Rate Of Transcription Initiation Research Articles

Inhibition of HIV-1 replication by 5'LTR decoy RNA

Long terminal repeats (5'-LTR) contain essential enhancer and promoter elements that act as targets for cellular and viral regulatory proteins. The interplay of these cis-acting sequences with specific transcription factors (NF kappa B, Ap1, and Sp1) present in infected cells determines the rate of transcription initiation from the 5'-LTR promoter element, and thus strongly influences the level of viral replication. Furthermore, Tat functions to enhance transcriptional elongation by binding to the trans-activation response element (TAR). These transcriptional factors enhance the transcriptional elongation from the HIV-1 promoter. HIV-1 genomic RNA packaging is a highly specific process involving interactions between the viral Gag precursor and cis acting packaging signals in the 5' leader sequence of HIV-1 genomic RNA. We examined the suppression effect of HIV-1 expression by the decoy RNAs targeted to these HIV-1 transcriptional and packaging regulatory regions. HIV-1 expression was inhibited by the 5'-LTR decoy RNA, and measured by the amount of HIV-1 gag p24 protein, but no inhibition of HIV-1 packaging could be detected.

Signal transduction cascade for regulation of RpoS: temperature regulation of DsrA.

Many environmental parameters modulate the amount of the RpoS sigma factor in Escherichia coli. Temperature control of RpoS depends on the untranslated RNA DsrA. DsrA activates RpoS translation by pairing with the leader of the mRNA. We find that temperature affects both the rate of transcription initiation of the dsrA gene and the stability of DsrA RNA. Both are increased at low temperature (25 degrees C) compared to 37 or 42 degrees C. The combination of these results is 25-fold-less DsrA at 37 degrees C and 30-fold less at 42 degrees C than at 25 degrees C. Using an adapted lacZ-based reporter system, we show that temperature control of transcription initiation of dsrA requires only the minimal promoter of 36 bp. Overall, transcription responses to temperature lead to a sixfold increase in DsrA synthesis at 25 degrees C over that at 42 degrees C. Furthermore, two activating regions and a site for LeuO negative regulation were identified in the dsrA promoter. The activating regions also activate transcription in vitro. DsrA decays with a half-life of 23 min at 25 degrees C and 4 min at 37 and 42 degrees C. These results demonstrate that the dsrA promoter and the stability of DsrA RNA are the thermometers for RpoS temperature sensing. Multiple inputs to DsrA accumulation allow sensitive modulation of changes in the synthesis of the downstream targets of DsrA such as RpoS.

Activation and repression of transcription initiation in bacteria.

Transcription initiation is the principal step at which bacterial gene expression is regulated. Bacterial transcription is due to a single multisubunit RNA polymerase. The potential transcription initiation rate of any promoter is set by the efficiency with which RNA polymerase recognizes the different promoter sequence elements. The sigma subunit plays the major role in the process of promoter recognition. Different RNA polymerase sigma subunits can guide RNA polymerase to different promoters. The E. coli genome encodes seven different sigma subunits, each of which allows the cell to respond to different environmental stimuli. A large number of transcription factors up-regulate and down-regulate expression from different promoters in response to environmental signals. Many transcription activators function by making a direct interaction with RNA polymerase. Some activators function by altering the conformation of promoter DNA. Most transcription repressors function by blocking access of RNA polymerase to their target promoter. In some cases, optimal repression depends on multiply bound repressor molecules that interact in complex ways. Many promoters are regulated by more than one transcription factor. A variety of mechanisms whereby a promoter can be regulated by a repressor and an activator, or by two activators, is known.

Construction of protein overproducer strains in Bacillus subtilis by an integrative approach.

