, but not to NFKB1 homodimers or heterodimers of NFKB1-RelA. Transient transfection analysis demonstrated a kappa B-dependent expression of the IL-8 promoter in a human fibrosarcoma cell line (8387) and in Jurkat T lymphocytes. Cotransfection with various NF-kappa B subunits indicated that RelA and c-Rel, but neither NFKB1 nor heterodimeric NFKB1-RelA, was able to activate transcription from the IL-8 promoter. Furthermore, cotransfection of NFKB1 and RelA, although able to support activation from the human immunodeficiency virus type 1 long terminal repeat, failed to activate expression from the IL-8 promoter. Antisense oligonucleotides to RelA, but not NFKB1, inhibited phorbol myristate acetate-induced IL-8 production in Jurkat T lymphocytes. These data demonstrate the differential ability of members of the kappa B/Rel family to bind to, and activate transcription from, the IL-8 promoter. Furthermore, while providing a novel example of a kappa B-regulated promoter in which the classical NF-kappa B complex is unable to activate transcription from the kappa B element, these data provide direct evidence for the role of RelA in regulation of IL-8 gene expression.
TL;DR: The amounts of the ilvBNC and ilvNC transcripts increased in response to the addition of alpha-ketobutyrate to the growth medium, correlated to an increase in specific AHAS activity, whereas IR activity was not increased because of the relatively large amount of theIlvC transcript present under all conditions assayed.
Abstract: Acetohydroxy acid synthase (AHAS) and isomeroreductase (IR) catalyze subsequent reactions in the flux of metabolites towards isoleucine, valine, leucine, and pantothenate. A 4,705-bp DNA fragment from Corynebacterium glutamicum known to code for AHAS and IR was sequenced and analyzed by Northern (RNA blot) analysis. As in other bacteria, the AHAS of this gram-positive organism is encoded by two genes, ilvB and ilvN. Gene disruption verified that these genes encode the single AHAS activity in C. glutamicum. The start of ilvB was determined by amino-terminal sequencing of a fusion peptide. By Northern analysis of the ilvBNC cluster, three in vivo transcripts of 3.9, 2.3, and 1.1 kb were identified, corresponding to ilvBNC, ilvNC, and ilvC messages, respectively. The ilvC transcript (encoding IR) was by far the most abundant one. With a clone from which the ilvB upstream regions had been deleted, only the ilvNC and ilvC transcripts were synthesized, and with a clone from which the ilvN upstream regions had been deleted, only the smallest ilvC transcript was formed. It is therefore concluded that in the ilv operon of C. glutamicum, three promoters are active. The amounts of the ilvBNC and ilvNC transcripts increased in response to the addition of alpha-ketobutyrate to the growth medium. This was correlated to an increase in specific AHAS activity, whereas IR activity was not increased because of the relatively large amount of the ilvC transcript present under all conditions assayed. Therefore, the steady-state level of the ilvBNC and ilvNC messages contributes significantly to the total activity of the single AHAS. The ilvC transcript of this operon, however, is regulated independently and present in a large excess, which is in accord with the constant IR activities determined.
TL;DR: A cDNA encoding a novel member of the G‐protein‐coupled receptor (GCR) superfamily, an ATP receptor, has been isolated from an embryonic chick whole brain cDNA library by hybridization screening.
TL;DR: It is shown that a cellular reducing catalyst thioredoxin (Trx) plays a major role in activation of the DNA binding of NF kappa B in vitro and stimulation of transcription from the NFKappa B-dependent gene expression and the redox control mechanism mediated by Trx might have a regulatory role in the NF k Kappa B-mediated gene expression.
TL;DR: The isolation of a gene (LAZ3) is reported that may act as a transcription regulator and play an important role in lymphomagenesis and the third most common specific translocations in non–Hodgkin's lymphoma.
Abstract: We have shown previously that chromosomal translocations involving chromosome 3q27 and immunoglobulin gene regions are the third most common specific translocations in non–Hodgkin's lymphoma (NHL). We now report the isolation of a gene that is disrupted in two cases by t(3;14) and t(3;4) translocations. The gene (LAZ3) encodes a 79 kDa protein containing six zinc–finger motifs and sharing amino–terminal homology with several transcription factors including the Drosophila tramtrack and Broad–complex genes, both of which are developmental transcription regulators. LAZ3 is transcribed as a 3.8 kb message predominantly in normal adult skeletal muscle and in several NHL carrying 3q27 chromosomal defects. We suggest that it may act as a transcription regulator and play an important role in lymphomagenesis.
TL;DR: A series of defective Sind Bis virus helper RNAs are described which can be used for packaging Sindbis virus RNA replicons and would be useful under conditions in which extensive amplification is advantageous.