We evaluated the effect of several genetic factors reported as having a role in the induction of the expression of significant levels of recombinant protein in Bacillus subtilis. We utilized the beta-galactosidase reporter protein from Escherichia coli as our model for measuring the overproduction of heterologous proteins in B. subtilis. The lacZ gene was expressed in B. subtilis using the regulatory region of the subtilisin gene aprE. In this study, we considered factors known to modulate the transcription and translation initiation rates and genetic and mRNA stability. We also consider the effects of different genetic backgrounds, such as degU32 and hpr2, that until now have been studied independently. By changing the native -35 promoter box to the consensus TTGACA sequence of the aprE promoter, a significant 100-fold increase in the beta-galactosidase activity was obtained. On the other hand, changes such as the GTG to ATG start codon, the construction of a consensus AAGGAGG ribosome binding site, and the addition of the cryIIIA transcription terminator at the 3' end of the lacZ gene, produced only marginal effects on the final beta-galactosidase activity.

A transcription reinitiation intermediate that is stabilized by activator.

High levels of gene transcription by RNA polymerase II depend on high rates of transcription initiation and reinitiation. Initiation requires recruitment of the complete transcription machinery to a promoter, a process facilitated by activators and chromatin remodelling factors. Reinitiation probably occurs through a different pathway. After initiation, a subset of the transcription machinery remains at the promoter, forming a platform for assembly of a second transcription complex. Here we describe the isolation of a reinitiation intermediate that includes transcription factors TFIID, TFIIA, TFIIH, TFIIE and Mediator. This intermediate can act as a scaffold for formation of a functional reinitiation complex. Formation of this scaffold is dependent on ATP and TFIIH. The scaffold is stabilized in the presence of the activator Gal4-VP16, but not Gal4-AH, suggesting a new role for some activators and Mediator in promoting high levels of transcription.

Transcriptional Regulation of L-type Calcium Channel Expression in Cardiac Myocytes

The L-type calcium channel is a heteromultimeric protein complex, which is expressed in the cardiac sarcolemma. Although post-translational regulation of its subunits by protein kinase A (PKA) has been widely reported, little is known about molecular processes that regulate expression of calcium channel subunits (α1C,α2 - δ, and β2Asubunits). Previous studies from our group demonstrate that the steady-state mRNA level of the α1Cunit is increased by treatment of myocytes with β -adrenergic agonists. The current study is designed to determine whether the mRNA levels for all subunits of the L-type calcium channel are coordinately controlled by a β -adrenergic agonist, and whether this occurs predominantly through control of rate of transcription. Nuclear run-on assays were used to determine the transcription initiation rate of these genes in cultured neonatal rat cardiac myocytes. In isoproterenol (10−7m)-treated myocytes, transcription of genes encoding the α1C, α2- δ, and β2Asubunits was enhanced. The increases in transcription initiation rate for α1C, α2- δ, andβ2A subunits genes were 404%, 367%, and 240% of control, respectively. Pretreatment with the β -adrenergic antagonist propranolol (10−5m) or PKA inhibitor H-89 (10−6m) blocked the effects of isoproterenol, while either drug alone did not affect the gene transcription rate significantly. Steady state mRNA levels of the subunits increased following isoproterenol treatment. These results suggest that β -adrenergic stimulation and the PKA signaling pathway play an important role in transcriptional regulation of the L-type calcium channel in myocyte. The expression of all the subunits of this ion channel is under coordinate transcriptional control.

A novel negative cis-regulatory element on the hepatitis B virus S-(+)-strand.

Hepatitis B virus (HBV) has a double-stranded DNA genome. The minus-strand contains coding regions for all known HBV proteins and most of the cis-regulatory elements. Little is known about transcription from the S-(+)-strand and its regulation. Thus, the presence of regulatory elements located on the S-(+)-strand was investigated by inserting nt 1038-1783 of HBV in both orientations between the human cytomegalovirus (HCMV) promoter and a luciferase gene. Transfection experiments revealed that the plasmid containing this HBV DNA fragment in an orientation allowing expression from the S-(+)-strand (antisense) led to inhibition of luciferase gene expression compared to the plasmid containing this sequence in an orientation that allows gene expression from the L-(-)-strand (sense). Deletion analyses delimit the sequence essential for the inhibitory effect to a 150 bp region that also carries part of the enhancerII/core promoter complex. However, the possible influence of this regulatory element has been excluded in various experiments. The repressing HBV sequence acts in an orientation- and position-dependent manner; no inhibition was observed when this DNA element was inserted upstream of the HCMV promoter or downstream of the luciferase gene. Northern blot analyses revealed reduced luciferase mRNA steady-state levels in cells transfected with constructs containing the essential HBV sequence in antisense orientation compared to plasmids containing this sequence in sense orientation. Since nuclear run-on experiments showed similar transcription initiation rates with these plasmids, the diminished luciferase mRNA steady-state levels must be due to altered stabilities, suggesting that nt 1783-1638 of HBV encode an RNA-destabilizing element.

Virus-inducible expression of a host chemokine gene relies on replication-linked mRNA stabilization.

Analysis of the host response to viral infection generally has focused on the capacity of viruses to activate or repress transcription of cellular genes, and this approach is also characteristic of work on RNA viruses such as respiratory syncytial virus (RSV). In the present study, it appeared initially that RSV-driven expression of a critical immune regulator, the beta-chemokine RANTES (regulated upon activation, normal T cell expressed and secreted), in primary-culture airway epithelial cells also depended on inducible gene transcription because expression was accompanied by coordinate increases in transcriptional initiation rate and gene promoter activity. However, RSV-driven increases in RANTES gene transcription and promoter activity were small and transient relative to RANTES expression, and they were no different in size and duration than for inactivated RSV that was incapable of fully inducing RANTES expression. These findings suggested that the increase in RANTES gene transcription was not sufficient for inducible expression and that critical regulatory effects occurred at a posttranscriptional level. This type of mechanism for virus-inducible expression of RANTES was established when we found that replicating (but not inactivated) RSV markedly increased RANTES mRNA half-life (from 0.8 to 6.8 h). In addition, RNase protection assays of heterologous promoter/reporter plasmids indicate that basal instability of RANTES mRNA is mediated at least in part by nucleotides 11-389 of the RANTES gene, and this region is also the target for induction by virus. The distinct pathway for production of RANTES (in combination with cytokine-dependent expression of RANTES and related immune-response genes) may more effectively coordinate immune cell interaction with epithelial barrier cells to mediate host defense.

Mechanism of Regulation of Complement Receptor Type 1 Transcription by Cytosine Arabinoside in a Pre‐Erythroid Model

Binding to erythrocyte complement receptor type 1 (CR1) clears immune complexes from blood and tissues, preventing complement-mediated pathological inflammation in disease. Previous work has demonstrated that Ara-C, a cytosine analogue, induces an 11-fold increase in CR1 mRNA expression in K-562 erythroleukaemia cells. In this work we therefore investigated whether the Ara-C/K-562 system could be used as a model for studying the pre-erythroid regulation of CR1. We demonstrated that increased CR1 expression could be induced independently of increased haemoglobin expression. Increases in CR1 mRNA levels produced by Ara-C treatment were not a function of increased stability of the message. However, Ara-C induced a protein synthesis-dependent increase in transcription initiation rate as early as 12h after treatment. Further data suggest that the effect of Ara-C on transcription is not a result of its direct DNA-damaging or DNA polymerase-inhibition activities. Induction of receptor transcription was inhibited by tyrosine kinase (TK) and protein kinase C (PKC) inhibitors. These data suggest that TK, PKC and dCTP-adducted phospholipid signalling pathways may all play a role in the mechanism of Ara-C-induced CR1 transcription.

Chromatin control of HIV-1 gene expression.