Abstract: Since the recovery of infectious RNA transcripts from full-length cDNA clones, alphavirus genome RNAs have been engineered to allow expression of heterologous RNAs and proteins. The highest levels of expression of heterologous products are achieved when the viral structural genes are replaced by the heterologous coding sequences. Such recombinant RNAs are self-replicating (replicons) and can be introduced into cells as naked RNA, but they require trans complementation to be packaged and released from cells as infectious virion particles. In this report, we describe a series of defective Sindbis virus helper RNAs which can be used for packaging Sindbis virus RNA replicons. The defective helper RNAs contain the cis-acting sequences required for replication as well as the subgenomic RNA promoter which drives expression of the structural protein genes. In cells cotransfected with both the replicon and defective helper RNAs, viral nonstructural proteins translated from the replicon RNA allow replication and transcription of the defective helper RNA to produce the virion structural proteins. A series of defective helper RNAs were compared for the ability to package the replicon RNA as well as for the ability to be replicated and packaged. One defective helper RNA not only packaged the replicon but also was itself encapsidated and would be useful under conditions in which extensive amplification is advantageous. Other defective helper RNAs were able to package the replicon efficiently but were packaged very poorly themselves. These helpers should be useful for applications in which expression of the viral structural proteins or virus spread is not desired.
TL;DR: The transcription and processing site of the fibronectin gene localized to the nuclear interior and was associated with larger transcript domains in over 88 percent of the cells, which support a view of nuclear function closely integrated with structure.
Abstract: Visualization of fibronectin and neurotensin messenger RNAs within mammalian interphase nuclei was achieved by fluorescence hybridization with genomic, complementary DNA, and intron-specific probes. Unspliced transcripts accumulated in one or two sites per nucleus. Fibronectin RNA frequently accumulated in elongated tracks that overlapped and extended well beyond the site of transcription. Splicing appears to occur directly within this RNA track, as evidenced by an unambiguous spatial separation of intron-containing and spliced transcripts. Excised introns for neurotensin RNA appear free to diffuse. The transcription and processing site of the fibronectin gene localized to the nuclear interior and was associated with larger transcript domains in over 88 percent of the cells. These results support a view of nuclear function closely integrated with structure.
TL;DR: Strains deleted for both MBP1 and SWI4 were inviable, demonstrating that transcriptional activation by MBF and SBF has an important role in the transition from G1 to S phase.
Abstract: In budding yeast genes that encode G1 cyclins and proteins involved in DNA synthesis are transcriptionally activated in late G1. A transcription factor, called SBF, is composed of Swi4 and Swi6 proteins and activates transcription of G1 cyclin genes. A different, but related, complex called MBF binds to MCB elements (Mlu I cell cycle box) found in the promoter of most DNA synthesis genes. MBF contains Swi6 and a 120-kilodalton protein (p120). MBF was purified and the gene encoding p120 (termed MBP1) was cloned. A deletion of MBP1 was not lethal but led to deregulated expression of DNA synthesis genes, indicating a direct regulatory role for MBF in MCB-driven transcription. Mbp1 is related to Swi4. Strains deleted for both MBP1 and SWI4 were inviable, demonstrating that transcriptional activation by MBF and SBF has an important role in the transition from G1 to S phase.
TL;DR: These experiments provide direct evidence that SRF and Elk-1 functionally cooperate in the ternary complex at the SRE to regulate transcription.
TL;DR: This study examines pausing on hsp70 and two of the small heat shock genes at high resolution using a technique that utilizes paramagnetic particle-mediated selection of terminated run-on transcripts, which provides precise information on the distribution of RNA polymerase within each transcription unit.
Abstract: The regulation of many eukaryotic genes occurs at the level of transcriptional elongation. On the uninduced hsp70 gene of Drosophila melanogaster, for example, an RNA polymerase II complex has initiated transcription but has paused early in elongation. In this study, we examine pausing on hsp70 and two of the small heat shock genes (hsp27 and hsp26) at high resolution, using a technique that utilizes paramagnetic particle-mediated selection of terminated run-on transcripts. This technique provides precise information on the distribution of RNA polymerase within each transcription unit. It also details the progression of 5' cap formation on the elongating transcripts. For each gene, we find polymerases paused over a relatively narrow promoter-proximal region. The regions are generally around 20 nucleotides wide, with two preferred pausing positions spaced roughly 10 nucleotides apart or about one turn of the helix. The bulk of capping occurs as transcripts pass between 20 and 30 nucleotides in length. Interestingly, in the three genes examined here, elongational pausing and 5' cap formation appear largely coincident.
TL;DR: Two factors, Dr2 and ACF, necessary for high levels of transcription in response to an activator have been identified and can enhance basal and activated transcription.
Abstract: Reconstituted transcription reactions containing the seven general transcription factors, in addition to RNA polymerase II, respond poorly to transcriptional activators. Two factors, Dr2 and ACF, necessary for high levels of transcription in response to an activator have been identified. ACF can enhance basal and activated transcription. Dr2 represses basal transcription, but this can be overcome by transcriptional activators or TFIIA. Dr2 is human DNA topoisomerase I. The DNA relaxation activity of topoisomerase I is dispensable for transcriptional repression. The effect of Dr2 is specific for TATA-box-containing promoters and is mediated by the TATA-binding protein.