Upon infection of susceptible cells, the RNA genome of the human immunodeficiency virus type 1 (HIV-1) is reverse transcribed into double-stranded DNA, which can be subsequently integrated into the cellular genome. After integration, the viral long terminal repeat (LTR) promoter is present in a nucleosome-bound conformation and is transcriptionally silent in the absence of stimulation. Activation of HIV-1 gene expression is concomitant with an acetylation-dependent rearrangement of the nucleosome positioned at the viral transcription start site. Thus, similar to most cellular genes, the transcriptional state of the integrated HIV-1 provirus is closely linked to histone acetylation. This enzymatic activity results from the function of histone-specific nuclear acetyltransferase (HAT) enzymes. Efficient viral transcription is strongly dependent on the virally-encoded Tat protein. the mechanism by which Tat increases the rate of transcriptional initiation has been recently demonstrated and involves the interaction of Tat with the transcriptional coactivator p300 and the closely related CREB-binding protein (CBP), having histone acetyltransferase activity.

Regulation of human IP-10 gene expression in astrocytoma cells by inflammatory cytokines

Because of its prominent expression in central nervous system inflammatory pathology by astrocytes, we examined the mechanism of human IP-10 (hIP-10) gene induction by interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) in astrocytoma cells. When present together, IFN-gamma and TNF-alpha induced robust accumulation of hIP-10 mRNA, but hIP-10 mRNA was minimally induced when astrocytoma cells were treated with individual cytokines. This pattern of expression resembled that previously described for murine IP-10 (mIP-10) gene induction in fibroblasts and in rat astroglia. Nuclear run-on experiments showed that the synergistic effect of the cytokines resulted from an increased rate of IP-10 transcriptional initiation. Functional analysis of the hIP-10 promoter after deletion and substitution mutagenesis indicated that an interferon-stimulated response element (ISRE) governed both simple response to IFN-gamma and synergy with TNF-alpha. Synergistic induction of hIP-10 also required an ISRE-proximal nuclear factor kappa-B (NFkappaB) binding site. TNF-alpha-induced NFkappaB binding activity at this site was composed of RelA (p65) homodimers. Our results document that cis-elements through which cytokines mediate synergistic induction of IP-10 in mouse and human are strictly conserved despite divergence elsewhere within the proximal 5'-flanking region.

Mechanism of Action of RNA Polymerase II Elongation Factor Elongin: MAXIMAL STIMULATION OF ELONGATION REQUIRES CONVERSION OF THE EARLY ELONGATION COMPLEX TO AN ELONGIN-ACTIVABLE FORM

We previously identified and purified Elongin by its ability to stimulate the rate of elongation by RNA polymerase II in vitro (Bradsher, J. N., Jackson, K. W., Conaway, R. C., and Conaway, J. W. (1993) J. Biol. Chem. 268, 25587-25593). In this report, we present evidence that stimulation of elongation by Elongin requires that the early RNA polymerase II elongation complex undergoes conversion to an Elongin-activable form. We observe (i) that Elongin does not detectably stimulate the rate of promoter-specific transcription initiation by the fully assembled preinitiation complex and (ii) that early RNA polymerase II elongation intermediates first become susceptible to stimulation by Elongin after synthesizing 8-9-nucleotide-long transcripts. Furthermore, we show that the relative inability of Elongin to stimulate elongation by early elongation intermediates correlates not with the lengths of their associated transcripts but, instead, with the presence of transcription factor IIF (TFIIF) in transcription reactions. By exploiting adenovirus 2 major late promoter derivatives that contain premelted transcriptional start sites and do not require TFIIF, TFIIE, or TFIIH for transcription initiation, we observe (i) that Elongin is capable of strongly stimulating the rate of synthesis of trinucleotide transcripts by a subcomplex of RNA polymerase II, TBP, and TFIIB and (ii) that the ability of Elongin to stimulate synthesis of these short transcripts is substantially reduced by addition of TFIIF to transcription reactions. Here we present these findings, which are consistent with the model that maximal stimulation of elongation by Elongin requires that early elongation intermediates undergo a structural transition that includes loss of TFIIF.

Promoter regions of four Balbiani ring genes in Chironomus tentans exhibit a common salivary gland-specific chromatin organisation, which is independent of the rate of transcriptional initiation.

The Balbiani ring (BR) gene family consists of four closely related genes which show some differences in transcriptional regulation. Here we analyse the chromatin structure of the promoter regions of the four BR genes. In cells in which the genes are inactive, digestion of chromatin with micrococcal nuclease suggests for the BR1 gene that precisely positioned nucleosomes are present from position +200 to -600. In salivary gland cells, a distinctly different organisation of the promoter chromatin leads to the appearance of two sites that are hypersensitive to both DNase I and micrococcal nuclease, located at the approximate positions -50 and -240. Nuclease digestion experiments suggest the presence of a nucleosome in the region between the two hypersensitive sites, and nucleosome disruption at the transcription initiation site. The positions of these hypersensitive sites in all BR genes is unaffected by extensive modulation of the rate of transcriptional initiation, although some alterations in the intensity of the hypersensitive sites were observed. We also show that total nucleosome displacement from the transcribing template is seen only when the RNA polymerase density exceeds one polymerase molecule per 100 bp.

Visualization of single RNA transcripts in situ.

Fluorescence in situ hybridization (FISH) and digital imaging microscopy were modified to allow detection of single RNA molecules. Oligodeoxynucleotide probes were synthesized with five fluorochromes per molecule, and the light emitted by a single probe was calibrated. Points of light in exhaustively deconvolved images of hybridized cells gave fluorescent intensities and distances between probes consistent with single messenger RNA molecules. Analysis of beta-actin transcription sites after serum induction revealed synchronous and cyclical transcription from single genes. The rates of transcription initiation and termination and messenger RNA processing could be determined by positioning probes along the transcription unit. This approach extends the power of FISH to yield quantitative molecular information on a single cell.

Gap junction Cx26 gene modulation by phorbol esters in benign and malignant human mammary cells

Connexin (Cx) 26, a major gap junction protein expressed in mammary epithelial cells, has been considered to be a tumor suppressor gene candidate. This study investigated the molecular mechanism of transcriptional up-regulation of Cx26 by phorbol ester (TPA) in human immortalized MCF-10 mammary epithelial cells and MDA-MB-231 mammary cancer cells. Such up-regulation was mediated through the protein kinase C pathway and could be blocked by the PKC inhibitor, calphostin C. Based on the results of the nuclear run-on assay, there was a TPA-induced increase in the rate of transcriptional initiation. We identified a TPA-induced DNase I hypersensitivity (DH) region approximately 1 kb 5′ upstream of the ATG translation starting site. Sequence analysis revealed that this DH region was located in intron 1 and contained two TRE-like TGAT/ATCA elements, two 5′TTCA3′ motifs and a 5′AGGAAG3′ PEA3 motif. Both TRE-like elements were capable of binding AP1. TPA inducibility of this DH region was seen by the CAT reporter assay and appeared to be direction-dependent suggesting a functional cooperation between PEA3/TTCA and TRE.

Mechanism of induction of rat hepatic CYP2B and 3A by the pesticide methoxychlor.

Our earlier investigation demonstrated that methoxychlor treatment induced hepatic cytochrome P450 2B1/2B2 and 3A proteins in rats and enhanced their respective enzymatic activities. To determine the mechanism of the methoxychlor-mediated induction and whether methoxychlor acts as a frank inducer and not merely by causing stabilization of the cytochrome P450 proteins or of their mRNAs, we assessed the effect of methoxychlor treatment on the transcriptional rate and mRNA levels of these hemeproteins by nuclear run-on and northern blot analyses. Methoxychlor treatment markedly elevated the transcriptional rates of cytochrome P4502B1/2B2 and 3A. Also, higher levels of mRNA of these CYPs were observed. By contrast, mRNA levels of CYP1A1/1A2 were not affected. Using a nuclear run-on method that apparently measures specifically the transcription initiation rate of CYP2B1 and 2B2, we observed that the transcriptional rate of 2B2 was increased substantially more than that of CYP2B1. This differential induction of CYP2B2 versus 2B1 may account for the discrepancy between the levels of induced CYP2B protein and the much lower-than-expected CYP2B-mediated enzymatic activity observed in our earlier study.

Enhanced expression of the shape Bacillus thuringiensis cry9Aa2 gene in transgenic plants by nucleotide sequence modification confers resistance to potato tuber moth

The Bacillus thuringiensis cry9Aa2 gene encodes a 129 kDa protein with insecticidal activity against Lepidoptera, including the larvae of potato tuber moth (Phthorimaea operculella). The insecticidal moiety of Cry9Aa2 resides within the N-terminal 665 amino acids. Site-directed mutagenesis was used to modify a truncated version of the gene (cry9Aa2T nucleotides 1–1995), removing motifs likely to be deleterious to full-length transcription and transcript stability in plants. The codon usage of the gene was also altered to that more similar to the codon bias of dicotyledonous plant genes. The native gene and three modified versions of cry9Aa2T, with incremental modifications from the 5' end, were each transformed into Nicotiana tabacum, under the control of the CaMV 35S promoter. Plants transformed with the native gene did not show resistance to potato tuber moth larvae. In contrast, significant levels of larval mortality and reductions in larval growth and leaf damage were observed on many of the plants transformed with the modified genes. The cry9Aa2T mRNA was barely detectable in plants transformed with the native gene, whereas significant accumulation of full-length cry9Aa2T transcript was seen in plants transformed with modified genes. Modifications in the 5'-terminal 693 nucleotides of cry9Aa2T had the most significant effect on increasing the steady-state levels of cry mRNA. Transcription initiation rates of both the native and modified cry9Aa2T genes were similar, suggesting that the lack of native transcript accumulation was a consequence of transcript instability and that the sequence modifications had significantly improved the stability of the cry9Aa2T transcript. This improvement in steady-state full-length transcript levels resulted in expression of the insecticidal gene in N. tabacum to levels which conferred significant resistance to potato tuber moth larvae. Abbreviations: CaMV, cauliflower mosaic virus; IPTG, isoprophylthio-β-galactoside; PCR, polymerase chain reaction; poly(A), polyadenylation; PTM, potato tuber moth; UTR, untranslated region.

Role of open complex instability in kinetic promoter selection by bacteriophage T7 RNA polymerase

By measuring steady-state rates of dinucleotide synthesis on double-stranded (d.s.) and partially single-stranded (p.s.s.) promoters, and topological unwinding due to open complex formation on plasmids, we have obtained evidence that open complex formation in bacteriophage T7 RNA polymerase:promoter binary complexes is thermodynamically disfavored and that the rate of collapse of the open complex is competitive with the rate of transcription initiation. It is suggested that open complex instability is a kinetic mechanism that allows T7 RNA polymerase (RNAP) to achieve promoter specificity while still allowing for efficient promoter release. Open complex instability could also provide a mechanism for modulating the KMfor the initiating NTPs so as to allow different promoters to respond differently to physiological changes in NTP concentration.

Activation of gene expression by herpes simplex virus type 1 ICP0 occurs at the level of mRNA synthesis.

ICP0 is a nuclear phosphoprotein involved in the activation of herpes simplex virus type 1 (HSV-1) gene expression during lytic infection and reactivation from viral latency. Although available evidence suggests that ICP0 acts at the level of transcription, definitive studies specifically addressing this issue have not been reported. In the present study we measured the ability of ICP0 to activate gene expression (i) from promoters representing the major kinetic classes of viral genes in transient expression assays and (ii) from the same promoters during viral infection at multiplicities of infection ranging from 0.1 to 5.0 PFU/cell. The levels of synthesis and steady-state accumulation of mRNA, mRNA stability, and levels of protein synthesis were compared in cells transfected with a reporter plasmid in the presence and absence of ICP0 and in cells infected with wild-type HSV-1 or an ICP0 null mutant, n212. In transient expression assays and during viral infection at all multiplicities tested, the levels of steady-state mRNA and protein were significantly lower in the absence of ICP0, indicating that ICP0 activates gene expression at the level of mRNA accumulation. In transient expression assays and during infection at low multiplicities (< 1 PFU/cell) in the presence or absence of ICP0, marked increases in the levels of viral mRNAs accompanied by proportional increases in the levels of protein synthesis were observed with increasing multiplicity. At a high multiplicity (5 PFU/cell) in the presence or absence of ICP0, mRNA levels did not increase as a function of multiplicity and changes in the levels of protein were no longer related to changes in the levels of mRNA. Collectively, these tests indicate that transcription of viral genes is rate limiting at low multiplicities and that translation is rate limiting at high multiplicities, independent of ICP0. Consistent with the lower levels of mRNA detected in the absence of ICP0, the rates of transcription initiation measured by nuclear run-on assays were uniformly lower in cells infected with the ICP0 null mutant at all multiplicities tested, implying that ICP0 enhances transcription at or before initiation or both. No evidence was found of posttranscriptional effects of ICP0 (i.e., effects on the stability of mRNA, nuclear-cytoplasmic distribution, polyribosomal mRNA distribution, or rates of protein synthesis). Taken together, these results suggest that ICP0 activates gene expression prior to or at the level of initiation of mRNA synthesis in transient expression assays and during viral infection. Based on these findings; we hypothesize that the exaggerated multiplicity-dependent growth phenotype characteristic of ICP0 null mutants reflects the requirement for ICP0 under conditions where the steady-state level of mRNA is rate limiting, such as during low-multiplicity infection and reactivation from latency.

Distribution and configuration of c-myc RNA during transcriptional attenuation in differentiating cells in-situ.

Previous northern studies of c-myc RNA downregulation during early cellular differentiation have shown reduced levels of mature transcript within 6-24 h, attributed to attenuation of transcription at pause sites downstream of the P2 promoter. The transcription initiation rate has been shown to be decreased in some and increased in other such studies. We assessed the contribution of RNA trafficking to c-myc reduction during differentiation by examining the localisation and configuration of exon-specific transcripts, using oligonucleotide probes and fluorescent in-situ hybridisation, in HL60 cells induced to differentiate with 12-O-tetradecanoylphorbol-13-acetate. A 2,4-dinitrophenyl-labelled probe to c-myc exon 3 sequences gave a strong cytoplasmic and nucleolar hybridisation signal in undifferentiated cells, which decreased markedly after 24 h of differentiation. Nucleolar staining for c-myc RNA colocalised with that from a probe for ribosomal 28 S RNA. The signal from an exon 1 probe specific for sequences upstream of the c-myc P2 promoter was much weaker, but increased in the nuclei of differentiating cells, which possessed unusual ring-like or lamellar deposits, outside the nucleolus. These deposits appeared faintly together with nuclear staining with the exon 1 sense probe but not the exon 3 sense probe in differentiating cells. These findings demonstrate that within the first 24 h of differentiation, full-length c-myc RNA, which is compartmentalised as expected for a mature transcript, is considerably downregulated but nuclear primary RNA continues to be transcribed from exon 1. This is in a configuration similar to that reported for unspliced transcripts and is not elongated into exon 3. Antisense transcription with these RNA morphological features also occurs in exon 1 during differentiation. These results indicate significant changes in the intracellular dynamics of c-myc RNA during differentiation and support transcriptional attenuation and post-transcriptional processes, such as splicing, rather than reduced transcription initiation as the primary mechanism of c-myc downregulation in the early phases of differentiation.